Professor Kristian Helin
Group Leader: Epigenetics and Cancer
OrcID: 0000-0003-1975-6097
Email: [email protected]
OrcID: 0000-0003-1975-6097
Email: [email protected]
Biography
Professor Kristian Helin is the Chief Executive and President of The Institute of Cancer Research, London. He started at the ICR in September 2021.
See all news stories on our website that feature the work of Professor Helin and colleagues.
Kristian Helin obtained his MSc in Chemical Engineering from the Technical University of Denmark and his PhD from the University of Copenhagen. Between 1991 and 1993, he was a research fellow in the laboratory of Professor Ed Harlow at the Massachusetts General Hospital and Harvard Medical School in Boston. Here, he identified and cloned the first members of the E2F transcription factor family and showed their importance for understanding the function of the retinoblastoma protein.
He returned to Denmark in 1993 to start as a group leader at the Danish Cancer Society in Copenhagen and moved two years later to the European Institute of Oncology in Milan, Italy, to become a founding member of the Department of Experimental Oncology.
As a group leader in Copenhagen and in Italy, his lab focused on understanding the role of the E2F transcription factors in cell cycle control, DNA replication and apoptosis. Moreover, the group started projects related to DNA damage and mitosis, and took the first steps into studying the function of epigenetic proteins in cancer.
In 2003 Kristian Helin was appointed as the founding director of a new research centre in Copenhagen, named the Biotech Research and Innovation Centre (BRIC), with the task of establishing a centre of excellence. He remained the Director of BRIC for more than 15 years and in that period, BRIC developed into a world-class research centre with approximately 250 employees, funded primary on external competitive grants.
While at BRIC, Professor Helin also established a Center for Epigenetics, which was funded by the Danish National Research Foundation (2007-2017), was a founding member of the Danish Stem Cell Center (2012-2021), and was also interim Vice-Dean for Research at the Faculty of Health and Medical Sciences at the University of Copenhagen (2017). In 2018, he took up a position at the Memorial Sloan Kettering Cancer Center as the Chair of Cell Biology and Director of the Center for Epigenetics Research.
The Helin laboratory has made several seminal discoveries in the field of cell cycle control, epigenetics and cancer. In addition to providing novel insights into mechanisms regulating transcription, stem cell identity and differentiation, the work in Professor Helin’s lab has led to the establishment of biotech company EpiTherapeutics, which in 2015 was sold to Gilead Inc.
The lab continues studying the role of epigenetic factors in transcription, cell fate decisions and in cancer. In recent years, the group has developed a particular interest in acute myeloid leukemia, in part because there is an unmet need for the development of novel therapies and in part because many epigenetic regulators are altered in AML. The work on AML has led to the identification of potential novel targets for the treatment of AML and insights into how normal haematopoiesis is regulated.
Professor Helin is an elected member of European Molecular Biology Organisation (EMBO) and the Royal Danish Academy of Science and Letters. He has received several prestigious awards for outstanding biomedical research and serves on several editorial boards, advisory boards and grant committees.
Related pages
Types of Publications
Journal articles
Nuclear receptor-binding SET-domain protein 1 (NSD1), a methyltransferase that catalyzes H3K36me2, is essential for mammalian development and is frequently dysregulated in diseases, including Sotos syndrome. Despite the impacts of H3K36me2 on H3K27me3 and DNA methylation, the direct role of NSD1 in transcriptional regulation remains largely unknown. Here, we show that NSD1 and H3K36me2 are enriched at cis-regulatory elements, particularly enhancers. NSD1 enhancer association is conferred by a tandem quadruple PHD (qPHD)-PWWP module, which recognizes p300-catalyzed H3K18ac. By combining acute NSD1 depletion with time-resolved epigenomic and nascent transcriptomic analyses, we demonstrate that NSD1 promotes enhancer-dependent gene transcription by facilitating RNA polymerase II (RNA Pol II) pause release. Notably, NSD1 can act as a transcriptional coactivator independent of its catalytic activity. Moreover, NSD1 enables the activation of developmental transcriptional programs associated with Sotos syndrome pathophysiology and controls embryonic stem cell (ESC) multilineage differentiation. Collectively, we have identified NSD1 as an enhancer-acting transcriptional coactivator that contributes to cell fate transition and Sotos syndrome development.
Novel therapies for the treatment of acute myeloid leukemia (AML) are urgently needed, because current treatments do not cure most patients with AML. We report a domain-focused, kinome-wide CRISPR-Cas9 screening that identified protein kinase targets for the treatment of AML, which led to the identification of Rio-kinase 2 (RIOK2) as a potential novel target. Loss of RIOK2 led to a decrease in protein synthesis and to ribosomal instability followed by apoptosis in leukemic cells, but not in fibroblasts. Moreover, the ATPase function of RIOK2 was necessary for cell survival. When a small-molecule inhibitor was used, pharmacological inhibition of RIOK2 similarly led to loss of protein synthesis and apoptosis and affected leukemic cell growth in vivo. Our results provide proof of concept for targeting RIOK2 as a potential treatment of patients with AML.
Activation of interferon genes constitutes an important anticancer pathway able to restrict proliferation of cancer cells. Here, we demonstrate that the H3K9me3 histone methyltransferase (HMT) suppressor of variegation 3-9 homolog 1 (SUV39H1) is required for the proliferation of acute myeloid leukemia (AML) and find that its loss leads to activation of the interferon pathway. Mechanistically, we show that this occurs via destabilization of a complex composed of SUV39H1 and the two H3K9me2 HMTs, G9A and GLP. Indeed, loss of H3K9me2 correlated with the activation of key interferon pathway genes, and interference with the activities of G9A/GLP largely phenocopied loss of SUV39H1. Last, we demonstrate that inhibition of G9A/GLP synergized with DNA demethylating agents and that SUV39H1 constitutes a potential biomarker for the response to hypomethylation treatment. Collectively, we uncovered a clinically relevant role for H3K9me2 in safeguarding cancer cells against activation of the interferon pathway.
Two recurrent mutations, K27M and G34R/V, within histone variant H3.3 were recently identified in ∼50% of pHGGs. Both mutations define clinically and biologically distinct subgroups of pHGGs. Here, we provide further insight about the dominant-negative effect of K27M mutant H3.3, leading to a global reduction of the repressive histone mark H3K27me3. We demonstrate that this is caused by aberrant recruitment of the PRC2 complex to K27M mutant H3.3 and enzymatic inhibition of the H3K27me3-establishing methyltransferase EZH2. By performing chromatin immunoprecipitation followed by next-generation sequencing and whole-genome bisulfite sequencing in primary pHGGs, we show that reduced H3K27me3 levels and DNA hypomethylation act in concert to activate gene expression in K27M mutant pHGGs.
CRISPR interference (CRISPRi) represents a newly developed tool for targeted gene repression. It has great application potential for studying gene function and mapping gene regulatory elements. However, the optimal parameters for efficient single guide RNA (sgRNA) design for CRISPRi are not fully defined. In this study, we systematically assessed how sgRNA position affects the efficiency of CRISPRi in human cells. We analyzed 155 sgRNAs targeting 41 genes and found that CRISPRi efficiency relies heavily on the precise recruitment of the effector complex to the target gene transcription start site (TSS). Importantly, we demonstrate that the FANTOM5/CAGE promoter atlas represents the most reliable source of TSS annotations for this purpose. We also show that the proximity to the FANTOM5/CAGE-defined TSS predicts sgRNA functionality on a genome-wide scale. Moreover, we found that once the correct TSS is identified, CRISPRi efficiency can be further improved by considering sgRNA sequence preferences. Lastly, we demonstrate that CRISPRi sgRNA functionality largely depends on the chromatin accessibility of a target site, with high efficiency focused in the regions of open chromatin. In summary, our work provides a framework for efficient CRISPRi assay design based on functionally defined TSSs and features of the target site chromatin.
Protein arginine methyltransferase 5 (PRMT5) has emerged as a promising cancer drug target, and three PRMT5 inhibitors are currently in clinical trials for multiple malignancies. In this study, we investigated the role of PRMT5 in human acute myeloid leukemia (AML). Using an enzymatic dead version of PRMT5 and a PRMT5-specific inhibitor, we demonstrated the requirement of the catalytic activity of PRMT5 for the survival of AML cells. We then identified PRMT5 substrates using multiplexed quantitative proteomics and investigated their role in the survival of AML cells. We found that the function of the splicing regulator SRSF1 relies on its methylation by PRMT5 and that loss of PRMT5 leads to changes in alternative splicing of multiple essential genes. Our study proposes a mechanism for the requirement of PRMT5 for leukemia cell survival and provides potential biomarkers for the treatment response to PRMT5 inhibitors.
Pluripotent embryonic stem cells (ESCs) constitute an essential cellular niche sustained by epigenomic and transcriptional regulation. Any role of post-transcriptional processes remains less explored. Here, we identify a link between nuclear RNA levels, regulated by the poly(A) RNA exosome targeting (PAXT) connection, and transcriptional control by the polycomb repressive complex 2 (PRC2). Knockout of the PAXT component ZFC3H1 impairs mouse ESC differentiation. In addition to the upregulation of bona fide PAXT substrates, Zfc3h1<sup>-/-</sup> cells abnormally express developmental genes usually repressed by PRC2. Such de-repression is paralleled by decreased PRC2 binding to chromatin and low PRC2-directed H3K27 methylation. PRC2 complex stability is compromised in Zfc3h1<sup>-/-</sup> cells with elevated levels of unspecific RNA bound to PRC2 components. We propose that excess RNA hampers PRC2 function through its sequestration from DNA. Our results highlight the importance of balancing nuclear RNA levels and demonstrate the capacity of bulk RNA to regulate chromatin-associated proteins.
Acetylation of lysine 16 on histone H4 (H4K16ac) is catalyzed by histone acetyltransferase KAT8 and can prevent chromatin compaction in vitro. Although extensively studied in Drosophila, the functions of H4K16ac and two KAT8-containing protein complexes (NSL and MSL) are not well understood in mammals. Here, we demonstrate a surprising complex-dependent activity of KAT8: it catalyzes H4K5ac and H4K8ac as part of the NSL complex, whereas it catalyzes the bulk of H4K16ac as part of the MSL complex. Furthermore, we show that MSL complex proteins and H4K16ac are not required for cell proliferation and chromatin accessibility, whereas the NSL complex is essential for cell survival, as it stimulates transcription initiation at the promoters of housekeeping genes. In summary, we show that KAT8 switches catalytic activity and function depending on its associated proteins and that, when in the NSL complex, it catalyzes H4K5ac and H4K8ac required for the expression of essential genes.
We investigated the role of PRMT5 in myeloproliferative neoplasm (MPN) pathogenesis and aimed to elucidate key PRMT5 targets contributing to MPN maintenance. PRMT5 is overexpressed in primary MPN cells, and PRMT5 inhibition potently reduced MPN cell proliferation <i>ex vivo</i>. PRMT5 inhibition was efficacious at reversing elevated hematocrit, leukocytosis, and splenomegaly in a model of JAK2<sup>V617F+</sup> polycythemia vera and leukocyte and platelet counts, hepatosplenomegaly, and fibrosis in the MPL<sup>W515L</sup> model of myelofibrosis. Dual targeting of JAK and PRMT5 was superior to JAK or PRMT5 inhibitor monotherapy, further decreasing elevated counts and extramedullary hematopoiesis <i>in vivo.</i> PRMT5 inhibition reduced expression of E2F targets and altered the methylation status of E2F1 leading to attenuated DNA damage repair, cell-cycle arrest, and increased apoptosis. Our data link PRMT5 to E2F1 regulatory function and MPN cell survival and provide a strong mechanistic rationale for clinical trials of PRMT5 inhibitors in MPN. SIGNIFICANCE: Expression of PRMT5 and E2F targets is increased in JAK2<sup>V617F+</sup> MPN. Pharmacologic inhibition of PRMT5 alters the methylation status of E2F1 and shows efficacy in JAK2<sup>V617F</sup>/MPL<sup>W515L</sup> MPN models and primary samples. PRMT5 represents a potential novel therapeutic target for MPN, which is now being clinically evaluated.<i>This article is highlighted in the In This Issue feature, p. 1611</i>.
Chromatin-associated proteins are essential for the specification and maintenance of cell identity. They exert these functions through modulating and maintaining transcriptional patterns. To elucidate the functions of the Jmjd2 family of H3K9/H3K36 histone demethylases, we generated conditional Jmjd2a/Kdm4a, Jmjd2b/Kdm4b and Jmjd2c/Kdm4c/Gasc1 single, double and triple knockout mouse embryonic stem cells (ESCs). We report that while individual Jmjd2 family members are dispensable for ESC maintenance and embryogenesis, combined deficiency for specifically Jmjd2a and Jmjd2c leads to early embryonic lethality and impaired ESC self-renewal, with spontaneous differentiation towards primitive endoderm under permissive culture conditions. We further show that Jmjd2a and Jmjd2c both localize to H3K4me3-positive promoters, where they have widespread and redundant roles in preventing accumulation of H3K9me3 and H3K36me3. Jmjd2 catalytic activity is required for ESC maintenance, and increased H3K9me3 levels in knockout ESCs compromise the expression of several Jmjd2a/c targets, including genes that are important for ESC self-renewal. Thus, continual removal of H3K9 promoter methylation by Jmjd2 demethylases represents a novel mechanism ensuring transcriptional competence and stability of the pluripotent cell identity.
The chronic skin inflammation psoriasis is crucially dependent on the IL-23/IL-17 cytokine axis. Although IL-23 is expressed by psoriatic keratinocytes and immune cells, only the immune cell-derived IL-23 is believed to be disease relevant. Here we use a genetic mouse model to show that keratinocyte-produced IL-23 is sufficient to cause a chronic skin inflammation with an IL-17 profile. Furthermore, we reveal a cell-autonomous nuclear function for the actin polymerizing molecule N-WASP, which controls IL-23 expression in keratinocytes by regulating the degradation of the histone methyltransferases G9a and GLP, and H3K9 dimethylation of the IL-23 promoter. This mechanism mediates the induction of IL-23 by TNF, a known inducer of IL-23 in psoriasis. Finally, in keratinocytes of psoriatic lesions a decrease in H3K9 dimethylation correlates with increased IL-23 expression, suggesting relevance for disease. Taken together, our data describe a molecular pathway where epigenetic regulation of keratinocytes can contribute to chronic skin inflammation.
Deciphering the mechanisms that control the pluripotent ground state is key for understanding embryonic development. Nonetheless, the epigenetic regulation of ground-state mouse embryonic stem cells (mESCs) is not fully understood. Here, we identify the epigenetic protein MPP8 as being essential for ground-state pluripotency. Its depletion leads to cell cycle arrest and spontaneous differentiation. MPP8 has been suggested to repress LINE1 elements by recruiting the human silencing hub (HUSH) complex to H3K9me3-rich regions. Unexpectedly, we find that LINE1 elements are efficiently repressed by MPP8 lacking the chromodomain, while the unannotated C-terminus is essential for its function. Moreover, we show that SETDB1 recruits MPP8 to its genomic target loci, whereas transcriptional repression of LINE1 elements is maintained without retaining H3K9me3 levels. Taken together, our findings demonstrate that MPP8 protects the DNA-hypomethylated pluripotent ground state through its association with the HUSH core complex, however, independently of detectable chromatin binding and maintenance of H3K9me3.
Protein degradation technologies represent a powerful functional genomics tool, allowing fast and controllable target protein depletion. Establishing these systems requires a knock-in of the degradation tag into both endogenous target gene alleles. Here, we provide a step-by-step protocol for the efficient generation of biallelic degradation tag knock-ins in mouse and human cell lines using CRISPR-Cas9. We use knockin of an endogenous <i>Kansl3</i> degradation tag in mouse embryonic stem (ES) cells as an example but provide modifications for application in other cell types. For complete details on the use and execution of this protocol, please refer to Radzisheuskaya et al. (2021).
Generation of functionally mature organs requires exquisite control of transcriptional programs governing cell state transitions during development. Despite advances in understanding the behavior of adult intestinal stem cells and their progeny, the transcriptional regulators that control the emergence of the mature intestinal phenotype remain largely unknown. Using mouse fetal and adult small intestinal organoids, we uncover transcriptional differences between the fetal and adult state and identify rare adult-like cells present in fetal organoids. This suggests that fetal organoids have an inherent potential to mature, which is locked by a regulatory program. By implementing a CRISPR-Cas9 screen targeting transcriptional regulators expressed in fetal organoids, we establish <i>Smarca4</i> and <i>Smarcc1</i> as important factors safeguarding the immature progenitor state. Our approach demonstrates the utility of organoid models in the identification of factors regulating cell fate and state transitions during tissue maturation and reveals that SMARCA4 and SMARCC1 prevent precocious differentiation during intestinal development.
Trimethylation of histone H3 lysine 4 (H3K4me3) is associated with transcriptional start sites and has been proposed to regulate transcription initiation<sup>1,2</sup>. However, redundant functions of the H3K4 SET1/COMPASS methyltransferase complexes complicate the elucidation of the specific role of H3K4me3 in transcriptional regulation<sup>3,4</sup>. Here, using mouse embryonic stem cells as a model system, we show that acute ablation of shared subunits of the SET1/COMPASS complexes leads to a complete loss of all H3K4 methylation. Turnover of H3K4me3 occurs more rapidly than that of H3K4me1 and H3K4me2 and is dependent on KDM5 demethylases. Notably, acute loss of H3K4me3 does not have detectable effects on transcriptional initiation but leads to a widespread decrease in transcriptional output, an increase in RNA polymerase II (RNAPII) pausing and slower elongation. We show that H3K4me3 is required for the recruitment of the integrator complex subunit 11 (INTS11), which is essential for the eviction of paused RNAPII and transcriptional elongation. Thus, our study demonstrates a distinct role for H3K4me3 in transcriptional pause-release and elongation rather than transcriptional initiation.
Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML. This identified the Histone 3 Lysine 4 (H3K4) demethylase KDM5C as a tumor suppressor, and we show that reduced Kdm5c/KDM5C expression results in accelerated growth both in human and murine AML cell lines, as well as in vivo in Cebpa mutant and inv(16) AML mouse models. Mechanistically, we show that KDM5C act as a transcriptional repressor through its demethylase activity at promoters. Specifically, KDM5C knockdown results in globally increased H3K4me3 levels associated with up-regulation of bivalently marked immature genes. This is accompanied by a de-differentiation phenotype that could be reversed by modulating levels of several direct and indirect downstream mediators. Finally, the association of KDM5C levels with long-term disease-free survival of female AML patients emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML.
Posttranslational modifications of histones (PTMs) are associated with specific chromatin and gene expression states<sup>1,2</sup>. Although studies in Drosophila melanogaster have revealed phenotypic associations between chromatin-modifying enzymes and their histone substrates, comparable studies in mammalian models do not exist<sup>3-5</sup>. Here, we use CRISPR base editing in mouse embryonic stem cells (mESCs) to address the regulatory role of lysine 27 of histone H3 (H3K27), a substrate for Polycomb repressive complex 2 (PRC2)-mediated methylation and CBP/EP300-mediated acetylation<sup>6,7</sup>. By generating pan-H3K27R (pK27R) mutant mESCs, where all 28 alleles of H3.1, H3.2 and H3.3 have been mutated, we demonstrate similarity in transcription patterns of genes and differentiation to PRC2-null mutants. Moreover, H3K27 acetylation is not essential for gene derepression linked to loss of H3K27 methylation, or de novo activation of genes during cell-fate transition to epiblast-like cells (EpiLCs). In conclusion, our results show that H3K27 is an essential substrate for PRC2 in mESCs, whereas other PTMs in addition to H3K27 acetylation are likely involved in mediating CBP/EP300 function. Our work demonstrates the feasibility of large-scale multicopy gene editing to interrogate histone PTM function in mammalian cells.
Leukemic transformation (LT) of myeloproliferative neoplasm (MPN) has a dismal prognosis and is largely fatal. Mutational inactivation of TP53 is the most common somatic event in LT; however, the mechanisms by which TP53 mutations promote LT remain unresolved. Using an allelic series of mouse models of Jak2/Trp53 mutant MPN, we identify that only biallelic inactivation of Trp53 results in LT (to a pure erythroleukemia [PEL]). This PEL arises from the megakaryocyte-erythroid progenitor population. Importantly, the bone morphogenetic protein 2/SMAD pathway is aberrantly activated during LT and results in abnormal self-renewal of megakaryocyte-erythroid progenitors. Finally, we identify that Jak2/Trp53 mutant PEL is characterized by recurrent copy number alterations and DNA damage. Using a synthetic lethality strategy, by targeting active DNA repair pathways, we show that this PEL is highly sensitive to combination WEE1 and poly(ADP-ribose) polymerase inhibition. These observations yield new mechanistic insights into the process of p53 mutant LT and offer new, clinically translatable therapeutic approaches.
The tumor immune microenvironment is influenced by the epigenetic landscape of the tumor. Here, we have identified the SETDB1-TRIM28 complex as a critical suppressor of antitumor immunity. An epigenetic CRISPR-Cas9 screen of 1,218 chromatin regulators identified TRIM28 as a suppressor of PD-L1 expression. We then revealed that expression of the SETDB1-TRIM28 complex negatively correlated with infiltration of effector CD8<sup>+</sup> T cells. Inhibition of SETDB1-TRIM28 simultaneously upregulated PD-L1 and activated the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) innate immune response pathway to increase infiltration of CD8<sup>+</sup> T cells. Mechanistically, SETDB1-TRIM28 inhibition led to micronuclei formation in the cytoplasm, which is known to activate the cGAS-STING pathway. Thus, SETDB1-TRIM28 inhibition bridges innate and adaptive immunity. Indeed, <i>SETDB1</i> knockout enhanced the antitumor effects of immune checkpoint blockade with anti-PD-L1 in a mouse model of ovarian cancer in a cGAS-dependent manner. Our findings establish the SETDB1-TRIM28 complex as a regulator of antitumor immunity and demonstrate that its loss activates cGAS-STING innate immunity to boost the antitumor effects of immune checkpoint blockade.
DNA methylation at enhancers and CpG islands usually leads to gene repression, which is counteracted by DNA demethylation through the TET protein family. However, how TET enzymes are recruited and regulated at these genomic loci is not fully understood. Here, we identify TET2, the glycosyltransferase OGT and a previously undescribed proline and serine rich protein, PROSER1 as interactors of UTX, a component of the enhancer-associated MLL3/4 complexes. We find that PROSER1 mediates the interaction between OGT and TET2, thus promoting TET2 O-GlcNAcylation and protein stability. In addition, PROSER1, UTX, TET1/2, and OGT colocalize on many genomic elements genome-wide. Loss of PROSER1 results in lower enrichment of UTX, TET1/2, and OGT at enhancers and CpG islands, with a concomitant increase in DNA methylation and transcriptional down-regulation of associated target genes and increased DNA hypermethylation encroachment at H3K4me1-predisposed CpG islands. Furthermore, we provide evidence that PROSER1 acts as a more general regulator of OGT activity by controlling O-GlcNAcylation of multiple other chromatin signaling pathways. Taken together, this study describes for the first time a regulator of TET2 O-GlcNAcylation and its implications in mediating DNA demethylation at UTX-dependent enhancers and CpG islands and supports an important role for PROSER1 in regulating the function of various chromatin-associated proteins via OGT-mediated O-GlcNAcylation.
Polycomb group proteins are important for maintaining gene expression patterns and cell identity in metazoans. The mammalian Polycomb repressive deubiquitinase (PR-DUB) complexes catalyze removal of monoubiquitination on lysine 119 of histone H2A (H2AK119ub1) through a multiprotein core comprised of BAP1, HCFC1, FOXK1/2, and OGT in combination with either of ASXL1, 2, or 3. Mutations in PR-DUB components are frequent in cancer. However, mechanistic understanding of PR-DUB function in gene regulation is limited. Here, we show that BAP1 is dependent on the ASXL proteins and FOXK1/2 in facilitating gene activation across the genome. Although PR-DUB was previously shown to cooperate with PRC2, we observed minimal overlap and functional interaction between BAP1 and PRC2 in embryonic stem cells. Collectively, these results demonstrate that PR-DUB, by counteracting accumulation of H2AK119ub1, maintains chromatin in an optimal configuration ensuring expression of genes important for general functions such as cell metabolism and homeostasis.
The mutant protein FOXL2<sup>C134W</sup> is expressed in at least 95% of adult-type ovarian granulosa cell tumors (AGCT) and is considered to be a driver of oncogenesis in this disease. However, the molecular mechanism by which FOXL2<sup>C134W</sup> contributes to tumorigenesis is not known. Here, we show that mutant FOXL2<sup>C134W</sup> acquires the ability to bind SMAD4, forming a FOXL2<sup>C134W</sup>/SMAD4/SMAD2/3 complex that binds a novel hybrid DNA motif AGHCAHAA, unique to the FOXL2<sup>C134W</sup> mutant. This binding induced an enhancer-like chromatin state, leading to transcription of nearby genes, many of which are characteristic of epithelial-to-mesenchymal transition. FOXL2<sup>C134W</sup> also bound hybrid loci in primary AGCT. Ablation of SMAD4 or SMAD2/3 resulted in strong reduction of FOXL2<sup>C134W</sup> binding at hybrid sites and decreased expression of associated genes. Accordingly, inhibition of TGFβ mitigated the transcriptional effect of FOXL2<sup>C134W</sup>. Our results provide mechanistic insight into AGCT pathogenesis, identifying FOXL2<sup>C134W</sup> and its interaction with SMAD4 as potential therapeutic targets to this condition. SIGNIFICANCE: FOXL2<sup>C134W</sup> hijacks SMAD4 and leads to the expression of genes involved in EMT, stemness, and oncogenesis in AGCT, making FOXL2<sup>C134W</sup> and the TGFβ pathway therapeutic targets in this condition. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3466/F1.large.jpg.
The importance of germline-inherited post-translational histone modifications on priming early mammalian development is just emerging<sup>1-4</sup>. Histone H3 lysine 9 (H3K9) trimethylation is associated with heterochromatin and gene repression during cell-fate change<sup>5</sup>, whereas histone H3 lysine 4 (H3K4) trimethylation marks active gene promoters<sup>6</sup>. Mature oocytes are transcriptionally quiescent and possess remarkably broad domains of H3K4me3 (bdH3K4me3)<sup>1,2</sup>. It is unknown which factors contribute to the maintenance of the bdH3K4me3 landscape. Lysine-specific demethylase 4A (KDM4A) demethylates H3K9me3 at promoters marked by H3K4me3 in actively transcribing somatic cells<sup>7</sup>. Here, we report that KDM4A-mediated H3K9me3 demethylation at bdH3K4me3 in oocytes is crucial for normal pre-implantation development and zygotic genome activation after fertilization. The loss of KDM4A in oocytes causes aberrant H3K9me3 spreading over bdH3K4me3, resulting in insufficient transcriptional activation of genes, endogenous retroviral elements and chimeric transcripts initiated from long terminal repeats during zygotic genome activation. The catalytic activity of KDM4A is essential for normal epigenetic reprogramming and pre-implantation development. Hence, KDM4A plays a crucial role in preserving the maternal epigenome integrity required for proper zygotic genome activation and transfer of developmental control to the embryo.
Sclerosing stromal tumor (SST) of the ovary is a rare type of sex cord-stromal tumor (SCST), whose genetic underpinning is currently unknown. Here, using whole-exome, targeted capture and RNA-sequencing, we report recurrent FHL2-GLI2 fusion genes in 65% (17/26) of SSTs and other GLI2 rearrangements in additional 15% (4/26) SSTs, none of which are detected in other types of SCSTs (n = 48) or common cancer types (n = 9,950). The FHL2-GLI2 fusions result in transcriptomic activation of the Sonic Hedgehog (SHH) pathway in SSTs. Expression of the FHL2-GLI2 fusion in vitro leads to the acquisition of phenotypic characteristics of SSTs, increased proliferation, migration and colony formation, and SHH pathway activation. Targeted inhibition of the SHH pathway results in reversal of these oncogenic properties, indicating its role in the pathogenesis of SSTs. Our results demonstrate that the FHL2-GLI2 fusion is likely the oncogenic driver of SSTs, defining a genotypic-phenotypic correlation in ovarian neoplasms.
KDM4/JMJD2 are H3K9- and H3K36-specific demethylases, which are considered promising therapeutic targets for the treatment of acute myeloid leukemia (AML) harboring MLL translocations. Here, we investigate the long-term effects of depleting KDM4 activity on normal hematopoiesis to probe potential side effects of continuous inhibition of these enzymes. Utilizing conditional <i>Kdm4a/Kdm4b</i>/<i>Kdm4c</i> triple-knockout mice, we show that KDM4 activity is required for hematopoietic stem cell (HSC) maintenance in vivo. The knockout of the KDM4 demethylases leads to accumulation of H3K9me3 on transcription start sites and the corresponding downregulation of expression of several genes in HSCs. We show that 2 of these genes, <i>Taf1b</i> and <i>Nom1</i>, are essential for the maintenance of hematopoietic cells. Taken together, our results show that the KDM4 demethylases are required for the expression of genes essential for the long-term maintenance of normal hematopoiesis.
The epigenetic regulator <i>TET2</i> is frequently mutated in hematological diseases. Mutations have been shown to arise in hematopoietic stem cells early in disease development and lead to altered DNA methylation landscapes and an increased risk of hematopoietic malignancy. Here, we show by genome-wide mapping of TET2 binding sites in different cell types that TET2 localizes to regions of open chromatin and cell-type-specific enhancers. We find that deletion of <i>Tet2</i> in native hematopoiesis as well as fully transformed acute myeloid leukemia (AML) results in changes in transcription factor (TF) activity within these regions, and we provide evidence that loss of TET2 leads to attenuation of chromatin binding of members of the basic helix-loop-helix (bHLH) TF family. Together, these findings demonstrate that TET2 activity shapes the local chromatin environment at enhancers to facilitate TF binding and provides an example of how epigenetic dysregulation can affect gene expression patterns and drive disease development.
The PLZF transcription factor is essential for osteogenic differentiation of hMSCs; however, its regulation and molecular function during this process is not fully understood. Here, we revealed that the <i>ZBTB16</i> locus encoding PLZF, is repressed by Polycomb (PcG) and H3K27me3 in naive hMSCs. At the pre-osteoblast stage of differentiation, the locus lost PcG binding and H3K27me3, gained JMJD3 recruitment, and H3K27ac resulting in high expression of PLZF. Subsequently, PLZF was recruited to osteogenic enhancers, influencing H3K27 acetylation and expression of nearby genes important for osteogenic function. Furthermore, we identified a latent enhancer within the <i>ZBTB16/PLZF</i> locus itself that became active, gained PLZF, p300 and Mediator binding and looped to the promoter of the nicotinamide N-methyltransferase (<i>NNMT</i>) gene. The increased expression of NNMT correlated with a decline in SAM levels, which is dependent on PLZF and is required for osteogenic differentiation.
Polycomb repressive complex 2 (PRC2) catalyzes methylation on lysine 27 of histone H3 (H3K27) and is required for maintaining transcriptional patterns and cellular identity, but the specification and maintenance of genomic PRC2 binding and H3K27 methylation patterns remain incompletely understood. Epigenetic mechanisms have been proposed, wherein pre-existing H3K27 methylation directs recruitment and regulates the catalytic activity of PRC2 to support its own maintenance. Here we investigate whether such mechanisms are required for specifying H3K27 methylation patterns in mouse embryonic stem cells (mESCs). Through re-expression of PRC2 subunits in PRC2-knockout cells that have lost all H3K27 methylation, we demonstrate that methylation patterns can be accurately established de novo. We find that regional methylation kinetics correlate with original methylation patterns even in their absence, and specification of the genomic PRC2 binding pattern is retained and specifically dependent on the PRC2 core subunit SUZ12. Thus, the H3K27 methylation patterns in mESCs are not dependent on self-autonomous epigenetic inheritance.
Regulation of chromatin composition through post-translational modifications of histones contributes to transcriptional regulation and is essential for many cellular processes, including differentiation and development. KDM4A (JMJD2A) is a lysine demethylase with specificity towards di- and tri-methylated lysine 9 and lysine 36 of histone H3 (H3K9me2/me3 and H3K36me2/me3). Here, we report that <i>Kdm4a</i> as a maternal factor plays a key role in embryo survival and is vital for female fertility. <i>Kdm4a<sup>-/-</sup></i> female mice ovulate normally with comparable fertilization but poor implantation rates, and cannot support healthy transplanted embryos to term. This is due to a role for Kdm4a in uterine function, where its loss causes reduced expression of key genes involved in ion transport, nutrient supply and cytokine signalling, which impact embryo survival. In addition, a significant proportion of Kdm4a-deficient oocytes displays a poor intrinsic ability to develop into blastocysts. These embryos cannot compete with healthy embryos for implantation <i>in vivo</i>, highlighting <i>Kdm4a</i> as a maternal effect gene. Thus, our study dissects an important dual role for maternal Kdm4a in determining faithful early embryonic development and the implantation process.
Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor that is located in the pons and primarily affects children. Nearly 80% of DIPGs harbor mutations in histone H3 genes, wherein lysine 27 is substituted with methionine (H3K27M). H3K27M has been shown to inhibit polycomb repressive complex 2 (PRC2), a multiprotein complex responsible for the methylation of H3 at lysine 27 (H3K27me), by binding to its catalytic subunit EZH2. Although DIPGs with the H3K27M mutation show global loss of H3K27me3, several genes retain H3K27me3. Here we describe a mouse model of DIPG in which H3K27M potentiates tumorigenesis. Using this model and primary patient-derived DIPG cell lines, we show that H3K27M-expressing tumors require PRC2 for proliferation. Furthermore, we demonstrate that small-molecule EZH2 inhibitors abolish tumor cell growth through a mechanism that is dependent on the induction of the tumor-suppressor protein p16<sup>INK4A</sup>. Genome-wide enrichment analyses show that the genes that retain H3K27me3 in H3K27M cells are strong polycomb targets. Furthermore, we find a highly significant overlap between genes that retain H3K27me3 in the DIPG mouse model and in human primary DIPGs expressing H3K27M. Taken together, these results show that residual PRC2 activity is required for the proliferation of H3K27M-expressing DIPGs, and that inhibition of EZH2 is a potential therapeutic strategy for the treatment of these tumors.
The Polycomb repressive complexes PRC1 and PRC2 play a central role in developmental gene regulation in multicellular organisms. PRC1 and PRC2 modify chromatin by catalysing histone H2A lysine 119 ubiquitylation (H2AK119u1), and H3 lysine 27 methylation (H3K27me3), respectively. Reciprocal crosstalk between these modifications is critical for the formation of stable Polycomb domains at target gene loci. While the molecular mechanism for recognition of H3K27me3 by PRC1 is well defined, the interaction of PRC2 with H2AK119u1 is poorly understood. Here we demonstrate a critical role for the PRC2 cofactor Jarid2 in mediating the interaction of PRC2 with H2AK119u1. We identify a ubiquitin interaction motif at the amino-terminus of Jarid2, and demonstrate that this domain facilitates PRC2 localization to H2AK119u1 both in vivo and in vitro. Our findings ascribe a critical function to Jarid2 and define a key mechanism that links PRC1 and PRC2 in the establishment of Polycomb domains.
Acute myeloid leukemias (AMLs) with a rearrangement of the mixed-linage leukemia (MLL) gene are aggressive hematopoietic malignancies. Here, we explored the feasibility of using the H3K9- and H3K36-specific demethylases Jmjd2/Kdm4 as putative drug targets in MLL-AF9 translocated leukemia. Using Jmjd2a, Jmjd2b, and Jmjd2c conditional triple-knockout mice, we show that Jmjd2/Kdm4 activities are required for MLL-AF9 translocated AML in vivo and in vitro. We demonstrate that expression of the interleukin 3 receptor α (Il3ra also known as Cd123) subunit is dependent on Jmjd2/Kdm4 through a mechanism involving removal of H3K9me3 from the promoter of the Il3ra gene. Importantly, ectopic expression of Il3ra in Jmjd2/Kdm4 knockout cells alleviates the requirement of Jmjd2/Kdm4 for the survival of AML cells, showing that Il3ra is a critical downstream target of Jmjd2/Kdm4 in leukemia. These results suggest that the JMJD2/KDM4 proteins are promising drug targets for the treatment of AML.
ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICRBS) and many other loci (non-ICRBS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICRBS and non-ICRBS suggesting that different cis-acting regulatory functions are repressed by ZFP57 at these two classes of target loci. Overall, these data demonstrate that ZFP57 is pivotal to maintain the allele-specific epigenetic modifications of ICRs that in turn are necessary for maintaining the imprinted expression over long distances. At non-ICRBS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet required.
The pattern of DNA methylation at cytosine bases in the genome is tightly linked to gene expression, and DNA methylation abnormalities are often observed in diseases. The ten eleven translocation (TET) enzymes oxidize 5-methylcytosines (5mCs) and promote locus-specific reversal of DNA methylation. TET genes, and especially TET2, are frequently mutated in various cancers, but how the TET proteins contribute to prevent the onset and maintenance of these malignancies is largely unknown. Here, we highlight recent advances in understanding the physiological function of the TET proteins and their role in regulating DNA methylation and transcription. In addition, we discuss some of the key outstanding questions in the field.
TEN-ELEVEN-TRANSLOCATION-2 (TET2) and DNA-METHYLTRANSFERASE-3A (DNMT3A), both encoding proteins involved in regulating DNA methylation, are mutated in hematological malignancies affecting both myeloid and lymphoid lineages. We previously reported an association of TET2 and DNMT3A mutations in progenitors of patients with angioimmunoblastic T-cell lymphomas (AITL). Here, we report on the cooperative effect of Tet2 inactivation and DNMT3A mutation affecting arginine 882 (DNMT3A(R882H)) using a murine bone marrow transplantation assay. Five out of eighteen primary recipients developed hematological malignancies with one mouse developing an AITL-like disease, two mice presenting acute myeloid leukemia (AML)-like and two others T-cell acute lymphoblastic leukemia (T-ALL)-like diseases within 6 months following transplantation. Serial transplantations of DNMT3A(R882H) Tet2(-/-) progenitors led to a differentiation bias toward the T-cell compartment, eventually leading to AITL-like disease in 9/12 serially transplanted recipients. Expression profiling suggested that DNMT3A(R882H) Tet2(-/-) T-ALLs resemble those of NOTCH1 mutant. Methylation analysis of DNMT3A(R882H) Tet2(-/-) T-ALLs showed a global increase in DNA methylation affecting tumor suppressor genes and local hypomethylation affecting genes involved in the Notch pathway. Our data confirm the transformation potential of DNMT3A(R882H) Tet2(-/-) progenitors and represent the first cooperative model in mice involving Tet2 inactivation driving lymphoid malignancies.
Oncogene-induced senescence (OIS) can occur in response to oncogenic insults and is considered an important tumour suppressor mechanism. Here we identify the lncRNA MIR31HG as upregulated in OIS and find that knockdown of MIR31HG promotes a strong p16(INK4A)-dependent senescence phenotype. Under normal conditions, MIR31HG is found in both nucleus and cytoplasm, but following B-RAF expression MIR31HG is located mainly in the cytoplasm. We show that MIR31HG interacts with both INK4A and MIR31HG genomic regions and with Polycomb group (PcG) proteins, and that MIR31HG is required for PcG-mediated repression of the INK4A locus. We further identify a functional enhancer, located between MIR31HG and INK4A, which becomes activated during OIS and interacts with the MIR31HG promoter. Data from melanoma patients show a negative correlation between MIR31HG and p16(INK4A) expression levels, suggesting a role for this transcript in cancer. Hence, our data provide a new lncRNA-mediated regulatory mechanism for the tumour suppressor p16(INK4A).
Adult stem cells maintain regenerative tissue structure and function by producing tissue-specific progeny, but the factors that preserve their tissue identities are not well understood. The small and large intestines differ markedly in cell composition and function, reflecting their distinct stem cell populations. Here we show that SATB2, a colon-restricted chromatin factor, singularly preserves LGR5<sup>+</sup> adult colonic stem cell and epithelial identity in mice and humans. Satb2 loss in adult mice leads to stable conversion of colonic stem cells into small intestine ileal-like stem cells and replacement of the colonic mucosa with one that resembles the ileum. Conversely, SATB2 confers colonic properties on the mouse ileum. Human colonic organoids also adopt ileal characteristics upon SATB2 loss. SATB2 regulates colonic identity in part by modulating enhancer binding of the intestinal transcription factors CDX2 and HNF4A. Our study uncovers a conserved core regulator of colonic stem cells able to mediate cross-tissue plasticity in mature intestines.
Transposable elements (TEs) are widespread genetic parasites known to be kept under tight transcriptional control. Here, we describe a functional connection between the mouse-orthologous "nuclear exosome targeting" (NEXT) and "human silencing hub" (HUSH) complexes, involved in nuclear RNA decay and the epigenetic silencing of TEs, respectively. Knocking out the NEXT component ZCCHC8 in embryonic stem cells results in elevated TE RNA levels. We identify a physical interaction between ZCCHC8 and the MPP8 protein of HUSH and establish that HUSH recruits NEXT to chromatin at MPP8-bound TE loci. However, while NEXT and HUSH both dampen TE RNA expression, their activities predominantly affect shorter non-polyadenylated and full-length polyadenylated transcripts, respectively. Indeed, our data suggest that the repressive action of HUSH promotes a condition favoring NEXT RNA decay activity. In this way, transcriptional and post-transcriptional machineries synergize to suppress the genotoxic potential of TE RNAs.
Polycomb group (PcG) proteins are essential for post-implantation development by depositing repressive histone modifications at promoters, mainly CpG islands (CGIs), of developmental regulator genes. However, promoter PcG marks are erased after fertilization and de novo established in peri-implantation embryos, coinciding with the transition from naive to primed pluripotency. Nevertheless, the molecular basis for this establishment remains unknown. In this study, we show that the expression of the long KDM2B isoform (KDM2BLF), which contains the demethylase domain, is specifically induced at peri-implantation and that its H3K36me2 demethylase activity is required for PcG enrichment at CGIs. Moreover, KDM2BLF interacts with BRG1/BRM-associated factor (BAF) and stabilizes BAF occupancy at CGIs for subsequent gain of accessibility, which precedes PcG enrichment. Consistently, KDM2BLF inactivation results in significantly delayed post-implantation development. In summary, our data unveil dynamic chromatin configuration of CGIs during exit from naive pluripotency and provide a conceptual framework for the spatiotemporal establishment of PcG functions.
Mutations in the epigenetic modifier TET2 are frequent in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). Here, we investigate associations between TET2 mutations and DNA methylation in whole blood in 305 elderly twins, 15 patients with CCUS and 18 healthy controls. We find that TET2 mutations are associated with DNA hypermethylation at enhancer sites in whole blood in CHIP and in both granulocytes and mononuclear cells in CCUS. These hypermethylated sites are associated with leukocyte function and immune response and ETS-related and C/EBP-related transcription factor motifs. While the majority of TET2-associated hypermethylation sites are shared between CHIP and in AML, we find a set of AML-specific hypermethylated loci at active enhancer elements in hematopoietic stem cells. In summary, we show that TET2 mutations is associated with hypermethylated enhancers involved in myeloid differentiation in both CHIP, CCUS and AML patients.
A disproportional large number of neurodevelopmental disorders (NDDs) is caused by variants in genes encoding transcription factors and chromatin modifiers. However, the functional interactions between the corresponding proteins are only partly known. Here, we show that KDM5C, encoding a H3K4 demethylase, is at the intersection of transcriptional axes under the control of three regulatory proteins ARX, ZNF711 and PHF8. Interestingly, mutations in all four genes (KDM5C, ARX, ZNF711 and PHF8) are associated with X-linked NDDs comprising intellectual disability as a core feature. in vitro analysis of the KDM5C promoter revealed that ARX and ZNF711 function as antagonist transcription factors that activate KDM5C expression and compete for the recruitment of PHF8. Functional analysis of mutations in these genes showed a correlation between phenotype severity and the reduction in KDM5C transcriptional activity. The KDM5C decrease was associated with a lack of repression of downstream target genes Scn2a, Syn1 and Bdnf in the embryonic brain of Arx-null mice. Aiming to correct the faulty expression of KDM5C, we studied the effect of the FDA-approved histone deacetylase inhibitor suberanilohydroxamic acid (SAHA). In Arx-KO murine ES-derived neurons, SAHA was able to rescue KDM5C depletion, recover H3K4me3 signalling and improve neuronal differentiation. Indeed, in ARX/alr-1-deficient Caenorhabditis elegans animals, SAHA was shown to counteract the defective KDM5C/rbr-2-H3K4me3 signalling, recover abnormal behavioural phenotype and ameliorate neuronal maturation. Overall, our studies indicate that KDM5C is a conserved and druggable effector molecule across a number of NDDs for whom the use of SAHA may be considered a potential therapeutic strategy.
Colorectal cancer (CRC) is the fourth cause of death from cancer worldwide mainly due to the high incidence of drug-resistance. During a screen for new actionable targets in drug-resistant tumours we recently identified p65BTK - a novel oncogenic isoform of Bruton's tyrosine kinase. Studying three different cohorts of patients here we show that p65BTK expression correlates with histotype and cancer progression. Using drug-resistant TP53-null colon cancer cells as a model we demonstrated that p65BTK silencing or chemical inhibition overcame the 5-fluorouracil resistance of CRC cell lines and patient-derived organoids and significantly reduced the growth of xenografted tumours. Mechanistically, we show that blocking p65BTK in drug-resistant cells abolished a 5-FU-elicited TGFB1 protective response and triggered E2F-dependent apoptosis. Taken together, our data demonstrated that targeting p65BTK restores the apoptotic response to chemotherapy of drug-resistant CRCs and gives a proof-of-concept for suggesting the use of BTK inhibitors in combination with 5-FU as a novel therapeutic approach in CRC patients. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Transcription factors (TFs) regulate many cellular processes and can therefore serve as readouts of the signaling and regulatory state. Yet for many TFs, the mode of action-repressing or activating transcription of target genes-is unclear. Here, we present diffTF (https://git.embl.de/grp-zaugg/diffTF) to calculate differential TF activity (basic mode) and classify TFs into putative transcriptional activators or repressors (classification mode). In basic mode, it combines genome-wide chromatin accessibility/activity with putative TF binding sites that, in classification mode, are integrated with RNA-seq. We apply diffTF to compare (1) mutated and unmutated chronic lymphocytic leukemia patients and (2) two hematopoietic progenitor cell types. In both datasets, diffTF recovers most known biology and finds many previously unreported TFs. It classifies almost 40% of TFs based on their mode of action, which we validate experimentally. Overall, we demonstrate that diffTF recovers known biology, identifies less well-characterized TFs, and classifies TFs into transcriptional activators or repressors.
The Polycomb repressive complex 2 (PRC2) catalyzes H3K27 methylation across the genome, which impacts transcriptional regulation and is critical for establishment of cell identity. Because of its essential function during development and in cancer, understanding the delineation of genome-wide H3K27 methylation patterns has been the focus of intense investigation. PRC2 methylation activity is abundant and dispersed throughout the genome, but the highest activity is specifically directed to a subset of target sites that are stably occupied by the complex and highly enriched for H3K27me3. Here, we show, by systematically knocking out single and multiple non-core subunits of the PRC2 complex in mouse embryonic stem cells, that they each contribute to directing PRC2 activity to target sites. Furthermore, combined knockout of six non-core subunits reveals that, while dispensable for global H3K27 methylation levels, the non-core PRC2 subunits are collectively required for focusing H3K27me3 activity to specific sites in the genome.
<h4>Aims</h4>Posttranslational modifications of histones and transcription factors regulate gene expression and are implicated in beta-cell failure and diabetes. We have recently shown that preserving H3K27 and H3K4 methylation using the lysine demethylase inhibitor GSK-J4 reduces cytokine-induced destruction of beta-cells and improves beta-cell function. Here, we investigate the therapeutic potential of GSK-J4 to prevent diabetes development and examine the importance of H3K4 methylation for islet function.<h4>Materials and methods</h4>We used two mouse models of diabetes to investigate the therapeutic potential of GSK-J4. To clarify the importance of H3K4 methylation, we characterized a mouse strain with knockout (KO) of the H3K4 demethylase KDM5B.<h4>Results</h4>GSK-J4 administration failed to prevent the development of experimental diabetes induced by multiple low-dose streptozotocin or adoptive transfer of splenocytes from acutely diabetic NOD to NODscid mice. KDM5B-KO mice were growth retarded with altered body composition, had low IGF-1 levels, and exhibited reduced insulin secretion. Interestingly, despite secreting less insulin, KDM5B-KO mice were able to maintain normoglycemia following oral glucose tolerance test, likely via improved insulin sensitivity, as suggested by insulin tolerance testing and phosphorylation of proteins belonging to the insulin signaling pathway. When challenged with high-fat diet, KDM5B-deficient mice displayed similar weight gain and insulin sensitivity as wild-type mice.<h4>Conclusion</h4>Our results show a novel role of KDM5B in metabolism, as KDM5B-KO mice display growth retardation and improved insulin sensitivity.
Recurrent inactivating mutations have been identified in the X-linked plant homeodomain finger protein 6 (<i>PHF6</i>) gene, encoding a chromatin-binding transcriptional regulator protein, in various hematological malignancies. However, the role of PHF6 in normal hematopoiesis and its tumor-suppressor function remain largely unknown. We herein generated mice carrying a floxed <i>Phf6</i> allele and inactivated <i>Phf6</i> in hematopoietic cells at various developmental stages. The <i>Phf6</i> deletion in embryos augmented the capacity of hematopoietic stem cells (HSCs) to proliferate in cultures and reconstitute hematopoiesis in recipient mice. The <i>Phf6</i> deletion in neonates and adults revealed that cycling HSCs readily acquired an advantage in competitive repopulation upon the <i>Phf6</i> deletion, whereas dormant HSCs only did so after serial transplantations. <i>Phf6</i>-deficient HSCs maintained an enhanced repopulating capacity during serial transplantations; however, they did not induce any hematological malignancies. Mechanistically, Phf6 directly and indirectly activated downstream effectors in tumor necrosis factor α (TNFα) signaling. The <i>Phf6</i> deletion repressed the expression of a set of genes associated with TNFα signaling, thereby conferring resistance against the TNFα-mediated growth inhibition on HSCs. Collectively, these results not only define Phf6 as a novel negative regulator of HSC self-renewal, implicating inactivating <i>PHF6</i> mutations in the pathogenesis of hematological malignancies, but also indicate that a <i>Phf6</i> deficiency alone is not sufficient to induce hematopoietic transformation.
Targeting of histone methylation has therapeutic potential in oncology. Here, we provide proof-of-principle that pharmacological inhibition of KDM5 histone-demethylases is a new strategy for the personalized treatment of HER2<sup>+</sup> breast cancer. The anti-proliferative effects of the prototype of a new class of selective KDM5-inhibitors (KDM5-inh1) are evaluated in 40 cell lines, recapitulating the heterogeneity of breast cancer. This analysis demonstrates that HER2<sup>+</sup> cells are particularly sensitive to KDM5 inhibition. The results are confirmed in an appropriate in vivo model with a close structural analog (KDM5-inh1A). RNA-seq data obtained in HER2<sup>+</sup> BT-474 cells exposed to KDM5-Inh1 indicate that the compound alters expression of numerous genes downstream of the ERBB2 gene-product, HER2. In selected HER2-positive breast-cancer cells, we demonstrate synergistic interactions between KDM5-inh1 and HER2-targeting agents (trastuzumab and lapatinib). In addition, HER2<sup>+</sup> cell lines with innate and acquired resistance to trastuzumab show sensitivity to KDM5-inh1. The levels of KDM5A/B/C proteins, which are selectively targeted by the agent, have no significant association with KDM5-inh1 responsiveness across our panel of breast-cancer cell lines, suggesting the existence of other determinants of sensitivity. Using RNA-seq data of the breast-cancer cell lines we generate a gene-expression model that is a robust predictor of KDM5-inh1 sensitivity. In a test set of breast cancers, this model predicts sensitivity to the compound in a large fraction of HER2<sup>+</sup> tumors. In conclusion, KDM5 inhibition has potential in the treatment of HER2<sup>+</sup> breast cancer and our gene-expression model can be developed into a diagnostic tool for the selection of patients.
The polycomb repressive complex 2 (PRC2) is a chromatin-associated methyltransferase catalyzing mono-, di-, and trimethylation of lysine 27 on histone H3 (H3K27). This activity is required for normal organismal development and maintenance of gene expression patterns to uphold cell identity. PRC2 function is often deregulated in disease and is a promising candidate for therapeutic targeting in cancer. In this review, we discuss the molecular mechanisms proposed to take part in modulating PRC2 recruitment and shaping H3K27 methylation patterns across the genome. This includes consideration of factors influencing PRC2 residence time on chromatin and PRC2 catalytic activity with a focus on the mechanisms giving rise to regional preferences and differential deposition of H3K27 methylation. We further discuss existing evidence for functional diversity between distinct subsets of PRC2 complexes with the aim of extracting key concepts and highlighting major open questions toward a more complete understanding of PRC2 function.
<h4>Aim</h4>The histone demethylase Jarid1b limits gene expression by removing the active methyl mark from histone3 lysine4 at gene promoter regions. A vascular function of Jarid1b is unknown, but a vasoprotective function to inflammatory and hypertrophic stimuli, like angiotensin II (AngII) could be inferred. This hypothesis was tested using Jarid1b knockout mice and the inhibitor PBIT.<h4>Methods</h4>Mice or aortic segments were treated with AngII to induce endothelial dysfunction. Aortae from WT and Jarid1b knockout were studied in organ chambers and endothelium-dependent dilator responses to acetylcholine and endothelium-independent responses to DetaNONOate were recorded after pre-constriction with phenylephrine in the presence or absence of the NO-synthase inhibitor nitro-L-arginine. Molecular mechanisms were investigated with chromatin immunoprecipitation, RNA-Seq, RNA-3'-adaptor-ligation, actinomycin D and RNA-immunoprecipitation.<h4>Results</h4>Knockout or inhibition of Jarid1b prevented the development of endothelial dysfunction in response to AngII. This effect was not a consequence of altered nitrite oxide availability but accompanied by a loss of the inflammatory response to AngII. As Jarid1b mainly inhibits gene expression, an indirect effect should account for this observation. AngII induced the soluble epoxide hydrolase (sEH), which degrades anti-inflammatory lipids, and thus promotes inflammation. Knockout or inhibition of Jarid1b prevented the AngII-mediated sEH induction. Mechanistically, Jarid1b maintained the length of the 3'untranslated region of the sEH mRNA, thereby increasing its stability and thus sEH protein expression. Loss of Jarid1b activity therefore resulted in sEH mRNA destabilization.<h4>Conclusion</h4>Jarid1b contributes to the pro-inflammatory effects of AngII by stabilizing sEH expression. Jarid1b inhibition might be an option for future therapeutics against cardiovascular dysfunction.
Tumorigenesis is increasingly considered to rely on subclones of cells poised to undergo an epithelial to mesenchymal transition (EMT) program. We and others have provided evidence, however, that the tumorigenesis of human breast cancer is not always restricted to typical EMT cells but is also somewhat paradoxically conveyed by subclones of apparently differentiated, non-EMT cells. Here we characterize such non-EMT-like and EMT-like subclones. Through a loss-of-function screen we found that a member of the E3 ubiquitin ligase complexes, FBXO11, specifically fuels tumor formation of a non-EMT-like clone by restraining the p53/p21 pathway. Interestingly, in the related EMT-like clone, FBXO11 operates through the BCL2 pathway with little or no impact on tumorigenesis. These data command caution in attempts to assess tumorigenesis prospectively based on EMT profiling, and they emphasize the importance of next generation subtyping of tumors, that is at the level of clonal composition.
Oligodendrogliomas are brain tumors accounting for approximately 10% of all central nervous system cancers. CIC is a transcription factor that is mutated in most patients with oligodendrogliomas; these mutations are believed to be a key oncogenic event in such cancers. Analysis of the <i>Drosophila melanogaster</i> ortholog of CIC, Capicua, indicates that CIC loss phenocopies activation of the EGFR/RAS/MAPK pathway, and studies in mammalian cells have demonstrated a role for CIC in repressing the transcription of the PEA3 subfamily of ETS transcription factors. Here, we address the mechanism by which CIC represses transcription and assess the functional consequences of CIC inactivation. Genome-wide binding patterns of CIC in several cell types revealed that CIC target genes were enriched for MAPK effector genes involved in cell-cycle regulation and proliferation. CIC binding to target genes was abolished by high MAPK activity, which led to their transcriptional activation. CIC interacted with the SIN3 deacetylation complex and, based on our results, we suggest that CIC functions as a transcriptional repressor through the recruitment of histone deacetylases. Independent single amino acid substitutions found in oligodendrogliomas prevented CIC from binding its target genes. Taken together, our results show that CIC is a transcriptional repressor of genes regulated by MAPK signaling, and that ablation of CIC function leads to increased histone acetylation levels and transcription at these genes, ultimately fueling mitogen-independent tumor growth.<b>Significance:</b> Inactivation of CIC inhibits its direct repression of MAPK pathway genes, leading to their increased expression and mitogen-independent growth.<b>Graphical Abstract:</b> http://cancerres.aacrjournals.org/content/canres/78/15/4114/F1.large.jpg <i>Cancer Res; 78(15); 4114-25. ©2018 AACR</i>.
Global histone hyperacetylation is suggested to play a critical role for replacement of histones by transition proteins and protamines to compact the genome during spermiogenesis. However, the underlying mechanisms for hyperacetylation-mediated histone replacement remains poorly understood. Here, we report that EPC1 and TIP60, two critical components of the mammalian nucleosome acetyltransferase of H4 (NuA4) complexes, are coexpressed in male germ cells. Strikingly, genetic ablation of either <i>Epc1</i> or <i>Tip60</i> disrupts hyperacetylation and impairs histone replacement, in turn causing aberrant spermatid development. Taking these observations together, we reveal an essential role of the NuA4 complexes for histone hyperacetylation and subsequent compaction of the spermatid genome.
Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of Polycomb repressive complex 2 (PRC2), has attracted broad research attention in the past few years because of its involvement in the development and maintenance of many types of cancer and the use of specific EZH2 inhibitors in clinical trials. Several observations show that PRC2 can have both oncogenic and tumour-suppressive functions. We propose that these apparently opposing roles of PRC2 in cancer are a consequence of the molecular function of the complex in maintaining, rather than specifying, the transcriptional repression state of its several thousand target genes.
Limited perfusion of solid tumours produces a nutrient-deprived tumour core microenvironment. Low glutamine levels in the tumour core are now shown to lead to reduced levels of α-ketoglutarate and decreased histone demethylase activity, thereby promoting a less differentiated and more therapy-resistant state of the tumour cells.
The chromatin environment is modulated by a machinery of chromatin modifiers, required for the specification and maintenance of cell fate. Many mutations in the machinery have been linked to the development and progression of cancer. In this review, we give a brief introduction to Polycomb group (PcG) proteins, their assembly into Polycomb repressive complexes (PRCs) and the normal physiological roles of these complexes with a focus on the PRC2. We review the many findings of mutations in the PRC2 coding genes, both loss-of-function and gain-of-function, associated with human cancers and discuss potential molecular mechanisms involved in the contribution of PRC2 mutations to cancer development and progression. Finally, we discuss some of the recent advances in developing and testing drugs targeting the PRC2 as well as emerging results from clinical trials using these drugs in the treatment of human cancers.
Bruton's tyrosine kinase (BTK) is essential for B-cell proliferation/differentiation and it is generally believed that its expression and function are limited to bone marrow-derived cells. Here, we report the identification and characterization of p65BTK, a novel isoform abundantly expressed in colon carcinoma cell lines and tumour tissue samples. p65BTK protein is expressed, through heterogeneous nuclear ribonucleoprotein K (hnRNPK)-dependent and internal ribosome entry site-driven translation, from a transcript containing an alternative first exon in the 5'-untranslated region, and is post-transcriptionally regulated, via hnRNPK, by the mitogen-activated protein kinase (MAPK) pathway. p65BTK is endowed with strong transforming activity that depends on active signal-regulated protein kinases-1/2 (ERK1/2) and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. Our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising therapeutic approach.
Histones are abundant chromatin constituents carrying numerous post-translational modifications (PTMs). Such PTMs mediate a variety of biological functions, including recruitment of enzymatic readers, writers and erasers that modulate DNA replication, transcription and repair. Individual histone molecules contain multiple coexisting PTMs, some of which exhibit crosstalk, i.e. coordinated or mutually exclusive activities. Here, we present an integrated experimental and computational systems level molecular characterization of histone PTMs and PTM crosstalk. Using wild type and engineered mouse embryonic stem cells (mESCs) knocked out in components of the Polycomb Repressive Complex 2 (PRC2, Suz12(-/-)), PRC1 (Ring1A/B(-/-)) and (Dnmt1/3a/3b(-/-)) we performed comprehensive PTM analysis of histone H3 tails (50 aa) by utilizing quantitative middle-down proteome analysis by tandem mass spectrometry. We characterized combinatorial PTM features across the four mESC lines and then applied statistical data analysis to predict crosstalk between histone H3 PTMs. We detected an overrepresentation of positive crosstalk (codependent marks) between adjacent mono-methylated and acetylated marks, and negative crosstalk (mutually exclusive marks) among most of the seven characterized di- and tri-methylated lysine residues in the H3 tails. We report novel features of PTM interplay involving hitherto poorly characterized arginine methylation and lysine methylation sites, including H3R2me, H3R8me and H3K37me. Integration of the H3 data with RNAseq data by coabundance clustering analysis of histone PTMs and histone modifying enzymes revealed correlations between PTM and enzyme levels. We conclude that middle-down proteomics is a powerful tool to determine conserved or dynamic interdependencies between histone marks, which paves the way for detailed investigations of the histone code. Histone H3 PTM data is publicly available in the CrossTalkDB repository at http://crosstalkdb.bmb.sdu.dk.
Alterations in chromatin structure caused by deregulated epigenetic mechanisms collaborate with underlying genetic lesions to promote cancer. SMARCA4/BRG1, a core component of the SWI/SNF ATP-dependent chromatin-remodelling complex, has been implicated by its mutational spectrum as exerting a tumour-suppressor function in many solid tumours; recently however, it has been reported to sustain leukaemogenic transformation in MLL-rearranged leukaemia in mice. Here we further explore the role of SMARCA4 and the two SWI/SNF subunits SMARCD2/BAF60B and DPF2/BAF45D in leukaemia. We observed the selective requirement for these proteins for leukaemic cell expansion and self-renewal in-vitro as well as in leukaemia. Gene expression profiling in human cells of each of these three factors suggests that they have overlapping functions in leukaemia. The gene expression changes induced by loss of the three proteins demonstrate that they are required for the expression of haematopoietic stem cell associated genes but in contrast to previous results obtained in mouse cells, the three proteins are not required for the expression of c-MYC regulated genes.
Genome-wide analyses in Caenorhabditis elegans show that post-translational modifications (PTMs) of histones are evolutionary conserved and distributed along functionally distinct genomic domains. However, a global profile of PTMs and their co-occurrence on the same histone tail has not been described in this organism. We used mass spectrometry based middle-down proteomics to analyze histone H3 N-terminal tails from C. elegans embryos for the presence, the relative abundance and the potential cross-talk of co-existing PTMs. This analysis highlighted that the lysine 23 of histone H3 (H3K23) is extensively modified by methylation and that tri-methylated H3K9 (H3K9me3) is exclusively detected on histone tails with di-methylated H3K23 (H3K23me2). Chromatin immunoprecipitation approaches revealed a positive correlation between H3K23me2 and repressive marks. By immunofluorescence analyses, H3K23me2 appears differentially regulated in germ and somatic cells, in part by the action of the histone demethylase JMJD-1.2. H3K23me2 is enriched in heterochromatic regions, localizing in H3K9me3 and heterochromatin protein like-1 (HPL-1)-positive foci. Biochemical analyses indicated that HPL-1 binds to H3K23me2 and interacts with a conserved CoREST repressive complex. Thus, our study suggests that H3K23me2 defines repressive domains and contributes to organizing the genome in distinct heterochromatic regions during embryogenesis.
ASXL1 mutations are frequently found in hematological tumors, and loss of Asxl1 promotes myeloid transformation in mice. Here we present data supporting a role for an ASXL1-BAP1 complex in the deubiquitylation of mono-ubiquitylated lysine 119 on Histone H2A (H2AK119ub1) in vivo. The Polycomb group proteins control the expression of the INK4B-ARF-INK4A locus during normal development, in part through catalyzing mono-ubiquitylation of H2AK119. Since the activation of the locus INK4B-ARF-INK4A plays a fail-safe mechanism protecting against tumorigenesis, we investigated whether ASXL1-dependent H2A deubiquitylation plays a role in its activation. Interestingly, we found that ASXL1 is specifically required for the increased expression of p15(INK4B) in response to both oncogenic signaling and extrinsic anti-proliferative signals. Since we found that ASXL1 and BAP1 both are enriched at the INK4B locus, our results suggest that activation of the INK4B locus requires ASXL1/BAP1-mediated deubiquitylation of H2AK119ub1. Consistently, our results show that ASXL1 mutations are associated with lower expression levels of p15(INK4B) and a proliferative advantage of hematopoietic progenitors in primary bone marrow cells, and that depletion of ASXL1 in multiple cell lines results in resistance to growth inhibitory signals. Taken together, this study links ASXL1-mediated H2A deubiquitylation and transcriptional activation of INK4B expression to its tumor suppressor functions.
<h4>Objective</h4>Altering endothelial biology through epigenetic modifiers is an attractive novel concept, which is, however, just in its beginnings. We therefore set out to identify chromatin modifiers important for endothelial gene expression and contributing to angiogenesis.<h4>Approach and results</h4>To identify chromatin modifying enzymes in endothelial cells, histone demethylases were screened by microarray and polymerase chain reaction. The histone 3 lysine 4 demethylase JARID1B was identified as a highly expressed enzyme at the mRNA and protein levels. Knockdown of JARID1B by shRNA in human umbilical vein endothelial cells attenuated cell migration, angiogenic sprouting, and tube formation. Similarly, pharmacological inhibition and overexpression of a catalytic inactive JARID1B mutant reduced the angiogenic capacity of human umbilical vein endothelial cells. To identify the in vivo relevance of JARID1B in the vascular system, Jarid1b knockout mice were studied. As global knockout results in increased mortality and developmental defects, tamoxifen-inducible and endothelial-specific knockout mice were generated. Acute knockout of Jarid1b attenuated retinal angiogenesis and endothelial sprout outgrowth from aortic segments. To identify the underlying mechanism, a microarray experiment was performed, which led to the identification of the antiangiogenic transcription factor HOXA5 to be suppressed by JARID1B. Importantly, downregulation or inhibition of JARID1B, but not of JARID1A and JARID1C, induced HOXA5 expression in human umbilical vein endothelial cells. Consistently, chromatin immunoprecipitation revealed that JARID1B occupies and reduces the histone 3 lysine 4 methylation levels at the HOXA5 promoter, demonstrating a direct function of JARID1B in endothelial HOXA5 gene regulation.<h4>Conclusions</h4>JARID1B, by suppressing HOXA5, maintains the endothelial angiogenic capacity in a demethylase-dependent manner.
Respiratory syncytial virus (RSV) infection can result in severe disease partially due to its ability to interfere with the initiation of Th1 responses targeting the production of type I interferons (IFN) and promoting a Th2 immune environment. Epigenetic modulation of gene transcription has been shown to be important in regulating inflammatory pathways. RSV-infected bone marrow-derived DCs (BMDCs) upregulated expression of Kdm5b/Jarid1b H3K4 demethylase. Kdm5b-specific siRNA inhibition in BMDC led to a 10-fold increase in IFN-β as well as increases in IL-6 and TNF-α compared to control-transfected cells. The generation of Kdm5bfl/fl-CD11c-Cre+ mice recapitulated the latter results during in vitro DC activation showing innate cytokine modulation. In vivo, infection of Kdm5bfl/fl-CD11c-Cre+ mice with RSV resulted in higher production of IFN-γ and reduced IL-4 and IL-5 compared to littermate controls, with significantly decreased inflammation, IL-13, and mucus production in the lungs. Sensitization with RSV-infected DCs into the airways of naïve mice led to an exacerbated response when mice were challenged with live RSV infection. When Kdm5b was blocked in DCs with siRNA or DCs from Kdm5bfl/fl-CD11c-CRE mice were used, the exacerbated response was abrogated. Importantly, human monocyte-derived DCs treated with a chemical inhibitor for KDM5B resulted in increased innate cytokine levels as well as elicited decreased Th2 cytokines when co-cultured with RSV reactivated CD4+ T cells. These results suggest that KDM5B acts to repress type I IFN and other innate cytokines to promote an altered immune response following RSV infection that contributes to development of chronic disease.
We previously reported that deficiency for Samd9L, which was cloned as a candidate gene for -7/7q- syndrome, accelerated leukemia cooperatively with enhanced expression of a histone demethylase: F-box and leucine-rich repeat protein 10 (Fbxl10, also known as Jhdm1b, Kdm2b, and Ndy1). To further investigate the role of Fbxl10 in leukemogenesis, we generated transgenic (Tg) mice that overexpress Fbxl10 in hematopoietic stem cells (HSCs). Interestingly, Fbxl10 Tg mice developed myeloid or B-lymphoid leukemia with complete penetrance. HSCs from the Tg mice exhibited an accelerated G0/G1-to-S transition with a normal G0 to G1 entry, resulting in pleiotropic progenitor cell expansion. Fbxl10 Tg HSCs displayed enhanced expression of neuron-specific gene family member 2 (Nsg2), and forced expression of Nsg2 in primary bone marrow cells resulted in expansion of immature cells. In addition, the genes involved in mitochondrial oxidative phosphorylation were markedly enriched in Fbxl10 Tg HSCs, coupled with increased cellular adenosine 5'-triphosphate levels. Moreover, chromatin immunoprecipitation followed by sequencing analysis demonstrated that Fbxl10 directly binds to the regulatory regions of Nsg2 and oxidative phosphorylation genes. These findings define Fbxl10 as a bona fide oncogene, whose deregulated expression contributes to the development of leukemia involving metabolic proliferative advantage and Nsg2-mediated impaired differentiation.
DNA methylation is tightly regulated throughout mammalian development, and altered DNA methylation patterns are a general hallmark of cancer. The methylcytosine dioxygenase TET2 is frequently mutated in hematological disorders, including acute myeloid leukemia (AML), and has been suggested to protect CG dinucleotide (CpG) islands and promoters from aberrant DNA methylation. In this study, we present a novel Tet2-dependent leukemia mouse model that closely recapitulates gene expression profiles and hallmarks of human AML1-ETO-induced AML. Using this model, we show that the primary effect of Tet2 loss in preleukemic hematopoietic cells is progressive and widespread DNA hypermethylation affecting up to 25% of active enhancer elements. In contrast, CpG island and promoter methylation does not change in a Tet2-dependent manner but increases relative to population doublings. We confirmed this specific enhancer hypermethylation phenotype in human AML patients with TET2 mutations. Analysis of immediate gene expression changes reveals rapid deregulation of a large number of genes implicated in tumorigenesis, including many down-regulated tumor suppressor genes. Hence, we propose that TET2 prevents leukemic transformation by protecting enhancers from aberrant DNA methylation and that it is the combined silencing of several tumor suppressor genes in TET2 mutated hematopoietic cells that contributes to increased stem cell proliferation and leukemogenesis.
Jarid1b/KDM5b is a histone demethylase that regulates self-renewal and differentiation in stem cells and cancer; however, its function in hematopoiesis is unclear. Here, we find that Jarid1b is highly expressed in primitive hematopoietic compartments and is overexpressed in acute myeloid leukemias. Constitutive genetic deletion of Jarid1b did not impact steady-state hematopoiesis. In contrast, acute deletion of Jarid1b from bone marrow increased peripheral blood T cells and, following secondary transplantation, resulted in loss of bone marrow reconstitution. Our results reveal that deletion of Jarid1b compromises hematopoietic stem cell (HSC) self-renewal capacity and suggest that Jarid1b is a positive regulator of HSC potential.
The polycomb repressive complex 2 (PRC2) exerts oncogenic effects in many tumour types. However, loss-of-function mutations in PRC2 components occur in a subset of haematopoietic malignancies, suggesting that this complex plays a dichotomous and poorly understood role in cancer. Here we provide genomic, cellular, and mouse modelling data demonstrating that the polycomb group gene SUZ12 functions as tumour suppressor in PNS tumours, high-grade gliomas and melanomas by cooperating with mutations in NF1. NF1 encodes a Ras GTPase-activating protein (RasGAP) and its loss drives cancer by activating Ras. We show that SUZ12 loss potentiates the effects of NF1 mutations by amplifying Ras-driven transcription through effects on chromatin. Importantly, however, SUZ12 inactivation also triggers an epigenetic switch that sensitizes these cancers to bromodomain inhibitors. Collectively, these studies not only reveal an unexpected connection between the PRC2 complex, NF1 and Ras, but also identify a promising epigenetic-based therapeutic strategy that may be exploited for a variety of cancers.
We present an integrated middle-down proteomics platform for sensitive mapping and quantification of coexisting PTMs in large polypeptides (5-7 kDa). We combined an RP trap column with subsequent weak cation exchange-hydrophilic interaction LC interfaced directly to high mass accuracy ESI MS/MS using electron transfer dissociation. This enabled automated and efficient separation and sequencing of hypermodified histone N-terminal tails for unambiguous localization of combinatorial PTMs. We present Histone Coder and IsoScale software to extract, filter, and analyze MS/MS data, including quantification of cofragmenting isobaric polypeptide species. We characterized histone tails derived from murine embryonic stem cells knockout in suppressor of zeste12 (Suz12(-/-) ) and quantified 256 combinatorial histone marks in histones H3, H4, and H2A. Furthermore, a total of 713 different combinatorial histone marks were identified in purified histone H3. We measured a seven-fold reduction of H3K27me2/me3 (where me2 and me3 are dimethylation and trimethylation, respectively) in Suz12(-) (/) (-) cells and detected significant changes of the relative abundance of 16 other single PTMs of histone H3 and other combinatorial marks. We conclude that the inactivation of Suz12 is associated with changes in the abundance of not only H3K27 methylation but also multiple other PTMs in histone H3 tails.
A dichotomous choice for metazoan cells is between proliferation and differentiation. Measuring tRNA pools in various cell types, we found two distinct subsets, one that is induced in proliferating cells, and repressed otherwise, and another with the opposite signature. Correspondingly, we found that genes serving cell-autonomous functions and genes involved in multicellularity obey distinct codon usage. Proliferation-induced and differentiation-induced tRNAs often carry anticodons that correspond to the codons enriched among the cell-autonomous and the multicellularity genes, respectively. Because mRNAs of cell-autonomous genes are induced in proliferation and cancer in particular, the concomitant induction of their codon-enriched tRNAs suggests coordination between transcription and translation. Histone modifications indeed change similarly in the vicinity of cell-autonomous genes and their corresponding tRNAs, and in multicellularity genes and their tRNAs, suggesting the existence of transcriptional programs coordinating tRNA supply and demand. Hence, we describe the existence of two distinct translation programs that operate during proliferation and differentiation.
Polycomb group (PcG) proteins are required for normal differentiation and development and are frequently deregulated in cancer. PcG proteins are involved in gene silencing; however, their role in initiation and maintenance of transcriptional repression is not well defined. Here, we show that knockout of the Polycomb repressive complex 2 (PRC2) does not lead to significant gene expression changes in mouse embryonic stem cells (mESCs) and that it is dispensable for initiating silencing of target genes during differentiation. Transcriptional inhibition in mESCs is sufficient to induce genome-wide ectopic PRC2 recruitment to endogenous PcG target genes found in other tissues. PRC2 binding analysis shows that it is restricted to nucleosome-free CpG islands (CGIs) of untranscribed genes. Our results show that it is the transcriptional state that governs PRC2 binding, and we propose that it binds by default to nontranscribed CGI genes to maintain their silenced state and to protect cell identity.
Ten-eleven-translocation 2 (TET2) belongs to the TET protein family that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine and plays a central role in normal and malignant adult hematopoiesis. Yet the role of TET2 in human hematopoietic development remains largely unknown. Here, we show that TET2 expression is low in human embryonic stem cell (ESC) lines and increases during hematopoietic differentiation. shRNA-mediated TET2 knockdown had no effect on the pluripotency of various ESCs. However, it skewed their differentiation into neuroectoderm at the expense of endoderm and mesoderm both in vitro and in vivo. These effects were rescued by reintroducing the targeted TET2 protein. Moreover, TET2-driven differentiation was dependent on NANOG transcriptional factor. Indeed, TET2 bound to NANOG promoter and in TET2-deficient cells the methylation of the NANOG promoter correlated with a decreased in NANOG expression. The altered differentiation resulting from TET2 knockdown in ESCs led to a decrease in both the number and the cloning capacities of hematopoietic progenitors. These defects were due to an increased apoptosis and an altered gene expression profile, including abnormal expression of neuronal genes. Intriguingly, when TET2 was knockdown in hematopoietic cells, it increased hematopoietic development. In conclusion, our work suggests that TET2 is involved in different stages of human embryonic development, including induction of the mesoderm and hematopoietic differentiation.
MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.
The chromatin environment is essential for the correct specification and preservation of cell identity through modulation and maintenance of transcription patterns. Many chromatin regulators are required for development, stem cell maintenance, and differentiation. Here, we review the roles of the polycomb repressive complexes, PRC1 and PRC2, and the HDAC1- and HDAC2-containing complexes, NuRD, Sin3, and CoREST, in stem cells, development, and cancer, as well as the ongoing efforts to develop therapies targeting these complexes in human cancer. Furthermore, we discuss the role of repressive complexes in modulating thresholds for gene activation and their importance for specification and maintenance of cell fate.
The ability of PRC1 and PRC2 to promote proliferation is a main feature that links polycomb (PcG) activity to cancer. PcGs silence the expression of the tumour suppressor locus Ink4a/Arf, whose products positively regulate pRb and p53 functions. Enhanced PcG activity is a frequent feature of human tumours, and PcG inhibition has been proposed as a strategy for cancer treatment. However, the recurrent inactivation of pRb/p53 responses in human cancers raises a question regarding the ability of PcG proteins to affect cellular proliferation independently from this checkpoint. Here we demonstrate that PRCs regulate cellular proliferation and transformation independently of the Ink4a/Arf-pRb-p53 pathway. We provide evidence that PRCs localize at replication forks, and that loss of their function directly affects the progression and symmetry of DNA replication forks. Thus, we have identified a novel activity by which PcGs can regulate cell proliferation independently of major cell cycle restriction checkpoints.
Oncogene-induced senescence (OIS) can occur in response to hyperactive oncogenic signals and is believed to be a fail-safe mechanism protecting against tumorigenesis. To identify new factors involved in OIS, we performed a screen for microRNAs that can overcome or inhibit OIS in human diploid fibroblasts. This screen led to the identification of miR-378a-5p and in addition several other miRNAs that have previously been shown to play a role in senescence. We show that ectopic expression of miR-378a-5p reduces the expression of several senescence markers, including p16(INK4A) and senescence-associated β-galactosidase. Moreover, cells with ectopic expression of miR-378a-5p retain proliferative capacity even in the presence of an activated Braf oncogene. Finally, we identified several miR-378a-5p targets in diploid fibroblasts that might explain the mechanism by which the microRNA can delay OIS. We speculate that miR-378a-5p might positively influence tumor formation by delaying OIS, which is consistent with a known pro-oncogenic function of this microRNA.
The JmjC domain-containing protein JMJD3/KDM6B catalyses the demethylation of H3K27me3 and H3K27me2. JMJD3 appears to be highly regulated at the transcriptional level and is upregulated in response to diverse stimuli such as differentiation inducers and stress signals. Accordingly, JMJD3 has been linked to the regulation of different biological processes such as differentiation of embryonic stem cells, inflammatory responses in macrophages, and induction of cellular senescence via regulation of the INK4A-ARF locus. Here we show here that JMJD3 interacts with the tumour suppressor protein p53. We find that the interaction is dependent on the p53 tetramerization domain. Following DNA damage, JMJD3 is transcriptionally upregulated and by performing genome-wide mapping of JMJD3, we demonstrate that it binds genes involved in basic cellular processes, as well as genes regulating cell cycle, response to stress and apoptosis. Moreover, we find that JMJD3 binding sites show significant overlap with p53 bound promoters and enhancer elements. The binding of JMJD3 to p53 target sites is increased in response to DNA damage, and we demonstrate that the recruitment of JMJD3 to these sites is dependent on p53 expression. Therefore, we propose a model in which JMJD3 is recruited to p53 responsive elements via its interaction with p53 and speculate that JMJD3 could act as a fail-safe mechanism to remove low levels of H3K27me3 and H3K27me2 to allow for efficient acetylation of H3K27.
The histone demethylase JMJD2C, also known as KDM4C/GASC1, has activity against methylated H3K9 and H3K36 and is amplified and/or overexpressed in human cancers. By the generation of Jmjd2c knockout mice, we demonstrate that loss of Jmjd2c is compatible with cellular proliferation, embryonic stem cell (ESC) self-renewal, and embryonic development. Moreover, we report that JMJD2C localizes to H3K4me3-positive transcription start sites in both primary cells and in the human carcinoma KYSE150 cell line containing an amplification of the JMJD2C locus. Binding is dependent on the double Tudor domain of JMJD2C, which recognizes H3K4me3 but not H4K20me2/me3 in vitro, showing a binding specificity different from that of the double Tudor domains of JMJD2A and JMJD2B. Depletion of JMJD2C in KYSE150 cells has a modest effect on H3K9me3 and H3K36me3 levels but impairs proliferation and leads to deregulated expression of a subset of target genes involved in cell cycle progression. Taking these findings together, we show that JMJD2C is targeted to H3K4me3-positive transcription start sites, where it can contribute to transcriptional regulation, and report that the putative oncogene JMJD2C generally is not required for cellular proliferation or embryonic development.
Epigenetic regulatory mechanisms are implicated in the pathogenesis of acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). Recent progress suggests that proteins involved in epigenetic control are amenable to drug intervention, but little is known about the cancer-specific dependency on epigenetic regulators for cell survival and proliferation. We used a mouse model of human AML induced by the MLL-AF9 fusion oncogene and an epigenetic short hairpin RNA (shRNA) library to screen for novel potential drug targets. As a counter-screen for general toxicity of shRNAs, we used normal mouse bone marrow cells. One of the best candidate drug targets identified in these screens was Jmjd1c. Depletion of Jmjd1c impairs growth and colony formation of mouse MLL-AF9 cells in vitro as well as establishment of leukemia after transplantation. Depletion of JMJD1C impairs expansion and colony formation of human leukemic cell lines, with the strongest effect observed in the MLL-rearranged ALL cell line SEM. In both mouse and human leukemic cells, the growth defect upon JMJD1C depletion appears to be primarily due to increased apoptosis, which implicates JMJD1C as a potential therapeutic target in leukemia.
The defective generation or function of regulatory T (Treg) cells in autoimmune disease contributes to chronic inflammation and tissue injury. We report the identification of FoxA1 as a transcription factor in T cells that, after ectopic expression, confers suppressive properties in a newly identified Treg cell population, herein called FoxA1(+) Treg cells. FoxA1 bound to the Pdl1 promoter, inducing programmed cell death ligand 1 (Pd-l1) expression, which was essential for the FoxA1(+) Treg cells to kill activated T cells. FoxA1(+) Treg cells develop primarily in the central nervous system in response to autoimmune inflammation, have a distinct transcriptional profile and are CD4(+)FoxA1(+)CD47(+)CD69(+)PD-L1(hi)FoxP3(-). Adoptive transfer of stable FoxA1(+) Treg cells inhibited experimental autoimmune encephalomyelitis in a FoxA1-and Pd-l1-dependent manner. The development of FoxA1(+) Treg cells is induced by interferon-β (IFN-β) and requires T cell-intrinsic IFN-α/β receptor (Ifnar) signaling, as the frequency of FoxA1(+) Treg cells was reduced in Ifnb(-/-) and Ifnar(-/-) mice. In individuals with relapsing-remitting multiple sclerosis, clinical response to treatment with IFN-β was associated with an increased frequency of suppressive FoxA1(+) Treg cells in the blood. These findings suggest that FoxA1 is a lineage-specification factor that is induced by IFN-β and supports the differentiation and suppressive function of FoxA1(+) Treg cells.
During X chromosome inactivation (XCI), the Polycomb Repressive Complex 2 (PRC2) is thought to participate in the early maintenance of the inactive state. Although Xist RNA is essential for the recruitment of PRC2 to the X chromosome, the precise mechanism remains unclear. Here, we demonstrate that the PRC2 cofactor Jarid2 is an important mediator of Xist-induced PRC2 targeting. The region containing the conserved B and F repeats of Xist is critical for Jarid2 recruitment via its unique N-terminal domain. Xist-induced Jarid2 recruitment occurs chromosome-wide independently of a functional PRC2 complex, unlike at other parts of the genome, such as CG-rich regions, where Jarid2 and PRC2 binding are interdependent. Conversely, we show that Jarid2 loss prevents efficient PRC2 and H3K27me3 enrichment to Xist-coated chromatin. Jarid2 thus represents an important intermediate between PRC2 and Xist RNA for the initial targeting of the PRC2 complex to the X chromosome during onset of XCI.
The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers and recent investigations have shown TET2 mutations in diffuse large B-cell lymphoma. However, the detection rates and the types of TET2 mutations vary, and the relation to global methylation patterns has not been investigated. Here, we show TET2 mutations in 12 of 100 diffuse large B-cell lymphomas with 7% carrying loss-of-function and 5% carrying missense mutations. Genome-wide methylation profiling using 450K Illumina arrays identified 315 differentially methylated genes between TET2 mutated and TET2 wild-type cases. TET2 mutations are primarily associated with hypermethylation within CpG islands (70%; P<0.0001), and at CpG-rich promoters (60%; P<0.0001) of genes involved in hematopoietic differentiation and cellular development. Hypermethylated loci in TET2 mutated samples overlap with the bivalent (H3K27me3/H3K4me3) silencing mark in human embryonic stem cells (P=1.5×10(-30)). Surprisingly, gene expression profiling showed that only 11% of the hypermethylated genes were down-regulated, among which there were several genes previously suggested to be tumor suppressors. A meta-analysis suggested that the 35 hypermethylated and down-regulated genes are associated with the activated B-cell-like type of diffuse large B-cell lymphoma in other studies. In conclusion, our data suggest that TET2 mutations may cause aberrant methylation mainly of genes involved in hematopoietic development, which are silenced but poised for activation in human embryonic stem cells.
It has recently been demonstrated that the genes controlling the epigenetic programmes that are required for maintaining chromatin structure and cell identity include genes that drive human cancer. This observation has led to an increased awareness of chromatin-associated proteins as potentially interesting drug targets. The successful introduction of DNA methylation and histone deacetylase (HDAC) inhibitors for the treatment of specific subtypes of cancer has paved the way for the use of epigenetic therapy. Here, we highlight key biological findings demonstrating the roles of members of the histone lysine demethylase class of enzymes in the development of cancers, discuss the potential and challenges of therapeutically targeting them, and highlight emerging small-molecule inhibitors of these enzymes.
A plethora of groundbreaking studies have demonstrated the importance of chromatin-associated proteins and post-translational modifications of histones, proteins and DNA (so-called epigenetic modifications) for transcriptional control and normal development. Disruption of epigenetic control is a frequent event in disease, and the first epigenetic-based therapies for cancer treatment have been approved. A generation of new classes of potent and specific inhibitors for several chromatin-associated proteins have shown promise in preclinical trials. Although the biology of epigenetic regulation is complex, new inhibitors such as these will hopefully be of clinical use in the coming years.
The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well as in several cancers, including brain tumors like medulloblastoma and glioblastoma. Inhibition of aberrant Hh-Gli signaling has, thus, emerged as an attractive approach for anticancer therapy; however, the mechanisms that mediate Hh-Gli signaling in vertebrates remain poorly understood. Here, we show that the histone acetyltransferase PCAF/KAT2B is an important factor of the Hh pathway. Specifically, we show that PCAF depletion impairs Hh activity and reduces expression of Hh target genes. Consequently, PCAF downregulation in medulloblastoma and glioblastoma cells leads to decreased proliferation and increased apoptosis. In addition, we found that PCAF interacts with GLI1, the downstream effector in the Hh-Gli pathway, and that PCAF or GLI1 loss reduces the levels of H3K9 acetylation on Hh target gene promoters. Finally, we observed that PCAF silencing reduces the tumor-forming potential of neural stem cells in vivo. In summary, our study identified the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, leading us to propose PCAF as a candidate therapeutic target for the treatment of patients with medulloblastoma and glioblastoma.
Polycomb group (PcG) proteins are epigenetic regulators of transcription that have key roles in stem-cell identity, differentiation and disease. Mechanistically, they function within multiprotein complexes, called Polycomb repressive complexes (PRCs), which modify histones (and other proteins) and silence target genes. The dynamics of PRC1 and PRC2 components has been the focus of recent research. Here we discuss our current knowledge of the PRC complexes, how they are targeted to chromatin and how the high diversity of the PcG proteins allows these complexes to influence cell identity.
Combinations of histone modifications have significant biological roles, such as maintenance of pluripotency and cancer development, but cannot be analyzed at the single cell level. Here, we visualized a combination of histone modifications by applying the in situ proximity ligation assay, which detects two proteins in close vicinity (∼30 nm). The specificity of the method [designated as imaging of a combination of histone modifications (iChmo)] was confirmed by positive signals from H3K4me3/acetylated H3K9, H3K4me3/RNA polymerase II and H3K9me3/H4K20me3, and negative signals from H3K4me3/H3K9me3. Bivalent modification was clearly visualized by iChmo in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell population, namely, ESCs at an early stage of differentiation, and this revealed that the bivalent modification disappeared in a highly concerted manner, whereas phenotypic differentiation proceeded with large variations among cells. Also, using this method, we were able to visualize a combination of repressive histone marks in tissue samples. The application of iChmo to samples with heterogeneous cell population and tissue samples is expected to clarify unknown biological and pathological significance of various combinations of epigenetic modifications.
The chromatin remodeler CHD5 is expressed in neural tissue and is frequently deleted in aggressive neuroblastoma. Very little is known about the function of CHD5 in the nervous system or its mechanism of action. Here we report that depletion of Chd5 in the developing neocortex blocks neuronal differentiation and leads to an accumulation of undifferentiated progenitors. CHD5 binds a large cohort of genes and is required for facilitating the activation of neuronal genes. It also binds a cohort of Polycomb targets and is required for the maintenance of H3K27me3 on these genes. Interestingly, the chromodomains of CHD5 directly bind H3K27me3 and are required for neuronal differentiation. In the absence of CHD5, a subgroup of Polycomb-repressed genes becomes aberrantly expressed. These findings provide insights into the regulatory role of CHD5 during neurogenesis and suggest how inactivation of this candidate tumor suppressor might contribute to neuroblastoma.
Post-translational modifications (PTMs) of histones play a major role in regulating chromatin dynamics and influence processes such as transcription and DNA replication. Here, we report 114 distinct combinations of coexisting PTMs of histone H3 obtained from mouse embryonic stem (ES) cells. Histone H3 N-terminal tail peptides (amino acids 1-50, 5-6 kDa) were separated by optimized weak cation exchange/hydrophilic interaction liquid chromatography (WCX/HILIC) and sequenced online by electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS). High mass accuracy and near complete sequence coverage allowed unambiguous mapping of the major histone marks and discrimination between isobaric and nearly isobaric PTMs such as trimethylation and acetylation. Hierarchical data analysis identified H3K27me2-H3K36me2 as the most frequently observed PTMs in H3. Modifications at H3 residues K27 and K36 often coexist with the abundant mark K23ac, and we identified two frequently occurring quadruplet marks 'K9me1K23acK27me2K36me2' and 'K9me3K23acK27me2K36me', which might indicate a role in crosstalk. Co-occurrence frequency analysis revealed also an interplay between methylations of K9, K27, and K36, suggesting interdependence between histone methylation marks. We hypothesize that the most abundant coexisting PTMs may provide a signature for the permissive state of mouse ES cells.
Cdc10-dependent transcript 1 (Cdt1) is an essential DNA replication protein whose accumulation at the end of the cell cycle promotes the formation of pre-replicative complexes and replication in the next cell cycle. Geminin is thought to be involved in licensing replication by promoting the accumulation of Cdt1 in mitosis, because decreasing the Geminin levels prevents Cdt1 accumulation and impairs DNA replication. Geminin is known to inhibit Cdt1 function; its depletion during G2 leads to DNA rereplication and checkpoint activation. Here we show that, despite rapid Cdt1 protein turnover in G2 phase, Geminin promotes Cdt1 accumulation by increasing its RNA and protein levels in the unperturbed cell cycle. Therefore, Geminin is a master regulator of cell-cycle progression that ensures the timely onset of DNA replication and prevents its rereplication.
<h4>Purpose</h4>Evasion from chemotherapy-induced apoptosis due to p53 loss strongly contributes to drug resistance. Identification of specific targets for the treatment of drug-resistant p53-null tumors would therefore increase the effectiveness of cancer therapy.<h4>Experimental design</h4>By using a kinase-directed short hairpin RNA library and HCT116p53KO drug-resistant colon carcinoma cells, glycogen synthase kinase 3 beta (GSK3B) was identified as a target whose silencing bypasses drug resistance due to loss of p53. p53-null colon cancer cell lines with different sets of mutations were used to validate the role of GSK3B in sustaining resistance and to characterize cell death mechanisms triggered by chemotherapy when GSK3B is silenced. In vivo xenograft studies were conducted to confirm resensitization of drug-resistant cells to chemotherapy upon GSK3 inhibition. Colon cancer samples from a cohort of 50 chemotherapy-treated stage II patients were analyzed for active GSK3B expression.<h4>Results</h4>Downregulation of GSK3B in various drug-resistant p53-null colon cancer cell lines abolished cell viability and colony growth after drug addition without affecting cell proliferation or cell cycle in untreated cells. Cell death of 5-fluorouracil (5FU)-treated p53-null GSK3B-silenced colon carcinoma cells occurred via PARP1-dependent and AIF-mediated but RIP1-independent necroptosis. In vivo studies showed that drug-resistant xenograft tumor mass was significantly reduced only when 5FU was given after GSK3B inhibition. Tissue microarray analysis of colon carcinoma samples from 5FU-treated patients revealed that GSK3B is significantly more activated in drug-resistant versus responsive patients.<h4>Conclusions</h4>Targeting GSK3B, in combination with chemotherapy, may represent a novel strategy for the treatment of chemotherapy-resistant tumors.
Embryonic development requires chromatin remodeling for dynamic regulation of gene expression patterns to ensure silencing of pluripotent transcription factors and activation of developmental regulators. Demethylation of H3K27me3 by the histone demethylases Utx and Jmjd3 is important for the activation of lineage choice genes in response to developmental signals. To further understand the function of Utx in pluripotency and differentiation we generated Utx knockout embryonic stem cells (ESCs). Here we show that Utx is not required for the proliferation of ESCs, however, Utx contributes to the establishment of ectoderm and mesoderm in vitro. Interestingly, this contribution is independent of the catalytic activity of Utx. Furthermore, we provide data showing that the Utx homologue, Uty, which is devoid of detectable demethylase activity, and Jmjd3 partly compensate for the loss of Utx. Taken together our results show that Utx is required for proper formation of ectoderm and mesoderm in vitro, and that Utx, similar to its C.elegans homologue, has demethylase dependent and independent functions.
Histone modifications are thought to regulate chromatin structure, transcription and other nuclear processes. Histone methylation was originally believed to be an irreversible modification that could only be removed by histone eviction or by dilution during DNA replication. However, the isolation of two families of enzymes that can demethylate histones has changed this notion. The biochemical activities of these histone demethylases towards specific Lys residues on histones, and in some cases non-histone substrates, have highlighted their importance in developmental control, cell-fate decisions and disease. Their ability to be regulated through protein-targeting complexes and post-translational modifications is also beginning to shed light on how they provide dynamic control during transcription.
Embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and are pluripotent, as they are able to differentiate into all cell types of the adult organism. Once established, the pluripotent ES cells can be maintained under defined culture conditions, but can also be induced rapidly to differentiate. Maintaining this balance of stability versus plasticity is a challenge, and extensive studies in recent years have focused on understanding the contributions of transcription factors and epigenetic enzymes to the "stemness" properties of these cells. Identifying the molecular switches that regulate ES cell self-renewal versus differentiation can provide insights into the nature of the pluripotent state and enhance the potential use of these cells in therapeutic applications. Here, we review the latest models for how changes in chromatin methylation can modulate ES cell fate, focusing on two major repressive pathways, Polycomb group (PcG) repressive complexes and promoter DNA methylation.
Embryonic development is tightly regulated by transcription factors and chromatin-associated proteins. H3K4me3 is associated with active transcription and H3K27me3 with gene repression, while the combination of both keeps genes required for development in a plastic state. Here we show that deletion of the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) results in major neonatal lethality due to respiratory failure. Jarid1b knockout embryos have several neural defects including disorganized cranial nerves, defects in eye development, and increased incidences of exencephaly. Moreover, in line with an overlap of Jarid1b and Polycomb target genes, Jarid1b knockout embryos display homeotic skeletal transformations typical for Polycomb mutants, supporting a functional interplay between Polycomb proteins and Jarid1b. To understand how Jarid1b regulates mouse development, we performed a genome-wide analysis of histone modifications, which demonstrated that normally inactive genes encoding developmental regulators acquire aberrant H3K4me3 during early embryogenesis in Jarid1b knockout embryos. H3K4me3 accumulates as embryonic development proceeds, leading to increased expression of neural master regulators like Pax6 and Otx2 in Jarid1b knockout brains. Taken together, these results suggest that Jarid1b regulates mouse development by protecting developmental genes from inappropriate acquisition of active histone modifications.
The balance between self-renewal and differentiation of adult stem cells is essential for tissue homeostasis. Here we show that in the haematopoietic system this process is governed by polycomb chromobox (Cbx) proteins. Cbx7 is specifically expressed in haematopoietic stem cells (HSCs), and its overexpression enhances self-renewal and induces leukaemia. This effect is dependent on integration into polycomb repressive complex-1 (PRC1) and requires H3K27me3 binding. In contrast, overexpression of Cbx2, Cbx4 or Cbx8 results in differentiation and exhaustion of HSCs. ChIP-sequencing analysis shows that Cbx7 and Cbx8 share most of their targets; we identified approximately 200 differential targets. Whereas genes targeted by Cbx8 are highly expressed in HSCs and become repressed in progenitors, Cbx7 targets show the opposite expression pattern. Thus, Cbx7 preserves HSC self-renewal by repressing progenitor-specific genes. Taken together, the presence of distinct Cbx proteins confers target selectivity to PRC1 and provides a molecular balance between self-renewal and differentiation of HSCs.
Polycomb repressive complex 1 (PRC1) catalyzes lysine 119 monoubiquitylation on H2A (H2AK119ub1) and regulates pluripotency in embryonic stem cells (ESCs). However, the mechanisms controlling the binding of PRC1 to genomic sites and its catalytic activity are poorly understood. Here, we show that Fbxl10 interacts with Ring1B and Nspc1, forming a noncanonical PRC1 that is required for H2AK119ub1 in mouse ESCs. Genome-wide analyses reveal that Fbxl10 preferentially binds to CpG islands and colocalizes with Ring1B on Polycomb target genes. Notably, Fbxl10 depletion causes a decrease in Ring1B binding to target genes and a major loss of H2AK119ub1. Furthermore, genetic analyses demonstrate that Fbxl10 DNA binding capability and integration into PRC1 are required for H2AK119 ubiquitylation. ESCs lacking Fbxl10, like previously characterized Polycomb mutants, cannot differentiate properly. These results demonstrate that Fbxl10 has a key role in regulating Ring1B recruitment to its target genes and H2AK119 ubiquitylation in ESCs.
While all-trans retinoic acid (ATRA) treatment in acute promyelocytic leukemia (APL) has been the paradigm of targeted therapy for oncogenic transcription factors, the underlying mechanisms remain largely unknown, and a significant number of patients still relapse and become ATRA resistant. We identified the histone demethylase PHF8 as a coactivator that is specifically recruited by RARα fusions to activate expression of their downstream targets upon ATRA treatment. Forced expression of PHF8 resensitizes ATRA-resistant APL cells, whereas its downregulation confers resistance. ATRA sensitivity depends on the enzymatic activity and phosphorylation status of PHF8, which can be pharmacologically manipulated to resurrect ATRA sensitivity to resistant cells. These findings provide important molecular insights into ATRA response and a promising avenue for overcoming ATRA resistance.
O-linked N-acetylglucosamine (O-GlcNAc) transferase (Ogt) activity is essential for embryonic stem cell (ESC) viability and mouse development. Ogt is present both in the cytoplasm and the nucleus of different cell types and catalyzes serine and threonine glycosylation. We have characterized the biochemical features of nuclear Ogt and identified the ten-eleven translocation (TET) proteins Tet1 and Tet2 as stable partners of Ogt in the nucleus of ESCs. We show at a genome-wide level that Ogt preferentially associates with Tet1 to genes promoters in close proximity of CpG-rich transcription start sites. These regions are characterized by low levels of DNA modification, suggesting a link between Tet1 and Ogt activities in regulating CpG island methylation. Finally, we show that Tet1 is required for binding of Ogt to chromatin affecting Tet1 activity. Taken together, our data characterize how O-GlcNAcylation is recruited to chromatin and interacts with the activity of 5-methylcytosine hydroxylases.
<h4>Background</h4>DNA methylation is a key epigenetic mechanism for driving and stabilizing cell-fate decisions. Local deposition and removal of DNA methylation are tightly coupled with transcription factor binding, although the relationship varies with the specific differentiation process. Conversion of monocytes to osteoclasts is a unique terminal differentiation process within the hematopoietic system. This differentiation model is relevant to autoimmune disease and cancer, and there is abundant knowledge on the sets of transcription factors involved.<h4>Results</h4>Here we focused on DNA methylation changes during osteoclastogenesis. Hypermethylation and hypomethylation changes took place in several thousand genes, including all relevant osteoclast differentiation and function categories. Hypomethylation occurred in association with changes in 5-hydroxymethylcytosine, a proposed intermediate toward demethylation. Transcription factor binding motif analysis revealed an over-representation of PU.1, NF-κB, and AP-1 (Jun/Fos) binding motifs in genes undergoing DNA methylation changes. Among these, only PU.1 motifs were significantly enriched in both hypermethylated and hypomethylated genes; ChIP-seq data analysis confirmed its association to both gene sets. Moreover, PU.1 interacts with both DNMT3b and TET2, suggesting its participation in driving hypermethylation and hydroxymethylation-mediated hypomethylation. Consistent with this, siRNA-mediated PU.1 knockdown in primary monocytes impaired the acquisition of DNA methylation and expression changes, and reduced the association of TET2 and DNMT3b at PU.1 targets during osteoclast differentiation.<h4>Conclusions</h4>The work described here identifies key changes in DNA methylation during monocyte-to-osteoclast differentiation and reveals novel roles for PU.1 in this process.
The methylcytosine hydroxylase Tet2 has been implicated in hematopoietic differentiation and the formation of myeloid malignancies when mutated. An ideal system to study the role of Tet2 in myelopoeisis is CEBPα-induced transdifferentiation of pre-B cells into macrophages. Here we found that CEBPα binds to upstream regions of Tet2 and that the gene becomes activated. Tet2 knockdowns impaired the upregulation of macrophage markers as well as phagocytic capacity, suggesting that the enzyme is required for both early and late stage myeloid differentiation. A slightly weaker effect was seen in primary cells with a Tet2 ablation. Expression arrays of transdifferentiating cells with Tet2 knockdowns permitted the identification of a small subset of myeloid genes whose upregulation was blunted. Activation of these target genes was accompanied by rapid increases of promoter hydroxy-methylation. Our observations indicate that Tet2 helps CEBPα rapidly derepress myeloid genes during the conversion of pre-B cells into macrophages.
Aberrant activation of the Wnt signaling pathway is causally involved in the formation of most colorectal cancers (CRCs). Although detailed knowledge exists regarding Wnt-regulated protein-coding genes, much less is known about the possible involvement of non-coding RNAs. Here we used TaqMan Array MicroRNA Cards, capable of detecting 664 unique human microRNAs (miRNAs), to describe changes of the miRNA transcriptome following disruption of beta-catenin/TCF4 activity in DLD1 CRC cells. Most miRNAs appeared to respond independent of host gene regulation and proximal TCF4 chromatin occupancy as inferred from expression microarray and ChIP-chip data. A module of miRNAs induced by abrogated Wnt signaling in vitro was downregulated in two independent series of human primary CRCs (n=76) relative to normal adjacent mucosa (n=34). Several of these miRNAs (miR-145, miR-126, miR-30e-3p and miR-139-5p) markedly inhibited CRC cell growth in vitro when ectopically expressed. By using an integrative approach of proteomics and expression microarrays, we found numerous mRNAs and proteins to be affected by ectopic miR-30e-3p levels. This included HELZ and PIK3C2A that were directly repressed by several miRNA binding sites as confirmed by luciferase reporter assays in combination with mutational analyses. Finally, small interfering RNA-mediated downregulation of PIK3C2A, but not HELZ, was sufficient on its own to restrict CRC cell growth. Collectively, our study demonstrates that multiple miRNAs are upregulated as a consequence of forced attenuation of Wnt signaling in CRC cells, and some of these miRNAs inhibit cell growth with concomitant suppression of several growth-stimulatory cancer-related genes.
Dynamic methylations and demethylations of histone lysine residues are important for gene regulation and are facilitated by histone methyltransferases and histone demethylases (HDMs). KDM5B/Jarid1B/PLU1 is an H3K4me3/me2-specific lysine demethylase belonging to the JmjC domain-containing family of histone demethylases (JHDMs). Several studies have linked KDM5B to breast, prostate and skin cancer, highlighting its potential as a drug target. However, most inhibitor studies have focused on other JHDMs, and inhibitors for KDM5B remain to be explored. Here, we report the expression, purification and characterization of the catalytic core of recombinant KDM5B (ccKDM5B, residues 1-769). We show that ccKDM5B, recombinantly expressed in insect cells, demethylates H3K4me3 and H3K4me2 in vitro. The kinetic characterization showed that ccKDM5B has an apparent Michaelis constant (K(m) (app) ) value of 0.5 μm for its trimethylated substrate H3(1-15)K4me3, a considerably increased apparent substrate affinity than reported for related HDMs. Despite the presence of a PHD domain, the catalytic activity was not affected by additional methylation at the H3K9 position, suggesting that in vitro chromatin cross-talk between H3K4 and H3K9 does not occur for ccKDM5B. Inhibition studies of ccKDM5B showed both in vitro and in cell inhibition of ccKDM5B by 2,4-pyridinedicarboxylic acid (2,4-PDCA) with a potency similar to that reported for the HDM KDM4C. Structure-guided sequence alignment indicated that the binding mode of 2,4-PDCA is conserved between KDM4A/C and KDM5B.
Identification of inhibitors of histone-lysine demethylase (HDM) enzymes is important because of their involvement in the development of cancer. An ELISA-based assay was developed for identification of inhibitors of the HDM KDM4C in a natural products library. Based on one of the hits with affinity in the low μM range (1, a catechol), a subset of structurally related compounds was selected and tested against a panel of HDMs. In this subset, two inhibitors (2 and 10) had comparable affinities towards KDM4C and KDM6A but no effect on PHF8. One inhibitor restored H3K9me3 levels in KDM4C transfected U2-OS cells.
HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene epigenetically silenced or deleted in many human cancers. HIC1 is involved in regulatory loops modulating p53- and E2F1-dependent cell survival, growth control, and stress responses. HIC1 is also essential for normal development because Hic1-deficient mice die perinatally and exhibit gross developmental defects throughout the second half of development. HIC1 encodes a transcriptional repressor with five C(2)H(2) zinc fingers mediating sequence-specific DNA binding and two repression domains: an N-terminal BTB/POZ domain and a central region recruiting CtBP and NuRD complexes. By yeast two-hybrid screening, we identified the Polycomb-like protein hPCL3 as a novel co-repressor for HIC1. Using multiple biochemical strategies, we demonstrated that HIC1 interacts with hPCL3 and its paralog PHF1 to form a stable complex with the PRC2 members EZH2, EED, and Suz12. Confirming the implication of HIC1 in Polycomb recruitment, we showed that HIC1 shares some of its target genes with PRC2, including ATOH1. Depletion of HIC1 by siRNA interference leads to a partial displacement of EZH2 from the ATOH1 promoter. Furthermore, in vivo, ATOH1 repression by HIC1 is associated with Polycomb activity during mouse cerebellar development. Thus, our results identify HIC1 as the first transcription factor in mammals able to recruit PRC2 to some target promoters through its interaction with Polycomb-like proteins.
In this issue, Mousavi et al. (2011) report a novel gene activation function of the H3K27 methyltransferase, Ezh1, in addition to its known role in transcriptional repression.
DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent discovery that the three members of the TET protein family can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has provided a potential mechanism leading to DNA demethylation. Moreover, the demonstration that TET2 is frequently mutated in haematopoietic tumours suggests that the TET proteins are important regulators of cellular identity. Here, we review the current knowledge regarding the function of the TET proteins, and discuss various mechanisms by which they contribute to transcriptional control. We propose that the TET proteins have an important role in regulating DNA methylation fidelity, and that their inactivation contributes to the DNA hypermethylation phenotype often observed in cancer.
H3K4 methylation is associated with active transcription and in combination with H3K27me3 thought to keep genes regulating development in a poised state. The contribution of enzymes regulating trimethylation of lysine 4 at histone 3 (H3K4me3) levels to embryonic stem cell (ESC) self-renewal and differentiation is just starting to emerge. Here, we show that the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) is dispensable for ESC self-renewal, but essential for ESC differentiation along the neural lineage. By genome-wide location analysis, we demonstrate that Jarid1b localizes predominantly to transcription start sites of genes encoding developmental regulators, of which more than half are also bound by Polycomb group proteins. Virtually all Jarid1b target genes are associated with H3K4me3 and depletion of Jarid1b in ESCs leads to a global increase of H3K4me3 levels. During neural differentiation, Jarid1b-depleted ESCs fail to efficiently silence lineage-inappropriate genes, specifically stem and germ cell genes. Our results delineate an essential role for Jarid1b-mediated transcriptional control during ESC differentiation.
<h4>Background</h4>Polycomb group (PcG) genes code for chromatin multiprotein complexes that are responsible for maintaining gene silencing of transcriptional programs during differentiation and in adult tissues. Despite the large amount of information on PcG function during development and cell identity homeostasis, little is known regarding the dynamics of PcG complexes and their role during terminal differentiation.<h4>Results</h4>We show that two distinct polycomb repressive complex (PRC)2 complexes contribute to skeletal muscle cell differentiation: the PRC2-Ezh2 complex, which is bound to the myogenin (MyoG) promoter and muscle creatine kinase (mCK) enhancer in proliferating myoblasts, and the PRC2-Ezh1 complex, which replaces PRC2-Ezh2 on MyoG promoter in post-mitotic myotubes. Interestingly, the opposing dynamics of PRC2-Ezh2 and PRC2-Ezh1 at these muscle regulatory regions is differentially regulated at the chromatin level by Msk1 dependent methyl/phospho switch mechanism involving phosphorylation of serine 28 of the H3 histone (H3S28ph). While Msk1/H3S28ph is critical for the displacement of the PRC2-Ezh2 complex, this pathway does not influence the binding of PRC2-Ezh1 on the chromatin. Importantly, depletion of Ezh1 impairs muscle differentiation and the chromatin recruitment of MyoD to the MyoG promoter in differentiating myotubes. We propose that PRC2-Ezh1 is necessary for controlling the proper timing of MyoG transcriptional activation and thus, in contrast to PRC2-Ezh2, is required for myogenic differentiation.<h4>Conclusions</h4>Our data reveal another important layer of epigenetic control orchestrating skeletal muscle cell terminal differentiation, and introduce a novel function of the PRC2-Ezh1 complex in promoter setting.
MMSET (WHSC1/NSD2) is a SET domain-containing histone lysine methyltransferase the expression of which is deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation associated with poor prognosis. Recent studies have shown that MMSET mRNA levels are increased in other tumor types as well. We have carried out immunohistochemical staining of tissue microarrays and found that MMSET protein is frequently and highly expressed in neuroblastoma (MMSET positive in 75% of neuroblastomas, n = 164). The expression level of MMSET in neuroblastomas was significantly associated with poor survival, negative prognostic factors, and metastatic disease. Moreover, a subset of neuroblastomas for which pre- and postchemotherapy biopsies were available displayed a strong decrease in MMSET protein levels after chemotherapy. In agreement with neuroblastomas becoming more differentiated after treatment, we show that retinoic acid-induced differentiation of human neuroblastoma cells in vitro also leads to a strong decrease in MMSET levels. Furthermore, we show that the high levels of MMSET in normal neural progenitor cells are strongly downregulated during differentiation. Importantly, we show that MMSET is required for proliferation of neuroblastoma cells and brain-derived neural stem cells. Taken together, our results suggest that MMSET is implicated in neuroblastomagenesis possibly by supporting proliferation of progenitor cells and negatively regulating their differentiation. In this respect, MMSET might be a strong candidate therapeutic target in a subset of neuroblastomas with unfavorable prognosis.
Methylated lysines are important epigenetic marks. The enzymes involved in demethylation have recently been discovered and found to be involved in cancer development and progression. Despite the relative recent discovery of these enzymes a number of inhibitors have already appeared. Most of the inhibitors are either previously reported inhibitors of related enzymes or compounds derived from these. Development in terms of selectivity and potency is still pertinent. Several reports on the development of functional assays have been published.
Histone modifications serve as regulatory marks that are instrumental for the control of transcription and chromatin architecture. Strict regulation of gene expression patterns is crucial during development and differentiation, where diverse cell types evolve from common predecessors. Since the first histone lysine demethylase was discovered in 2004, a number of demethylases have been identified and implicated in the control of gene expression programmes and cell fate decisions. Histone demethylases are now emerging as important players in developmental processes and have been linked to human diseases such as neurological disorders and cancer.
Epigenetic regulation of chromatin structure is essential for the expression of genes determining cellular specification and function. The Polycomb repressive complex 2 (PRC2) di- and trimethylates histone H3 on lysine 27 (H3K27me2/me3) to establish repression of specific genes in embryonic stem cells and during differentiation. How the Polycomb group (PcG) target genes are regulated by environmental cues and signaling pathways is quite unexplored. Here, we show that the mitogen- and stress-activated kinases (MSK), through a mechanism that involves promoter recruitment, histone H3K27me3S28 phosphorylation, and displacement of PcG proteins, lead to gene activation. We present evidence that the H3K27me3S28 phosphorylation is functioning in response to stress signaling, mitogenic signaling, and retinoic acid (RA)-induced neuronal differentiation. We propose that MSK-mediated H3K27me3S28 phosphorylation serves as a mechanism to activate a subset of PcG target genes determined by the biological stimuli and thereby modulate the gene expression program determining cell fate.
The E2F and MYC transcription factors are critical regulators of cell proliferation and contribute to the development of human cancers. Here, we report on the identification of a novel E2F target gene, ATAD2, the predicted protein product of which contains both a bromodomain and an ATPase domain. The pRB-E2F pathway regulates ATAD2 expression, which is limiting for the entry into the S phase of the cell cycle. We show that ATAD2 binds the MYC oncogene and stimulates its transcriptional activity. ATAD2 maps to chromosome 8q24, 4.3 Mb distal to MYC, in a region that is frequently found amplified in cancer. Consistent with this, we show that ATAD2 expression is high in several human tumors and that the expression levels correlate with clinical outcome of breast cancer patients. We suggest that ATAD2 links the E2F and MYC pathways and contributes to the development of aggressive cancer through the enhancement of MYC-dependent transcription.
The ARF protein, encoded by alternate exon usage within the CDKN2A locus, provides a link between the retinoblastoma (pRb) and p53 tumor suppressor pathways. Agents that disable pRb or otherwise impinge on the E2F family of transcription factors induce expression of ARF, resulting in stabilization of p53 and activation of p53-regulated genes. However, in some cell types ARF is not induced upon cell cycle re-entry, as expected of a conventional E2F target gene, leading to the suggestion that the ARF promoter only responds to supra-physiological or aberrant levels of E2F. These properties have recently been attributed to a variant E2F binding site but attempts to map specific response elements within the ARF promoter have generally yielded confusing answers. Here we show that in IL2-dependent T-lymphocytes, ARF expression is induced as cells progress from G(0) into S phase, in parallel with other bona fide E2F target genes. This is accompanied by increased association of E2F1 with the endogenous ARF promoter. Our findings suggest that the ability of ARF to register normal proliferative cues depends on the levels of E2F generated in different settings and argue against the idea that it reacts exclusively to oncogenic signals.
<h4>Purpose</h4>Multiple myeloma SET (Suppressor of variegation, Enhancer of zeste, and Trithorax) domain (MMSET) is a histone lysine methyltransferase deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation and poor prognosis. With the aim of understanding, if MMSET can be involved in other types of cancer we investigated the expression of MMSET protein in different types of human tumors.<h4>Experimental design</h4>A monoclonal antibody against MMSET was developed and immunohistochemical staining of tissue microarrays (TMA) containing a large number of tumor samples (n = 3774) and corresponding normal tissues (n = 904) was carried out. Further validations of MMSET expression were carried out on independent, tumor-specific sets of TMAs for urinary bladder (n = 1293) and colon cancer (n = 1206) with corresponding clinicopathological data and long-term follow-up.<h4>Results</h4>MMSET protein was highly expressed in different tumor types compared to normal counterparts. Particular frequent and/or high MMSET expression was found in carcinomas of the gastrointestinal tract (stomach, colon, anal canal), small cell lung carcinoma, tumors of the urinary bladder, female genitals, and skin. In bladder cancer, MMSET expression correlated with tumor aggressiveness. In contrast, MMSET expression was associated with good prognostic factors in colon cancer and was more pronounced in early stages of colon carcinogenesis (dysplasias) than in adenocarcinomas. However, colon cancer patients with high MMSET levels showed a worse 5-year survival.<h4>Conclusions</h4>Our data suggest that MMSET has a broader role in cancer than previously anticipated, and further analysis might qualify it as a prognostic marker and a target for the development of therapy against several types of cancer.
<h4>Background</h4>Polycomb group (PcG) proteins play a crucial role in cellular senescence as key transcriptional regulators of the Ink4a/Arf tumor suppressor gene locus. However, how PcG complexes target and contribute to stable gene silencing of the Ink4a/Arf locus remains little understood.<h4>Methodology/principal findings</h4>We examined the function of Zinc finger domain-containing protein 277 (Zfp277), a novel zinc finger protein that interacts with the PcG protein Bmi1. Zfp277 binds to the Ink4a/Arf locus in a Bmi1-independent manner and interacts with polycomb repressor complex (PRC) 1 through direct interaction with Bmi1. Loss of Zfp277 in mouse embryonic fibroblasts (MEFs) caused dissociation of PcG proteins from the Ink4a/Arf locus, resulting in premature senescence associated with derepressed p16(Ink4a) and p19(Arf) expression. Levels of both Zfp277 and PcG proteins inversely correlated with those of reactive oxygen species (ROS) in senescing MEFs, but the treatment of Zfp277(-/-) MEFs with an antioxidant restored the binding of PRC2 but not PRC1 to the Ink4a/Arf locus. Notably, forced expression of Bmi1 in Zfp277(-/-) MEFs did not restore the binding of Bmi1 to the Ink4a/Arf locus and failed to bypass cellular senescence. A Zfp277 mutant that could not bind Bmi1 did not rescue Zfp277(-/-) MEFs from premature senescence.<h4>Conclusions/significance</h4>Our findings implicate Zfp277 in the transcriptional regulation of the Ink4a/Arf locus and suggest that the interaction of Zfp277 with Bmi1 is essential for the recruitment of PRC1 to the Ink4a/Arf locus. Our findings also highlight dynamic regulation of both Zfp277 and PcG proteins by the oxidative stress pathways.
Enzymes catalysing the methylation of the 5-position of cytosine (mC) have essential roles in regulating gene expression and maintaining cellular identity. Recently, TET1 was found to hydroxylate the methyl group of mC, converting it to 5-hydroxymethyl cytosine (hmC). Here we show that TET1 binds throughout the genome of embryonic stem cells, with the majority of binding sites located at transcription start sites (TSSs) of CpG-rich promoters and within genes. The hmC modification is found in gene bodies and in contrast to mC is also enriched at CpG-rich TSSs. We provide evidence further that TET1 has a role in transcriptional repression. TET1 binds a significant proportion of Polycomb group target genes. Furthermore, TET1 associates and colocalizes with the SIN3A co-repressor complex. We propose that TET1 fine-tunes transcription, opposes aberrant DNA methylation at CpG-rich sequences and thereby contributes to the regulation of DNA methylation fidelity.
There is now evidence for both increased and decreased activity of the enzymes controlling the methylation of lysine 27 on histone 3 (H3K27) in cancer. One of these enzymes, KDM6B formally known as JMJD3, a histone demethylase, which removes the trimethyl mark from H3K27, is required for the lineage commitment and terminal differentiation of neural stem cells and of keratinocytes. Our results suggest that KDM6B may also have a role in antigen-driven B-cell differentiation. KDM6B expression increases in B-cell subsets with increasing stage of differentiation, and gene expression profiling shows that KDM6B transcriptional targets in germinal centre B (GC B) cells are significantly enriched for those differentially expressed during memory and plasma cell differentiation. Our results also suggest that aberrant expression of KDM6B may contribute to the pathogenesis of Hodgkin's Lymphoma (HL), an Epstein-Barr virus (EBV) associated malignancy. KDM6B is over-expressed in primary HL and induced by the EBV oncogene, latent membrane protein (LMP1) in GC B cells, the presumptive progenitors of HL. Consistent with these observations, we found that KDM6B transcriptional targets in GC B cells are enriched for genes differentially expressed in HL, and that KDM6B depletion can restore the tri-methylation of H3K27 on these genes.
The DNA damage induced G(2)/M checkpoint is an important guardian of the genome that prevents cell division when DNA lesions are present. The checkpoint prevents cells from entering mitosis by degrading CDC25A, a key CDK activator. CDC25A proteolysis is controlled by direct phosphorylation events that lead to its recognition by the ubiquitin ligase beta-TrCP. Recently we have identified NEK11, a member of NIMA-related kinase family, as the critical kinase triggering CDC25A degradation. NEK11 controls degradation of CDC25A by directly phosphorylating CDC25A on residues whose phosphorylation is required for beta-TrCP mediated CDC25A polyubiquitylation and degradation. The activity of NEK11 is in turn controlled by CHK1 that activates NEK11 via phosphorylation on serine 273. Since inhibition of NEK11 activity forces checkpoint-arrested cells into mitosis and cell death, NEK11 is, like CHK1, a strong candidate target for the development of novel anticancer drugs. Here we further support this notion by showing results suggesting that NEK11 expression increases during colon cancer development.
Cancer is perceived as a heterogeneous group of diseases that is characterized by aberrant patterns of gene expression. In the last decade, an increasing amount of data has pointed to a key role for epigenetic alterations in human cancer. In this review, we focus on a subclass of epigenetic regulators, namely histone methyltransferases (HMTs). Several HMTs have been linked to different types of cancer; however, in most cases we only have limited knowledge regarding the molecular mechanisms by which the HMTs contribute to disease development. We summarize the current knowledge regarding some of the best-validated examples of HMTs contributing to tumorigenesis and discuss their potential mechanisms of action.
The eukaryotic cell cycle is regulated by multiple ubiquitin-mediated events, such as the timely destruction of cyclins and replication licensing factors. The histone H4 methyltransferase SET8 (Pr-Set7) is required for chromosome compaction in mitosis and for maintenance of genome integrity. In this study, we show that SET8 is targeted for degradation during S phase by the CRL4(CDT2) ubiquitin ligase in a proliferating cell nuclear antigen (PCNA)-dependent manner. SET8 degradation requires a conserved degron responsible for its interaction with PCNA and recruitment to chromatin where ubiquitylation occurs. Efficient degradation of SET8 at the onset of S phase is required for the regulation of chromatin compaction status and cell cycle progression. Moreover, the turnover of SET8 is accelerated after ultraviolet irradiation dependent on the CRL4(CDT2) ubiquitin ligase and PCNA. Removal of SET8 supports the modulation of chromatin structure after DNA damage. These results demonstrate a novel regulatory mechanism, linking for the first time the ubiquitin-proteasome system with rapid degradation of a histone methyltransferase to control cell proliferation.
The Polycomb group (PcG) proteins are transcriptional repressors that regulate lineage choices during development and differentiation. Recent studies have advanced our understanding of how the PcG proteins regulate cell fate decisions and how their deregulation potentially contributes to cancer. In this Review we discuss the emerging roles of long non-coding RNAs (ncRNAs) and a subset of transcription factors, which we call cell fate transcription factors, in the regulation of PcG association with target genes. We also speculate about how their deregulation contributes to tumorigenesis.
DNA damage-induced cell-cycle checkpoints have a critical role in maintaining genomic stability. A key target of the checkpoints is the CDC25A (cell division cycle 25 homologue A) phosphatase, which is essential for the activation of cyclin-dependent kinases and cell-cycle progression. To identify new genes involved in the G2/M checkpoint we performed a large-scale short hairpin RNA (shRNA) library screen. We show that NIMA (never in mitosis gene A)-related kinase 11 (NEK11) is required for DNA damage-induced G2/M arrest. Depletion of NEK11 prevents proteasome-dependent degradation of CDC25A, both in unperturbed and DNA-damaged cells. We show that NEK11 directly phosphorylates CDC25A on residues whose phosphorylation is required for beta-TrCP (beta-transducin repeat-containing protein)-mediated polyubiquitylation and degradation of CDC25A. Furthermore, we demonstrate that CHK1 (checkpoint kinase 1) directly activates NEK11 by phosphorylating it on Ser 273, indicating that CHK1 and NEK11 operate in a single pathway that controls proteolysis of CDC25A. Taken together, these results demonstrate that NEK11 is an important component of the pathway enforcing the G2/M checkpoint, suggesting that genetic mutations in NEK11 may contribute to the development of human cancer.
The polycomb group (PcG) proteins are essential for the normal development of multicellular organisms. They form multi-protein complexes that work as transcriptional repressors of several thousand genes controlling differentiation pathways during development. How the PcG proteins work as transcriptional repressors is incompletely understood, but involves post-translational modifications of histones by two major PcG protein complexes: polycomb repressive complex 1 and polycomb repressive complex 2.
Maintenance of gene expression through epigenetic mechanisms such as DNA- and histone-methylation is essential for preserving cellular identity and function. Multiplication of eukaryotic cells requires that the DNA content of the cell is duplicated through replication, which is coupled to incorporation of de novo synthesized core histones into nucleosomal structures. One of the challenging questions in biology is to explain how the organism ensures that regulatory epigenetic marks, once established, are transferred from one cell generation to the next. Based on studies in our laboratory, we have recently proposed a model for how the methylated lysine 27 of histone H3 (H3K27) can be stably transmitted through the cell division cycle. We found that the Polycomb Repressive Complex 2 (PRC2), which is responsible for di- and trimethylation of H3K27 (H3K27me2/me3), binds to its own site of methylation. Moreover, our results suggested that maintenance of transcriptional repression by PRC2 requires the binding of the PRC2 complex to H3K27me3/me2. Based on these two key observations we propose that PRC2 is able to copy the mark from an old parental H3 molecule to a newly synthesized H3 molecule as DNA replication proceeds. In addition, our results support a model for how the H3K27me3 mark could be preserved in the interphase of the cell cycle, where other events such as histone exchange and demethylation could counteract PRC2 function. Here we discuss the implications of our results in further detail.
The enzymes catalyzing lysine and arginine methylation of histones are essential for maintaining transcriptional programs and determining cell fate and identity. Until recently, histone methylation was regarded irreversible. However, within the last few years, several families of histone demethylases erasing methyl marks associated with gene repression or activation have been identified, underscoring the plasticity and dynamic nature of histone methylation. Recent discoveries have revealed that histone demethylases take part in large multiprotein complexes synergizing with histone deacetylases, histone methyltransferases, and nuclear receptors to control developmental and transcriptional programs. Here we review the emerging biochemical and biological functions of the histone demethylases and discuss their potential involvement in human diseases, including cancer.
Polycomb group (PcG) proteins are transcriptional repressors, which regulate proliferation and cell fate decisions during development, and their deregulated expression is a frequent event in human tumours. The Polycomb repressive complex 2 (PRC2) catalyzes trimethylation (me3) of histone H3 lysine 27 (K27), and it is believed that this activity mediates transcriptional repression. Despite the recent progress in understanding PcG function, the molecular mechanisms by which the PcG proteins repress transcription, as well as the mechanisms that lead to the activation of PcG target genes are poorly understood. To gain insight into these mechanisms, we have determined the global changes in histone modifications in embryonic stem (ES) cells lacking the PcG protein Suz12 that is essential for PRC2 activity. We show that loss of PRC2 activity results in a global increase in H3K27 acetylation. The methylation to acetylation switch correlates with the transcriptional activation of PcG target genes, both during ES cell differentiation and in MLL-AF9-transduced hematopoietic stem cells. Moreover, we provide evidence that the acetylation of H3K27 is catalyzed by the acetyltransferases p300 and CBP. Based on these data, we propose that the PcG proteins in part repress transcription by preventing the binding of acetyltransferases to PcG target genes.
ARHI is a maternally imprinted tumor suppressor gene whose expression is markedly downregulated in breast cancer. Reactivation of ARHI expression in breast cancer cells is associated with increased histone H3 acetylation and decreased lysine 9 methylation of histone H3. An ARHI promoter segment that spanned bases -420 to +58 (designated the P2 region) exhibits significantly higher promoter activity in normal cells than in cancer cells. To better understand the molecular mechanisms contributing to this differential transcriptional activity, we sought to identify transcription factors that bind to the P2 region of the ARHI promoter and regulate its activity. Sequence analysis and oligonucleotide competition in electrophoretic mobility shift assays identified an A2 fragment containing an E2F-binding site. Using specific antibodies in supershift assays, we have shown that anti-E2F1 and 4 antibodies can supershift the A2-protein complexes, whereas anti-E2F2 and 6 antibodies cannot, demonstrating that the A2 fragment interacts with specific members of the E2F family proteins. When compared with normal breast epithelial cells, breast cancer cells have significantly elevated expression of E2F1, 4 and increased E2F DNA-binding activity. Moreover, chromatin immunoprecipitation experiments revealed that both E2F1 and 4 bind to the ARHI promoter in breast cancer cells in vivo. This binding was reduced when the cells were treated with the histone deacetylase (HDAC) inhibitor--trichostatin A (TSA). When SKBr3 cells were cotransfected with an ARHI/luciferase reporter and E2F-expression vectors, E2F1 and 4 reduced ARHI promoter activity 2-3-fold, and this reduction could be reversed by TSA treatment. The negative regulation by E2F-HDAC complexes could also be reduced by small interfering RNA of E2F1 and 4. While the retinoblastoma protein, pRB, alone had no effect on ARHI promoter activity, repression by E2F1, but not E2F4, was enhanced by the coexpression of pRB. Taken together, our results suggest that E2F1, 4 and their complexes with HDAC play an important role in downregulating the expression of the tumor suppressor gene ARHI in breast cancer cells.
Eukaryotes have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. These mechanisms prevent relicensing of origins of replication after initiation of DNA replication in S phase until the end of mitosis. Most of our knowledge of mechanisms controlling prereplication complex (preRC) formation are based on studies from yeast and Xenopus, while much less is known for mammalian cells. Here we discuss our recent data demonstrating that Geminin is required for preventing rereplication in human normal and cancer cells.
X-linked mental retardation (XLMR) is an inherited disorder that mostly affects males and is caused by mutations in genes located on the X chromosome. Here, we show that the XLMR protein PHF8 and a C. elegans homolog F29B9.2 catalyze demethylation of di- and monomethylated lysine 9 of histone H3 (H3K9me2/me1). The PHD domain of PHF8 binds to H3K4me3 and colocalizes with H3K4me3 at transcription initiation sites. Furthermore, PHF8 interacts with another XMLR protein, ZNF711, which binds to a subset of PHF8 target genes, including the XLMR gene JARID1C. Of interest, the C. elegans PHF8 homolog is highly expressed in neurons, and mutant animals show impaired locomotion. Taken together, our results functionally link the XLMR gene PHF8 to two other XLMR genes, ZNF711 and JARID1C, indicating that MR genes may be functionally linked in pathways, causing the complex phenotypes observed in patients developing MR.
The Polycomb group (PcG) proteins have an important role in controlling the expression of genes essential for development, differentiation and maintenance of cell fates. The Polycomb repressive complex 2 (PRC2) is believed to regulate transcriptional repression by catalysing the di- and tri-methylation of lysine 27 on histone H3 (H3K27me2/3). At present, it is unknown how the PcG proteins are recruited to their target promoters in mammalian cells. Here we show that PRC2 forms a stable complex with the Jumonji- and ARID-domain-containing protein, JARID2 (ref. 4). Using genome-wide location analysis, we show that JARID2 binds to more than 90% of previously mapped PcG target genes. Notably, we show that JARID2 is sufficient to recruit PcG proteins to a heterologous promoter, and that inhibition of JARID2 expression leads to a major loss of PcG binding and to a reduction of H3K27me3 levels on target genes. Consistent with an essential role for PcG proteins in early development, we demonstrate that JARID2 is required for the differentiation of mouse embryonic stem cells. Thus, these results demonstrate that JARID2 is essential for the binding of PcG proteins to target genes and, consistent with this, for the proper differentiation of embryonic stem cells and normal development.
Polycomb complexes establish chromatin modifications for maintaining gene repression and are essential for embryonic development in mice. Here we use pluripotent embryonic stem (ES) cells to demonstrate an unexpected redundancy between Polycomb-repressive complex 1 (PRC1) and PRC2 during the formation of differentiated cells. ES cells lacking the function of either PRC1 or PRC2 can differentiate into cells of the three germ layers, whereas simultaneous loss of PRC1 and PRC2 abrogates differentiation. On the molecular level, the differentiation defect is caused by the derepression of a set of genes that is redundantly repressed by PRC1 and PRC2 in ES cells. Furthermore, we find that genomic repeats are Polycomb targets and show that, in the absence of Polycomb complexes, endogenous murine leukemia virus elements can mobilize. This indicates a contribution of the Polycomb group system to the defense against parasitic DNA, and a potential role of genomic repeats in Polycomb-mediated gene regulation.
p53 regulates the expression of genes involved in cell cycle control, apoptosis and DNA damage repair. Here we demonstrate that DUSP11 (dual specificity phosphatase 11), a member of the protein tyrosine phosphatase family that binds to RNA-RNP complexes and RNA splicing factors, is a p53 target gene. Consistent with this, the expression of DUSP11 is induced in a p53-dependent manner after treatment with DNA damaging agents. Chromatin immunoprecipitation analysis showed that p53 binds to 2 putative p53 DNA binding sites in the promoter region of DUSP11. Colony formation and proliferation assays demonstrated that the ectopic expression of wildtype, but not catalytical inactive, DUSP11 leads to growth arrest. Furthermore inhibition of DUSP11 expression by shRNA increases the proliferation of normal and DNA damaged cells in tissue culture. Finally we show that the splicing factor SAM68 (Src-associated protein in mitotic cells) binds to DUSP11 in vitro and in vivo. Taken together these results suggest that DUSP11 contributes to p53-dependent inhibition of cell proliferation and that it might be involved in regulating RNA splicing through SAM68.
The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that cooperates with p53 to suppress cancer development. We and others recently showed that HIC1 is a transcriptional target of p53. To identify additional transcriptional regulators of HIC1, we screened a set of transcription factors for regulation of a human HIC1 promoter reporter. We found that E2F1 strongly activates the full-length HIC1 promoter reporter. Promoter deletions and mutations identified two E2F responsive elements in the HIC1 core promoter region. Moreover, in vivo binding of E2F1 to the HIC1 promoter was shown by chromatin immunoprecipitation assays in human TIG3 fibroblasts expressing tamoxifen-activated E2F1. In agreement, activation of E2F1 in TIG3-E2F1 cells markedly increased HIC1 expression. Interestingly, expression of E2F1 in the p53(-/-) hepatocellular carcinoma cell line Hep3B led to an increase of endogenous HIC1 mRNA, although bisulfite genomic sequencing of the HIC1 promoter revealed that the region bearing the two E2F1 binding sites is hypermethylated. In addition, endogenous E2F1 induced by etoposide treatment bound to the HIC1 promoter. Moreover, inhibition of E2F1 strongly reduced the expression of etoposide-induced HIC1. In conclusion, we identified HIC1 as novel E2F1 transcriptional target in DNA damage responses.
The tumor suppressor proteins p16INK4A and p14ARF, encoded by the INK4A-ARF locus, are key regulators of cellular senescence. The locus is epigenetically silenced by the repressive H3K27me3 mark in normally growing cells, but becomes activated in response to oncogenic stress. Here, we show that expression of the histone H3 Lys 27 (H3K27) demethylase JMJD3 is induced upon activation of the RAS-RAF signaling pathway. JMJD3 is recruited to the INK4A-ARF locus and contributes to the transcriptional activation of p16INK4A in human diploid fibroblasts. Additionally, inhibition of Jmjd3 expression in mouse embryonic fibroblasts results in suppression of p16Ink4a and p19Arf expression and in their immortalization.
The finding that tumours, like normal tissues, are endowed with varying degrees of cellular heterogeneity has far-reaching consequences for our understanding of cancer. The cancer stem cell and clonal evolution models have both been proposed to explain tumour-associated cellular heterogeneity. Here, we briefly review these two non-exclusive models with special emphasis on how they aid our understanding of cancer and their implications for therapeutic strategies. Finally, we discuss the close association between basic stem cell biology and cancer, focusing on the role of self-renewal.
The initiation of DNA replication is a tightly controlled process that involves the formation of distinct complexes at origins of DNA replication at specific periods of the cell cycle. Pre-replicative complexes are formed during telophase and early G(1). They rearrange at the start of S phase to form pre-initiation complexes, which are a prerequisite for DNA replication. The CDT1 protein is required for the formation of the pre-replicative complexes. Here we show that human CDT1 associates with the CDC7 kinase and recruits CDC45 to chromatin. Moreover, we show that the amount of CDT1 bound to chromatin is regulated by CDC7. We propose a model in which chromatin-bound CDT1 is first stabilized and subsequently displaced by CDC7 activity, thereby ensuring the timely execution of DNA replication.
Death-associated protein kinase 2 (DAPK2) belongs to a family of proapoptotic Ca(2+)/calmodulin-regulated serine/threonine kinases. We recently identified DAPK2 as an enhancing factor during granulocytic differentiation. To identify transcriptional DAPK2 regulators, we cloned 2.7 kb of the 5'-flanking region of the DAPK2 gene. We found that E2F1 and Krüppel-like factor 6 (KLF6) strongly activate the DAPK2 promoter. We mapped the E2F1 and KLF6 responsive elements to a GC-rich region 5' of exon 1 containing several binding sites for KLF6 and Sp1 but not for E2F. Moreover, we showed that transcriptional activation of DAPK2 by E2F1 and KLF6 is dependent on Sp1 using Sp1/KLF6-deficient insect cells, mithramycin A treatment to block Sp1-binding or Sp1 knockdown cells. Chromatin immunoprecipitation revealed recruitment of Sp1 and to lesser extent that of E2F1 and KLF6 to the DAPK2 promoter. Activation of E2F1 in osteosarcoma cells led to an increase of endogenous DAPK2 paralleled by cell death. Inhibition of DAPK2 expression resulted in significantly reduced cell death upon E2F1 activation. Similarly, KLF6 expression in H1299 cells increased DAPK2 levels accompanied by cell death that is markedly decreased upon DAPK2 knockdown. Moreover, E2F1 and KLF6 show cooperation in activating the DAPK2 promoter. In summary, our findings establish DAPK2 as a novel Sp1-dependent target gene for E2F1 and KLF6 in cell death response.
Epigenetic information refers to heritable changes in gene function that are stable between cell divisions but which is not a result of changes in the DNA sequence. Part of the epigenetic mechanism has been ascribed to modifications of histones or DNA that affects the transcription of specific genes. In this context, post-translational modifications of histone tails, in particular methylation of lysines, are regarded as important for the storage of epigenetic information. Regulation of this information plays an important role during cellular differentiation where cells with different characteristic features evolve from the same ancestor, despite identical genomic material. The characterization of several enzymes catalyzing histone lysine methylation have supported this concept by showing the requirement of these enzymes for normal development and their involvement in diseases such as cancer. The recent identification of proteins with histone demethylase activity has shown that the methylated mark is much more dynamic than previously anticipated, thereby potentially challenging the concept of histone-methylation in stable epigenetic programming.
The two known DP proteins, TFDP1 and -2, bind E2Fs to form heterodimers essential for high affinity DNA binding and efficient transcriptional activation/repression. Here we report the identification of a new member of the DP family, human TFDP3. Despite the high degree of sequence similarity, TFDP3 is apparently distinct from TFDP1 in function. Although TFDP3 retained the capacity to bind to E2F proteins, the resulting heterodimers failed to interact with the E2F consensus sequence. In contrast to the stimulatory effect of TFDP1, TFDP3 inhibited E2F-mediated transcriptional activation. Consistent with this observation, we found that ectopic expression of TFDP3 impaired cell cycle progression from G(1) to S phase instead of facilitating such a transition as TFDP1 does. Sequence substitution analysis indicated that the DNA binding domain of TFDP3 was primarily responsible for the lack of DNA binding ability of E2F-TFDP3 heterodimers and the inhibition of E2F-mediated transcriptional activation. Fine mapping further revealed four amino acids in this region, which were critical for the functional conversion from activation by TFDP1 to suppression by TFDP3. In conclusion, these studies identify a new DP protein and a novel mechanism whereby E2F function is regulated.
Loss of the retinoblastoma protein, pRB, leads to apoptosis, and several results have suggested that this is dependent on the E2F transcription factors. However, so far, the ability of the different E2F family members to contribute to apoptosis is controversial. Here, we show that ectopic expression of E2F3 results in apoptosis in both primary mouse fibroblasts and transgenic mice. Apoptosis induced by E2F3 is associated with the accumulation of E2F1 and, strikingly, we found that E2F3-induced apoptosis is dependent on E2F1. On the basis of these results, we propose that the accumulation of crucial levels of E2F1 activity, and not total E2F activity, is essential for the induction of apoptosis in response to a deregulated pRB pathway. These results are consistent with previous findings that E2F1, but not other E2Fs, can have tumour-suppressing activities.
Glioma, and in particular high-grade astrocytoma termed glioblastoma multiforme (GBM), is the most common primary tumor of the brain. Primarily because of its diffuse nature, there is no effective treatment for GBM, and relatively little is known about the processes by which it develops. Therefore, in order to design novel therapies and treatments for GBM, research has recently intensified to identify the cellular and molecular mechanisms leading to GBM formation. Modeling of astrocytomas by genetic manipulation of mice suggests that deregulation of the pathways that control gliogenesis during normal brain development, such as the differentiation of neural stem cells (NSCs) into astrocytes, might contribute to GBM formation. These pathways include growth factor-induced signal transduction routes and processes that control cell cycle progression, such as the p16-CDK4-RB and the ARF-MDM2-p53 pathways. The expression of several of the components of these signaling cascades has been found altered in GBM, and recent data indicate that combinations of mutations in these pathways may contribute to GBM formation, although the exact mechanisms are still to be uncovered. Use of novel techniques including large-scale genomics and proteomics in combination with relevant mouse models will most likely provide novel insights into the molecular mechanisms underlying glioma formation and will hopefully lead to development of treatment modalities for GBM.
The transcriptional repressor E2F6 has been identified as a component of two distinct polycomb group protein (PcG)-containing complexes, suggesting a mechanism for the recruitment of repressive complexes to target sequences in DNA. Whereas one complex is involved in the repression of classic E2F target genes in G0, a role for E2F6 within the cell cycle has yet to be defined. We searched for novel E2F6-binding proteins using a yeast two-hybrid screen and identified the PcG protein, EPC1. We showed that, both in vitro and in vivo, E2F6, DP1, and EPC1 form a stable core complex with repressive activity. Furthermore, we identified the proliferation-specific PcG, EZH2, as an EPC1-interacting protein. Using affinity purification, we showed that E2F6, DP1, EPC1, EZH2, and Sin3B co-elute, suggesting the identification of a novel E2F6 complex that exists in vivo in both normal and transformed human cell lines. EZH2 is required for cellular proliferation and consistent with this, EZH2 elutes with the E2F6-EPC1 complex only in proliferating cells. Thus we have identified a novel E2F6-PcG complex (E2F6-EPC1) that interacts with EZH2 and may regulate genes required for cell cycle progression.
Cyclin G1 is a transcriptional target of the tumor suppressor p53, and its expression is increased after DNA damage. Recent data show that cyclin G1 can regulate the levels of p53 by a mechanism that involves dephosphorylation of Mdm2 by protein phosphatase 2A. To understand the biologic role of cyclin G1, we have generated cyclin G1-deficient mice. In agreement with previous results, we showed that these mice develop normally, and that proliferation and induction of cellular senescence in cyclin G1-deficient mouse embryo fibroblasts are indistinguishable from wild-type fibroblasts. However, we found that the p53 levels in the cyclin G1-deficient mice are 2-fold higher that in wild-type mice. Moreover, we showed that treatment of mice with the alkylating agent 1,4-bis[N,N'-di(ethylene)-phosphamide]piperazine (Dipin), followed by partial hepatectomy, decreased G1-S transition in cyclin G1-null hepatocytes as compared with wild type. Finally, we found a significant decrease in tumor incidence, mass, and malignancy in both male and female cyclin G1-null mice after treatment with the potent hepatocarcinogen N-diethylnitrosamine. Taken with recent published data, our results suggest that cyclin G1, together with Mdm2, constitute a part of a negative feedback system that attenuates the activity of p53. In conclusion, our data suggest that the decreased tumor susceptibility after loss of cyclin G1 function is caused by the increased tumor suppressor action of p53.
Deregulation of the retinoblastoma protein (pRB) pathway is a hallmark of cancer. In the absence of other genetic alterations, this deregulation results in lack of differentiation, hyperproliferation and apoptosis. The pRB protein acts as a transcriptional repressor by targeting the E2F transcription factors, whose functions are required for entry into S phase. Increased E2F activity can induce S phase in quiescent cells--this is a central element of most models for the development of cancer. We show that although E2F1 alone is not sufficient to induce S phase in diploid mouse and human fibroblasts, increased E2F1 activity can result in S-phase entry in diploid fibroblasts in which the p53-mediated G1 checkpoint is suppressed. In addition, we show that E2F1 can induce S phase in primary mouse fibroblasts lacking pRB. These results indicate that, in addition to acting as an E2F-dependent transcriptional repressor, pRB is also required for the cells to retain the G1 checkpoint in response to unprogrammed proliferative signals.
Apoptosis Protease-Activating Factor 1, APAF1, was originally isolated four years ago and shown to be the mammalian homologue of the C. elegans pro-apoptotic ced4 gene. Since then, the expression of APAF1 has been demonstrated to be involved in several cell death pathways, including the induction of apoptosis by the p53 tumour suppressor protein and neuronal apoptosis. In this review we will focus on the regulation of APAF1 expression, in particular with regard to recent developments in our understanding of the role of APAF1 in both tumourigenesis and mammalian development.
Loss of function of both the p53 pathway and the retinoblastoma protein (pRB) pathway plays a significant role in the development of most human cancers. Loss of pRB results in deregulated cell proliferation and apoptosis, whereas loss of p53 desensitizes cells to checkpoint signals, including apoptosis. In the past two years, mouse genetics and gene expression profiling have led to major advances in our understanding of how the pRB and p53 pathways regulate apoptosis and thus the development of tumours.
Expression of the bovine papillomavirus E2 protein in cervical carcinoma cells represses expression of integrated human papillomavirus (HPV) E6/E7 oncogenes, followed by repression of the cdc25A gene and other cellular genes required for cell cycle progression, resulting in dramatic growth arrest. To explore the mechanism of repression of cell cycle genes in cervical carcinoma cells following E6/E7 repression, we analyzed regulation of the cdc25A promoter, which contains two consensus E2F binding sites and a consensus E2 binding site. The wild-type E2 protein inhibited expression of a luciferase gene linked to the cdc25A promoter in HT-3 cervical carcinoma cells. Mutation of the distal E2F binding site in the cdc25A promoter abolished E2-induced repression, whereas mutation of the proximal E2F site or the E2 site had no effect. None of these mutations affected the activity of the promoter in the absence of E2 expression. Expression of the E2 protein also led to posttranscriptional increase in the level of E2F4, p105(Rb), and p130 and induced the formation of nuclear E2F4-p130 and E2F4-p105(Rb) complexes. This resulted in marked rearrangement of the protein complexes that formed at the distal E2F site in the cdc25A promoter, including the replacement of free E2F complexes with E2F4-p105(Rb) complexes. These experiments indicated that repression of E2F-responsive promoters following HPV E6/E7 repression was mediated by activation of the Rb tumor suppressor pathway and the assembly of repressing E2F4-Rb DNA binding complexes. Importantly, these experiments revealed that HPV-induced alterations in E2F transcription complexes that occur during cervical carcinogenesis are reversed by repression of HPV E6/E7 expression.
To elicit the transcriptional response following intra- or extracellular stimuli, the signals need to be transmitted to their site of action within the nucleus. The nucleocytoplasmic shuttling of transcription factors is a mechanism mediating this process. The activation and inactivation of the transcriptional response is essential for cells to progress through the cell cycle in a normal manner. The involvement of cytoplasmic and nuclear accessory molecules, and the general nuclear membrane transport components, are essential for this process. Although nuclear import and export for different transcription factor families are regulated by similar mechanisms, there are several differences that allow for the specific activation of each transcription factor. This review discusses the general import and export pathways found to be common amongst many different transcription factors, and highlights a select group of transcription factors that demonstrate the diversity displayed in their mode of activation and inactivation.
The transcription factor complex E2F-1/DP-1 regulates the G1-to-S-phase transition and has been associated with sensitivity to the S-phase-specific anticancer agents camptothecin and etoposide, which poison DNA topoisomerase I and II, respectively. To investigate the relationship between E2F-1 and drug sensitivity in detail, we established human osteosarcoma U-20S-TA cells expressing full-length E2F-1/ DP-1 under the control of a tetracycline-responsive promoter, designated UE1DP-1 cells. Topoisomerase I levels and activity as well as the number of camptothecin-induced DNA single- and double-strand breaks were unchanged in UEIDP-1/tc- cells with >10-fold E2F-1/DP-1 overexpression. However, UE1DP-1/tc- cells were hypersensitive to camptothecin in both a clonogenic assay and four different apoptotic assays. This indicates that camptothecin-induced toxicity in this model is due to the activation of an E2F-1/ DP-1-induced post-DNA damage pathway rather than an increase in the number of replication forks caused by the S-phase initiation. In contrast, topoisomerase IIalpha levels (but not topoisomerase IIbeta levels), together with topoisomerase IIalpha promoter activity, increased 2--3-fold in UE1DP-1/tc-cells. Furthermore, the number of etoposide-induced DNA single- and double-strand breaks increased in UE1DP-1/tc-cells together with a rise in clonogenic sensitivity to etoposide, but an equal apoptotic sensitivity to etoposide. The increase in topoisomerase IIalpha promoter activity in UE1DP-1/tc--cells was shown to be due to S-phase initiation per se because it was blocked by ectopic expression of dominant negative cyclin-dependent kinase 2. In conclusion, overexpression of E2F-1/DP-1 in U-20S-TA cells is sufficient to increase clonogenic sensitivity to both topoisomerase I- and II-targeted anticancer drugs. However, the mechanism by which this occurs appears to be qualitatively different. The UE1DP-1 cell model may be used to elucidate post-DNA damage mechanisms of cell death induced by topoisomerase I-directed anticancer agents.
Abstract Glioblastoma multiforme (GBM), the highest-grade glioma, is the most frequent tumour of the brain with a very poor prognosis and limited therapeutic options. Although little is known about the molecular mechanisms that underlie glioblastoma formation, a number of signal transduction routes, such as the Notch and Ras signalling pathways, seem to play an important role in the formation of GBM. In the present study, we show by in situ hybridization on primary tumour material that the transcription factor HEY1, a target of the Notch signalling pathway, is specifically up-regulated in glioma and that expression of HEY1 in GBM correlates with tumour-grade and survival. In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways. Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture. Together, these data imply a role for HEY1 in the progression of GBM, and therefore we propose that HEY1 may be a therapeutic target for glioblastoma patients. Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk.
Organization of chromatin by epigenetic mechanisms is essential for establishing and maintaining cellular identity in developing and adult organisms. A key question that remains unresolved about this process is how epigenetic marks are transmitted to the next cell generation during cell division. Here we provide a model to explain how trimethylated Lys 27 of histone 3 (H3K27me3), which is catalysed by the EZH2-containing Polycomb Repressive Complex 2 (PRC2), is maintained in proliferating cells. We show that the PRC2 complex binds to the H3K27me3 mark and colocalizes with this mark in G1 phase and with sites of ongoing DNA replication. Efficient binding requires an intact trimeric PRC2 complex containing EZH2, EED and SUZ12, but is independent of the catalytic SET domain of EZH2. Using a heterologous reporter system, we show that transient recruitment of the PRC2 complex to chromatin, upstream of the transcriptional start site, is sufficient to maintain repression through endogenous PRC2 during subsequent cell divisions. Thus, we suggest that once the H3K27me3 is established, it recruits the PRC2 complex to maintain the mark at sites of DNA replication, leading to methylation of H3K27 on the daughter strands during incorporation of newly synthesized histones. This mechanism ensures maintenance of the H3K27me3 epigenetic mark in proliferating cells, not only during DNA replication when histones synthesized de novo are incorporated, but also outside S phase, thereby preserving chromatin structure and transcriptional programs.
The transcriptional factor Snail1 is a repressor of E-cadherin (CDH1) gene expression essential for triggering epithelial-mesenchymal transition. Snail1 represses CDH1, directly binding its promoter and inducing the synthesis of the Zeb1 repressor. In this article, we show that repression of CDH1 by Snail1, but not by Zeb1, is dependent on the activity of Polycomb repressive complex 2 (PRC2). Embryonic stem (ES) cells null for Suz12, one of the components of PRC2, show higher levels of Cdh1 mRNA than control ES cells. In tumor cells, interference of PRC2 activity prevents the ability of Snail1 to downregulate CDH1 and partially derepresses CDH1. Chromatin immunoprecipitation assays demonstrated that Snail1 increases the binding of Suz12 to the CDH1 promoter and the trimethylation of lysine 27 in histone H3. Moreover, Snail1 interacts with Suz12 and Ezh2, as shown by coimmunoprecipitation experiments. In conclusion, these results demonstrate that Snail1 recruits PRC2 to the CDH1 promoter and requires the activity of this complex to repress E-cadherin expression.
At the imprinted Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited to the paternal and maternal sequences, respectively. The mutual exclusion arises because one mark prevents placement of the other. We demonstrated this in five ways: using 5-azacytidine treatments and mutations at the endogenous locus that disrupt DNA methylation; using a transgenic model in which the maternal DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated antagonism between H3K27me3 and DNA methylation. When DNA methylation was lost, H3K27me3 encroached into sites where it had not been before; inappropriate acquisition of DNA methylation excluded normal placement of H3K27me3, and loss of factors needed for H3K27 methylation enabled DNA methylation to appear where it had been excluded. These data reveal the previously unknown antagonism between H3K27 and DNA methylation and identify a means by which epigenetic states may change during disease and development.
<h4>Background</h4>The Polycomb Repressive Complex 2 (PRC2) functions as a transcriptional repressor through a mechanism that involves methylation of Histone H3 at lysine 27. The PRC2 complex activity is essential for cellular proliferation, development, and cell fate decisions. PRC2 target genes include important regulators of development and proliferation as well as tumor suppressor genes. Consistent with this, the activity of several Polycomb group (PcG) proteins is deregulated in human cancer suggesting an important role for PcGs in tumor development. Whereas the downstream functions of PcGs are well characterized, the mechanisms of their recruitment to target genes and the regulation of their activity are not fully understood.<h4>Principal findings</h4>Here we show that the two PRC2 components SUZ12 and EZH2 are sumoylated in vitro and in vivo. Among several putative sumoylation sites we have mapped the major site of SUZ12 sumoylation. Furthermore, we show that SUZ12 interacts with the E2-conjugating enzyme UBC9 both in vitro and in vivo and that mutation of the SUZ12 sumoylation site does not abolish this binding. Finally, we provide evidence that the E3-ligase PIASXbeta interacts and enhances the sumoylation of SUZ12 in vivo suggesting that PIASXbeta could function as an E3-ligase for SUZ12.<h4>Conclusions</h4>Taken together, our data identify sumoylation as a novel post-translational modification of components of the PRC2 complex, which could suggest a potential new mechanism to modulate PRC2 repressive activity. Further work aimed to identify the physiological conditions for these modifications will be required to understand the role of SUZ12 and EZH2 sumoylation in PcG-mediated epigenetic regulation of transcription.
Polycomb group (PcG) proteins regulate important cellular processes such as embryogenesis, cell proliferation, and stem cell self-renewal through the transcriptional repression of genes determining cell fate decisions. The Polycomb-Repressive Complex 2 (PRC2) is highly conserved during evolution, and its intrinsic histone H3 Lys 27 (K27) trimethylation (me3) activity is essential for PcG-mediated transcriptional repression. Here, we show a functional interplay between the PRC2 complex and the H3K4me3 demethylase Rbp2 (Jarid1a) in mouse embryonic stem (ES) cells. By genome-wide location analysis we found that Rbp2 is associated with a large number of PcG target genes in mouse ES cells. We show that the PRC2 complex recruits Rbp2 to its target genes, and that this interaction is required for PRC2-mediated repressive activity during ES cell differentiation. Taken together, these results demonstrate an elegant mechanism for repression of developmental genes by the coordinated regulation of epigenetic marks involved in repression and activation of transcription.
The E2F transcription factors are key regulators of cell cycle progression and the E2F field has made rapid advances since its advent in 1986. Yet, while our understanding of the roles and functions of the E2F family has made enormous progress, with each discovery new questions arise. In this review, we summarise the most recent advances in the field and discuss the remaining key questions. In particular, we will focus on how specificity is achieved among the E2Fs.
The E2F transcription factors are downstream effectors of the retinoblastoma protein (pRB) pathway and are required for the timely regulation of numerous genes essential for DNA replication and cell cycle progression. Several laboratories have used genome-wide approaches to discover novel target genes of E2F, leading to the identification of several hundred such genes that are involved not only in DNA replication and cell cycle progression, but also in DNA damage repair, apoptosis, differentiation and development. These new findings greatly enrich our understanding of how E2F controls transcription and cellular homeostasis.
Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show that small interfering RNA inhibition of SET8 expression leads to decreased cell proliferation and accumulation of cells in S phase. This is accompanied by DNA double-strand break (DSB) induction and recruitment of the DNA repair proteins replication protein A, Rad51, and 53BP1 to damaged regions. SET8 depletion causes DNA damage specifically during replication, which induces a Chk1-mediated S-phase checkpoint. Furthermore, we find that SET8 interacts with proliferating cell nuclear antigen through a conserved motif, and SET8 is required for DNA replication fork progression. Finally, codepletion of Rad51, an important homologous recombination repair protein, abrogates the DNA damage after SET8 depletion. Overall, we show that SET8 is essential for genomic stability in mammalian cells and that decreased expression of SET8 results in DNA damage and Chk1-dependent S-phase arrest.
Geminin is an unstable inhibitor of DNA replication that negatively regulates the licensing factor CDT1 and inhibits pre-replicative complex (pre-RC) formation in Xenopus egg extracts. Here we describe a novel function of Geminin. We demonstrate that human Geminin protects CDT1 from proteasome-mediated degradation by inhibiting its ubiquitination. In particular, Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis. Consistently, inhibition of Geminin synthesis during M phase leads to impairment of pre-RC formation and DNA replication during the following cell cycle. Moreover, we show that inhibition of CDK1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre-RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells. Taken together with recent data from our laboratory, our results demonstrate that Geminin is both a negative and positive regulator of pre-RC formation in human cells, playing a positive role in allowing CDT1 accumulation in G2-M, and preventing relicensing of origins in S-G2.
The molecular mechanisms and the cell-of-origin leading to retinoblastoma are not well defined. In this issue of Cancer Cell, Bremner and colleagues describe the first inheritable model of retinoblastoma, revealing that loss of the pocket proteins pRb and p107 deregulates cell cycle exit in retinal precursors. The authors show that a subset of these precursors contain an inherent resistance to apoptosis, and that while most terminally differentiate, some are likely to acquire additional mutations, leading to tumor formation. Thus, this work defines the cell-of-origin of retinoblastoma and suggests that mutations giving increased proliferative capacity are required for retinoblastoma development.
c-Myc plays an essential role in proliferation, differentiation, and apoptosis. Because of its relevance to cancer, most studies have focused on the cellular consequences of c-Myc overexpression. Here, we address the role of physiological levels of c-Myc in drug-induced apoptosis. By using c-MYC null cells we confirm and extend recent reports showing a c-Myc requirement for the induction of apoptosis by a number of anticancer agents. In particular, we show that c-Myc is required for the induction of apoptosis by doxorubicin and etoposide, whereas it is not required for camptothecin-induced cell death. We have investigated the molecular mechanisms involved in executing doxorubicin-induced apoptosis and show caspase-3 activation by both mitochondria-dependent and -independent pathways. Moreover, serine proteases participate in doxorubicin-induced apoptosis partly by contributing to caspase-3 activation. Finally, a complete rescue from doxorubicin-induced apoptosis is obtained only when serine proteases, caspase-3, and mitochondrial activation are inhibited simultaneously. Interestingly, doxorubicin requires c-Myc for the activation of all of these pathways. Our findings therefore support a model in which doxorubicin simultaneously triggers multiple c-Myc-dependent apoptosis pathways.
The trithorax and the polycomb group proteins are chromatin modifiers, which play a key role in the epigenetic regulation of development, differentiation and maintenance of cell fates. The polycomb repressive complex 2 (PRC2) mediates transcriptional repression by catalysing the di- and tri-methylation of Lys 27 on histone H3 (H3K27me2/me3). Owing to the essential role of the PRC2 complex in repressing a large number of genes involved in somatic processes, the H3K27me3 mark is associated with the unique epigenetic state of stem cells. The rapid decrease of the H3K27me3 mark during specific stages of embryogenesis and stem-cell differentiation indicates that histone demethylases specific for H3K27me3 may exist. Here we show that the human JmjC-domain-containing proteins UTX and JMJD3 demethylate tri-methylated Lys 27 on histone H3. Furthermore, we demonstrate that ectopic expression of JMJD3 leads to a strong decrease of H3K27me3 levels and causes delocalization of polycomb proteins in vivo. Consistent with the strong decrease in H3K27me3 levels associated with HOX genes during differentiation, we show that UTX directly binds to the HOXB1 locus and is required for its activation. Finally mutation of F18E9.5, a Caenorhabditis elegans JMJD3 orthologue, or inhibition of its expression, results in abnormal gonad development. Taken together, these results suggest that H3K27me3 demethylation regulated by UTX/JMJD3 proteins is essential for proper development. Moreover, the recent demonstration that UTX associates with the H3K4me3 histone methyltransferase MLL2 (ref. 8) supports a model in which the coordinated removal of repressive marks, polycomb group displacement, and deposition of activating marks are important for the stringent regulation of transcription during cellular differentiation.
The E2F family of transcription factors play an essential role in the regulation of cell cycle progression. In a screen for E2F-regulated genes we identified a novel E2F family member, E2F7. Like the recently identified E2F-like proteins of Arabidopsis, E2F7 has two DNA binding domains and binds to the E2F DNA binding consensus site independently of DP co-factors. Consistent with being an E2F target gene, we found that the expression of E2F7 is cell cycle regulated. Ectopic expression of E2F7 results in suppression of E2F target genes and accumulation of cells in G1. Furthermore, E2F7 associates with E2F-regulated promoters in vivo, and this association increases in S phase. Interestingly, however, E2F7 binds only a subset of E2F-dependent promoters in vivo, and in agreement with this, inhibition of E2F7 expression results in specific derepression of these promoters. Taken together, these data demonstrate that E2F7 is a unique repressor of a subset of E2F target genes whose products are required for cell cycle progression.
Epigenetic changes are common alterations in cancer cells. Here, we have investigated the role of Polycomb group proteins in the establishment and maintenance of the aberrant silencing of tumor suppressor genes during transformation induced by the leukemia-associated PML-RARalpha fusion protein. We show that in leukemic cells knockdown of SUZ12, a key component of Polycomb repressive complex 2 (PRC2), reverts not only histone modification but also induces DNA demethylation of PML-RARalpha target genes. This results in promoter reactivation and granulocytic differentiation. Importantly, the epigenetic alterations caused by PML-RARalpha can be reverted by retinoic acid treatment of primary blasts from leukemic patients. Our results demonstrate that the direct targeting of Polycomb group proteins by an oncogene plays a key role during carcinogenesis.
Deregulation of the retinoblastoma protein (pRB) pathway is a hallmark of human cancer. The core members of this pathway include the tumor suppressor protein, pRB, which through binding to a number of cellular proteins, most notably members of the E2F transcription factor family, regulates progression through the cell division cycle. With the aim of identifying transcriptional changes provoked by deregulation of the pRB pathway, we have used cell lines that conditionally express a constitutively active phosphorylation site mutant of pRB (pRBDeltaCDK) or p16INK4A (p16). The expression of pRBDeltaCDK and p16 resulted in significant repression and activation of a large number of genes as measured by high density oligonucleotide array analysis. Transcriptional changes were found in genes that are essential for DNA replication and cell proliferation. In agreement with previous results, we found a high degree of overlap between genes regulated by p16 and pRB. Data we have obtained previously for E2F family members showed that 74 of the genes repressed by pRB and p16 were induced by the E2Fs and 23 genes that were induced by pRB and p16 were repressed by the E2Fs. Thus, we have identified 97 genes as physiological targets of the pRB pathway, and the further characterization of these genes should provide insights into how this pathway controls proliferation. We show that Gibbs sampling detects enrichment of several sequence motifs, including E2F consensus binding sites, in the upstream regions of these genes and use this enrichment in an in silico filtering process to refine microarray derived gene lists.
Polycomb group (PcG) proteins form multiprotein complexes, called Polycomb repressive complexes (PRCs). PRC2 contains the PcG proteins EZH2, SUZ12, and EED and represses transcription through methylation of lysine (K) 27 of histone H3 (H3). Suz12 is essential for PRC2 activity and its inactivation results in early lethality of mouse embryos. Here, we demonstrate that Suz12(-/-) mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12(-/-) ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation-specific genes. Moreover, Suz12(-/-) ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-specific genes. Finally, we demonstrate that the PcGs are actively recruited to several genes during ES cell differentiation, which despite an increase in H3K27me3 levels is not always sufficient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of specific expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different mechanisms to regulate transcription during cellular differentiation.
In the past couple of years, several very exciting studies have demonstrated the enormous power of gene-expression profiling for cancer classification and prediction of patient survival. In addition to promising a more accurate classification of cancer and therefore better treatment of patients, gene-expression profiling can result in the identification of novel potential targets for cancer therapy and a better understanding of the molecular mechanisms leading to cancer.
Methylation of histones has been regarded as a stable modification defining the epigenetic program of the cell, which regulates chromatin structure and transcription. However, the recent discovery of histone demethylases has challenged the stable nature of histone methylation. Here we demonstrate that the JARID1 proteins RBP2, PLU1, and SMCX are histone demethylases specific for di- and trimethylated histone 3 lysine 4 (H3K4). Consistent with a role for the JARID1 Drosophila homolog Lid in regulating expression of homeotic genes during development, we show that RBP2 is displaced from Hox genes during embryonic stem (ES) cell differentiation correlating with an increase of their H3K4me3 levels and expression. Furthermore, we show that mutation or RNAi depletion of the C. elegans JARID1 homolog rbr-2 leads to increased levels of H3K4me3 during larval development and defects in vulva formation. Taken together, these results suggest that H3K4me3/me2 demethylation regulated by the JARID1 family plays an important role during development.
Functional inactivation of the pRB pathway is a very frequent event in human cancer, resulting in deregulated activity of the E2F transcription factors. To understand the functional role of the E2Fs in cell proliferation, we have developed cell lines expressing E2F-1, E2F-2, and E2F-3 fused to the estrogen receptor ligand binding domain (ER). In this study, we demonstrated that activation of all three E2Fs could relieve the mitogen requirement for entry into S phase in Rat1 fibroblasts and that E2F activity leads to a shortening of the G(0)-G(1) phase of the cell cycle by 6 to 7 h. In contrast to the current assumption that E2F-1 is the only E2F capable of inducing apoptosis, we showed that deregulated E2F-2 and E2F-3 activities also result in apoptosis. Using the ERE2F-expressing cell lines, we demonstrated that several genes containing E2F DNA binding sites are efficiently induced by the E2Fs in the absence of protein synthesis. Furthermore, CDC25A is defined as a novel E2F target whose expression can be directly regulated by E2F-1. Data showing that CDC25A is an essential target for E2F-1, since its activity is required for efficient induction of S phase by E2F-1, are provided. Finally, our results show that expression of two E2F target genes, namely CDC25A and cyclin E, is sufficient to induce entry into S phase in quiescent fibroblasts. Taken together, our results provide an important step in defining how E2F activity leads to deregulated proliferation.
The Polycomb group (PcG) proteins are essential for embryogenesis, and their expression is often found deregulated in human cancer. The PcGs form two major protein complexes, called polycomb repressive complexes 1 and 2 (PRC1 and PRC2) whose function is to maintain transcriptional repression. Here, we demonstrate that the chromodomain-containing protein, CBX8, which is part of one of the PRC1 complexes, regulates proliferation of diploid human and mouse fibroblasts through direct binding to the INK4A-ARF locus. Furthermore, we demonstrate that CBX8 is limiting for the regulation of INK4A-ARF, and that ectopic expression of CBX8 leads to repression of the Ink4a-Arf locus and bypass of senescence, leading to cellular immortalization. Gene expression and location analysis demonstrate that besides the INK4A-ARF locus, CBX8 also regulates a number of other genes important for cell growth and survival. On the basis of these results, we conclude that CBX8 is an essential component of one of the PRC1 complexes, which directly regulate the expression of numerous target genes, including the INK4A-ARF locus, involved in cell-fate decisions.
The E2F family of transcription factors are essential for the regulation of genes required for appropriate progression through the cell cycle. Five members of the E2F family have been previously reported, namely E2F1-5. All five are key elements in transcriptional regulation of essential genes, and they can be divided into two functional groups, those that induce S-phase progression when overexpressed in quiescent cells (E2Fs 1-3), and those that do not (E2Fs 4-5). Here, we describe the identification of a novel member of this family, which we refer to as E2F-6. E2F-6 shares significant homology with E2Fs 1-5, especially within the DNA binding, heterodimerization and marked box domains. Unlike E2Fs 1-5, E2F-6 lacks a transactivation and a pocket protein binding domain, hence, forms a unique third group within the E2F family. E2F-6 is a nuclear protein that can form heterodimers with the DP proteins (both DP-I and DP-2) in vitro and in vivo. Our results show that the complex formed between E2F-6 and the DP proteins, possesses high DNA binding activity, displaying a preference for a TTTCCCGC E2F recognition site, which is slightly different to the E2F consensus site derived from the E2 promoter (TTTCGCGC). In contrast to the other members of the E2F family, ectopic expression of E2F-6 inhibits transcription from promoters possessing E2F recognition sites rather than activating transcription. In addition, overexpression of E2F-6 suppresses the transactivational effects of coexpression of E2F-1 and DP-1. The inhibitory effect of E2F-6 is dependent on its DNA binding activity and its ability to form heterodimers with the DPs. Interestingly, ectopic expression of E2F-6 leads to accumulation of cells in S-phase. Our data suggest that E2F-6 expression delays the exit from S-phase rather than inducing S-phase, which further emphasizes the functional difference between E2F-6 and the previously known E2F family members.
Experimental data generated in the past year have further emphasized the essential role for the E2F transcription factors in the regulation of cell proliferation. Genetic studies have shown that E2F activity is required for normal development in fruitflies, and the generation of E2F-1(-/-) mice has demonstrated that individual members of the E2F transcription factor family are likely to have distinct roles in mammalian development and homeostasis. Additional mechanisms regulating the activity of the E2F transcription factors have been reported, including subcellular localization and proteolysis of the E2Fs in the proteasomes. Novel target genes for the E2F transcription factors have been identified that link the E2Fs directly to the initiation of DNA replication.
The E2F transcription factors are essential for regulating the correct timing of activation of several genes whose products are implicated in cell proliferation and DNA replication. The E2Fs are targets for negative regulation by the retinoblastoma protein family, which includes pRB, p107, and p130, and they are in a pathway that is frequently found altered in human cancers. There are five members of the E2F family, and they can be divided into two functional subgroups. Whereas, upon overexpression, E2F-1, -2, and -3 induce S phase in quiescent fibroblasts and override G1 arrests mediated by the p16INK4A tumor suppressor protein or neutralizing antibodies to cyclin D1, E2F-4 and -5 do not. Using E2F-1 and E2F-4 as representatives of the two subgroups, we showed here, by constructing a set of chimeric proteins, that the amino terminus of E2F-1 is sufficient to confer S-phase-inducing potential as well as the ability to efficiently transactivate an E2F-responsive promoter to E2F-4. We found that the E2F-1 amino terminus directs chimeric proteins to the nucleus. Surprisingly, a short nuclear localization signal derived from simian virus 40 large T antigen could perfectly substitute for the presence of the E2F-1 amino terminus in these assays. Thus, nuclearly localized E2F-4, when overexpressed, displayed biological activities similar to those of E2F-1. Furthermore, we showed that nuclear localization of endogenous E2F-4 is cell cycle regulated, with E2F-4 being nuclear in the G0 and early G1 phases and mainly cytoplasmic after the pRB family members have become phosphorylated. We propose a novel mechanism for the regulation of E2F-dependent transcription in which E2F-4 regulates transcription only from G0 until mid- to late G1 phase whereas E2F-1 is active in late G1 and S phases, until it is inactivated by cyclin A-dependent kinase in late S phase.
The retinoblastoma gene family consists of the tumor suppressor protein pRB and its two relatives p107 and p130. These proteins have been implicated in the regulation of cell cycle progression, in part, through inactivation of members of the E2F transcription factor family. Overexpression of pRB, p107, or p130 leads to growth arrest in the G1 phase of the cell cycle, and this arrest is abolished by complex formation with the adenovirus E1A, human papilloma virus E7, or simian virus 40 T oncoproteins. Inactivation of pRB by gross structural alterations or point mutations in the RB-1 gene has been described in a variety of human tumors, including retinoblastomas, osteosarcomas, and small cell lung carcinomas. Despite the structural and functional similarity between pRB, p107, and p130, alterations in the latter two proteins have not been identified in human tumors. We have screened a panel of 17 small cell lung carcinoma cell lines for the presence of functional p107 and p130 by evaluating their ability to form complexes with E1A in vitro. In the GLC2 small cell lung carcinoma cells no p130 protein was detected. The loss of the p130 protein is the result of a single point mutation within a splice acceptor sequence in the GLC2 genomic DNA. This mutation eliminates exon 2, leading to an in-frame stop codon, and no detectable protein is produced. These data are, to our knowledge, the first to describe the loss of p130 as a consequence of a genetic alteration, suggesting that not only pRB but also the other members of the family may contribute to tumorigenesis, providing a rationale for the observation that the DNA tumor viruses selectively target all the members of the retinoblastoma protein family.
The p16INK4A and p14ARF proteins, encoded by the INK4A-ARF locus, are key regulators of cellular senescence, yet the mechanisms triggering their up-regulation are not well understood. Here, we show that the ability of the oncogene BMI1 to repress the INK4A-ARF locus requires its direct association and is dependent on the continued presence of the EZH2-containing Polycomb-Repressive Complex 2 (PRC2) complex. Significantly, EZH2 is down-regulated in stressed and senescing populations of cells, coinciding with decreased levels of associated H3K27me3, displacement of BMI1, and activation of transcription. These results provide a model for how the INK4A-ARF locus is activated and how Polycombs contribute to cancer.
Methylation of lysine and arginine residues on histone tails affects chromatin structure and gene transcription. Tri- and dimethylation of lysine 9 on histone H3 (H3K9me3/me2) is required for the binding of the repressive protein HP1 and is associated with heterochromatin formation and transcriptional repression in a variety of species. H3K9me3 has long been regarded as a 'permanent' epigenetic mark. In a search for proteins and complexes interacting with H3K9me3, we identified the protein GASC1 (gene amplified in squamous cell carcinoma 1), which belongs to the JMJD2 (jumonji domain containing 2) subfamily of the jumonji family, and is also known as JMJD2C. Here we show that three members of this subfamily of proteins demethylate H3K9me3/me2 in vitro through a hydroxylation reaction requiring iron and alpha-ketoglutarate as cofactors. Furthermore, we demonstrate that ectopic expression of GASC1 or other JMJD2 members markedly decreases H3K9me3/me2 levels, increases H3K9me1 levels, delocalizes HP1 and reduces heterochromatin in vivo. Previously, GASC1 was found to be amplified in several cell lines derived from oesophageal squamous carcinomas, and in agreement with a contribution of GASC1 to tumour development, inhibition of GASC1 expression decreases cell proliferation. Thus, in addition to identifying GASC1 as a histone trimethyl demethylase, we suggest a model for how this enzyme might be involved in cancer development, and propose it as a target for anti-cancer therapy.
HMGA2 gene amplification and overexpression in human prolactinomas and the development of pituitary adenomas in HMGA2 transgenic mice showed that HMGA2 plays a crucial role in pituitary tumorigenesis. We have explored the pRB/E2F1 pathway to investigate the mechanism by which HMGA2 acts. Here we show that HMGA2 interacts with pRB and induces E2F1 activity in mouse pituitary adenomas by displacing HDAC1 from the pRB/E2F1 complex-a process that results in E2F1 acetylation. We found that loss of E2F1 function (obtained by mating HMGA2 and E2F1(-/-) mice) suppressed pituitary tumorigenesis in HMGA2 mice. Thus, HMGA2-mediated E2F1 activation is a crucial event in the onset of these tumors in transgenic mice and probably also in human prolactinomas.
The Polycomb group (PcG) proteins form chromatin-modifying complexes that are essential for embryonic development and stem cell renewal and are commonly deregulated in cancer. Here, we identify their target genes using genome-wide location analysis in human embryonic fibroblasts. We find that Polycomb-Repressive Complex 1 (PRC1), PRC2, and tri-methylated histone H3K27 co-occupy >1000 silenced genes with a strong functional bias for embryonic development and cell fate decisions. We functionally identify 40 genes derepressed in human embryonic fibroblasts depleted of the PRC2 components (EZH2, EED, SUZ12) and the PRC1 component, BMI-1. Interestingly, several markers of osteogenesis, adipogenesis, and chrondrogenesis are among these genes, consistent with the mesenchymal origin of fibroblasts. Using a neuronal model of differentiation, we delineate two different mechanisms for regulating PcG target genes. For genes activated during differentiation, PcGs are displaced. However, for genes repressed during differentiation, we paradoxically find that they are already bound by the PcGs in nondifferentiated cells despite being actively transcribed. Our results are consistent with the hypothesis that PcGs are part of a preprogrammed memory system established during embryogenesis marking certain key genes for repressive signals during subsequent developmental and differentiation processes.
The Myc oncoprotein forms a binary activating complex with its partner protein, Max, and a ternary repressive complex that, in addition to Max, contains the zinc finger protein Miz1. Here we show that the E3 ubiquitin ligase HectH9 ubiquitinates Myc in vivo and in vitro, forming a lysine 63-linked polyubiquitin chain. Miz1 inhibits this ubiquitination. HectH9-mediated ubiquitination of Myc is required for transactivation of multiple target genes, recruitment of the coactivator p300, and induction of cell proliferation by Myc. HectH9 is overexpressed in multiple human tumors and is essential for proliferation of a subset of tumor cells. Our results suggest that site-specific ubiquitination regulates the switch between an activating and a repressive state of the Myc protein, and they suggest a strategy to interfere with Myc function in vivo.
Nucleophosmin (NPM) is a nucleolar phosphoprotein that binds the tumor suppressors p53 and p19(Arf) and is thought to be indispensable for ribogenesis, cell proliferation, and survival after DNA damage. The NPM gene is the most frequent target of genetic alterations in leukemias and lymphomas, though its role in tumorigenesis is unknown. We report here the first characterization of a mouse NPM knockout strain. Lack of NPM expression results in accumulation of DNA damage, activation of p53, widespread apoptosis, and mid-stage embryonic lethality. Fibroblasts explanted from null embryos fail to grow and rapidly acquire a senescent phenotype. Transfer of the NPM mutation into a p53-null background rescued apoptosis in vivo and fibroblast proliferation in vitro. Cells null for both p53 and NPM grow faster than control cells and are more susceptible to transformation by activated oncogenes, such as mutated Ras or overexpressed Myc. In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. Our data demonstrate that NPM regulates DNA integrity and, through Arf, inhibits cell proliferation and are consistent with a putative tumor-suppressive function of NPM.
The retinoblastoma gene, RB1, is one of the most frequently mutated genes in human cancer. Rb heterozygous mice develop pituitary tumors with 100% incidence, and the E2F transcription factors are required for this. To assess whether deregulated E2F activity is sufficient to induce pituitary tumors, we generated transgenic mice expressing an inducible E2F3 protein in the intermediate lobe of the pituitary gland. We found that short-term deregulation of E2F activity, similar to the earliest stages of Rb loss, is able to induce abnormal proliferation of otherwise quiescent melanotrophs. However, while long-term exposure to deregulated E2F activity results in hyperplasia of the intermediate lobe, it did not lead to tumor formation. In fact, melanotrophs become insensitive to sustained E2F stimulation and enter an irreversible senescence-like state. Thus, although deregulated E2F activity results in hyperproliferation, it is not sufficient to mimic loss of Rb, sustain proliferation of melanotrophs, and ultimately induce pituitary tumors. Similarly, we found that primary cells in tissue culture become insensitive to sustained E2F3 activation and undergo premature senescence in a pRB-, p16Ink4a-, and p19Arf-dependent manner. Thus, we conclude that deregulated E2F activity is not sufficient to fully mimic loss of Rb due to the engagement of a senescence response.
Whereas the stress-inducible heat-shock protein 70 (Hsp70) has gained plenty of attention as a putative target for tumor therapy, little is known about the role of other Hsp70 proteins in cancer. Here we present the first thorough analysis of the expression and function of the cytosolic Hsp70 proteins in human cancer cells and identify Hsp70-2, a protein essential for spermatogenesis, as an important regulator of cancer cell growth. Targeted knock-down of the individual family members by RNA interference revealed that both Hsp70 and Hsp70-2 were required for cancer cell growth, whereas the survival of tumorigenic as well as nontumorigenic cells depended on Hsc70. Cancer cells depleted for Hsp70 and Hsp70-2 displayed strikingly different morphologies (detached and round vs. flat senescent-like), cell cycle distributions (G2/M vs. G1 arrest) and gene expression profiles. Only Hsp70-2 depletion induced the expression of macrophage inhibitory cytokine-1 that was identified as a target of P53 tumor-suppressor protein and a mediator of the G1 arrest and the senescent phenotype. Importantly, concomitant depletion of Hsp70 and Hsp70-2 had a synergistic antiproliferative effect on cancer cells. Thus, highly homologous Hsp70 proteins bring about nonoverlapping functions essential for cell growth and survival.
E2F1 is a crucial downstream effector of the retinoblastoma protein (pRB) pathway. To address the consequences of short-term increase in E2F1 activity in adult tissues, we generated transgenic mice expressing the human E2F1 protein fused to the oestrogen receptor (ER) ligand-binding domain. The expression of the ER-E2F1 fusion protein, which is inactive in the absence of 4-hydroxy tamoxifen (OHT), was targeted to the testes. We show that short-term activation of E2F1 results in activation of E2F target genes and apoptosis of germ cells. Consistent with our previously published results, the apoptotic response was independent of p53. Persistent E2F1 activation for 3 weeks led to massive apoptosis and severe testicular atrophy with seminiferous tubules containing only Sertoli cells and clusters of undifferentiated spermatogonia. The latter showed high expression of ER-E2F1 and excessive mitotic activity, including atypical mitoses. In addition, gonocyte-like dysplastic germ cells, resembling carcinoma in situ (CIS) cells in humans, appeared. Our results show that a relatively short period of deregulated E2F1 activity in testicles can induce premalignant changes. Moreover, we demonstrate the feasibility of tissue-specific expression of conditional ER-E2F1 in transgenic mice.
The E2F family of transcription factors are downstream effectors of the retinoblastoma protein, pRB, pathway and are essential for the timely regulation of genes necessary for cell-cycle progression. Here we describe the characterization of human and murine E2F8, a new member of the E2F family. Sequence analysis of E2F8 predicts the presence of two distinct E2F-related DNA binding domains suggesting that E2F8 and, the recently, identified E2F7 form a subgroup within the E2F family. We show that E2F transcription factors bind the E2F8 promoter in vivo and that expression of E2F8 is being induced at the G1/S transition. Purified recombinant E2F8 binds specifically to a consensus E2F-DNA-binding site indicating that E2F8, like E2F7, binds DNA without the requirement of co-factors such as DP1. E2F8 inhibits E2F-driven promoters suggesting that E2F8 is transcriptional repressor like E2F7. Ectopic expression of E2F8 in diploid human fibroblasts reduces expression of E2F-target genes and inhibits cell growth consistent with a role for repressing E2F transcriptional activity. Taken together, these data suggest that E2F8 has an important role in turning of the expression of E2F-target genes in the S-phase of the cell cycle.
SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show that mice lacking Suz12, like Ezh2 and Eed mutant mice, are not viable and die during early postimplantation stages displaying severe developmental and proliferative defects. Consistent with this, we demonstrate that SUZ12 is required for proliferation of cells in tissue culture. Furthermore, we demonstrate that SUZ12 is essential for the activity and stability of the PRC2/3 complexes in mouse embryos, in tissue culture cells and in vitro. Strikingly, Suz12-deficient embryos show a specific loss of di- and trimethylated H3K27, demonstrating that Suz12 is indeed essential for EZH2 activity in vivo. In conclusion, our data demonstrate an essential role of SUZ12 in regulating the activity of the PRC2/3 complexes, which are required for regulating proliferation and embryogenesis.
Human tumors are believed to harbor a disabled p53 tumor suppressor pathway, either through direct mutation of the p53 gene or through aberrant expression of proteins acting in the p53 pathway, such as p14(ARF) or Mdm2. A role for Mdmx (or Mdm4) as a key negative regulator of p53 function in vivo has been established. However, a direct contribution of Mdmx to tumor formation remains to be demonstrated. Here we show that retrovirus-mediated Mdmx overexpression allows primary mouse embryonic fibroblast immortalization and leads to neoplastic transformation in combination with HRas(V12). Furthermore, the human Mdmx ortholog, Hdmx, was found to be overexpressed in a significant percentage of various human tumors and amplified in 5% of primary breast tumors, all of which retained wild-type p53. Hdmx was also amplified and highly expressed in MCF-7, a breast cancer cell line harboring wild-type p53, and interfering RNA-mediated reduction of Hdmx markedly inhibited the growth potential of these cells in a p53-dependent manner. Together, these results make Hdmx a new putative drug target for cancer therapy.
Strict regulation of DNA replication is essential to ensure proper duplication and segregation of chromosomes during the cell cycle, as its deregulation can lead to genomic instability and cancer. Thus, eukaryotic organisms have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. Here, we show that inactivation of Geminin, an inhibitor of origin licensing, leads to rereplication in human normal and tumor cells within the same cell cycle. We found a CHK1-dependent checkpoint to be activated in rereplicating cells accompanied by formation of gammaH2AX and RAD51 nuclear foci. Abrogation of the checkpoint leads to abortive mitosis and death of rereplicated cells. In addition, we demonstrate that the induction of rereplication is dependent on the replication initiation factors CDT1 and CDC6, and independent of the functional status of p53. These data show that Geminin is required for maintaining genomic stability in human cells.
Epigenetic deregulation of gene expression is emerging as key mechanism in tumorigenesis. Deregulated activity of the chromatin remodeling Polycomb Repressive Complex 2 (PRC2) has recently been shown to be a frequent event in human tumors. Here we discuss these findings and speculate on the role of the PRC2 complex in controlling gene expression during normal cellular proliferation and cancer development.
Gene expression of the plasminogen activation system is cell-cycle dependent. Previously, we showed that ectopic expression of E2F1 repressed the plasminogen activator inhibitor type 1 (PAI-1) promoter in a manner dependent on the presence of DNA-binding and transactivation domains of E2F1 but independent of binding to pocket-binding proteins, suggesting a novel mechanism for E2F-mediated negative gene regulation [Koziczak, M., Krek, W. & Nagamine, Y. (2000) Mol. Cell. Biol. 20, 2014-2022]. However, it remains to be seen whether endogenous E2F can exert a similar effect. We report here that down-regulation of PAI-1 gene expression correlates with an increase in endogenous E2F activity. When cells were treated with a cdk2/4-specific inhibitor, which maintains E2F in an inactive state, the decline of serum-induced PAI-1 mRNA levels was suppressed. In mutant U2OS cells expressing a temperature-sensitive retinoblastoma protein (pRB), a shift to a permissive temperature induced PAI-1 mRNA expression. In U2OS cells stably expressing an E2F1-estrogen receptor chimeric protein that could be activated by tamoxifen, PAI-1 gene transcription was markedly reduced by tamoxifen even in the presence of cycloheximide. These results all indicate that endogenous E2F can directly repress the PAI-1 gene. DNase I hypersensitive-site analysis of the PAI-1 promoter suggested the involvement of conformation changes in chromatin structure of the PAI-1 promoter. 5' deletion analysis of the PAI-1 promoter showed that multiple sites were responsible for the E2F negative regulation, some of which were promoter dependent. Interestingly, one of these sites is a p53-binding element.
The six members of the E2F family of transcription factors play a key role in the control of cell cycle progression by regulating the expression of genes involved in DNA replication and cell proliferation. E2F-1, -2, and -3 belong to a structural and functional subfamily distinct from those of the other E2F family members. Here we report that E2F-1, -2, and -3, but not E2F-4, -5, and -6, associate with and are acetylated by p300 and cAMP-response element-binding protein acetyltransferases. Acetylation occurs at three conserved lysine residues located at the N-terminal boundary of their DNA binding domains. Acetylation of E2F-1 in vitro and in vivo markedly increases its binding affinity for a consensus E2F DNA-binding site, which is paralleled by enhanced transactivation of an E2F-responsive promoter. Acetylation of E2F-1 can be reversed by histone deacetylase-1, indicating that reversible acetylation is a mechanism for regulation also of non-histone proteins.
CDC6 is conserved during evolution and is essential and limiting for the initiation of eukaryotic DNA replication. Human CDC6 activity is regulated by periodic transcription and CDK-regulated subcellular localization. Here, we show that, in addition to being absent from nonproliferating cells, CDC6 is targeted for ubiquitin-mediated proteolysis by the anaphase promoting complex (APC)/cyclosome in G(1). A combination of point mutations in the destruction box and KEN-box motifs in CDC6 stabilizes the protein in G(1) and in quiescent cells. Furthermore, APC, in association with CDH1, ubiquitinates CDC6 in vitro, and both APC and CDH1 are required and limiting for CDC6 proteolysis in vivo. Although a stable mutant of CDC6 is biologically active, overexpression of this mutant or wild-type CDC6 is not sufficient to induce multiple rounds of DNA replication in the same cell cycle. The APC-CDH1-dependent proteolysis of CDC6 in early G(1) and in quiescent cells suggests that this process is part of a mechanism that ensures the timely licensing of replication origins during G(1).
Terminal differentiation of cultured myocytes requires withdrawal of the cells from the cell cycle. Constitutive overexpression of several oncogenes in myoblasts can inhibit in vitro myogenesis. Here we studied the role of the cellular transcription factor E2F1 on myogenic differentiation. E2F1 expression is irreversibly down-regulated during differentiation of C2C12 myocytes. Furthermore, deregulated E2F1 expression in C2C12 cells prevented myogenic differentiation. This inhibition of myogenesis was associated with the repression of myogenin expression and an elevated cyclin D1 expression. Moreover, E2F1-overexpressing myocytes failed to exit the cell cycle under differentiation conditions. These results are consistent with the notion that E2F1 can function as an oncogene and further suggest that E2F1 down-regulation is required for myogenic differentiation.
An overlapping inverted repeat sequence that binds the eukaryotic transcription factor E2F is 100% conserved near the major transcription start sites in the promoters of three mammalian genes encoding dihydrofolate reductase, and is also found in the promoters of several other important cellular and viral genes. This element, 5'-TTTCGCGCCAAA-3', is comprised of two overlapping, oppositely oriented sites which match the consensus E2F site (5'-TTT(C/G)(C/G)CGC-3'). Recent work has shown that E2F binding activity is composed of at least six related cellular polypeptides which are capable of forming DNA-binding homo- and heterodimers. We have investigated the binding of cellular E2F activity and of homo- and heterodimers of cloned E2F proteins to the inverted repeat E2F element. We have demonstrated that mutations in this element that abolish its inverted repeat nature, while preserving a single consensus E2F site, significantly decrease the binding stability of all of the forms of E2F tested. The rate of association of E2F-1/DP-1 heterodimers with the inverted repeat wild type site was not significantly different from those with the two single site mutated probes. Furthermore, the mutations decrease in vitro transcription and transient reporter gene expression 2-5-fold, an effect equivalent to that of abolishing E2F binding altogether. These data suggest a functional role that may explain the conservation of inverted repeat E2F elements among the DHFR promoters and several other cellular and viral promoters.
The E2F transcription factor plays an important regulatory role in cell proliferation, mediating the expression of genes whose products are essential for inducing resting cells to enter the cell cycle and synthesize DNA. To investigate the possible involvement of E2F in hematopoietic malignancies, we isolated genomic clones encompassing the human E2F1 gene. We then used fluorescence in situ hybridization to localize E2F1 to human chromosome 20q11, telomeric to the p107 locus, a gene whose product is related to the retinoblastoma gene product (pRb). This finding contrasts with the 1p36 and 6q22 chromosomal locations previously assigned E2F2 and E2F3, two additional members of the E2F family. Although deletions or structural rearrangements of E2F1 were not detected in 14 primary acute leukemia or myelodysplasia samples with structural abnormalities of chromosome 20q11, the gene was amplified and overexpressed in HEL erythroleukemia cells and translocated to other chromosomes in several established human leukemia cell lines. This study provides the first evidence of gene amplification involving a member of the E2F family of transcription factors. We propose that E2F1 overexpression in erythroid progenitors may stimulate abnormal cell proliferation by overriding negative regulatory signals mediated by tumor suppressor proteins such as pRb.
The E2F transcription factor family participates in growth control presumably through transcriptional activation of genes that promote entry into S phase. E2F activity is believed to be controlled across the cell cycle by association with various cellular proteins, including the product of the retinoblastoma gene (pRB). We find that E2F-1 proteins are heterogeneously phosphorylated in insect cells, as a result of which they migrate as a doublet on SDS-polyacrylamide gels. This electrophoretic shift is shown to be dependent upon specific phosphorylation of E2F-1 on serine-375 (S375), near the pRB-binding site. Phosphorylation on S375 also occurs in human cells. E2F-1 was most efficiently phosphorylated on this residue by cyclin A/cdk2 kinase, and to a lesser extent by cyclin A/cdk2, irrespective of the presence of the pRB-related p107 protein. Phosphorylation of E2F-1 on S375 greatly enhanced its affinity of pRB in vitro. These results suggest a novel way of regulating E2F-1 activity, namely by cell-cycle-dependent phosphorylation of this transcription factor.
E2F-1 is a member of a family of transcription factors implicated in the activation of genes required for the progression through the S phase of the cell cycle. We have examined the biological activities of E2F-1 with short-term colony-forming assays and long-term immortalization assays. High levels of E2F-1, produced by transfection of the E2F-1 cDNA under the control of a strong promoter, reduced colony formation in normal human foreskin keratinocytes (NHFKs). This inhibition could not be overcome by wild-type human papillomavirus type 16 (HPV16) E6 and E7, two proteins which cooperate to immortalize NHFKs, or by a transdominant p53 mutant. High levels of E2F-1 also inhibited growth of primary and established fibroblasts. The growth-inhibitory activity required the DNA binding function of E2F-1 but not its transactivation or pRB binding activities. A positive role for lower levels of E2F-1 in NHFK immortalization was established by examining the ability of E2F-1 to complement HPV16 E7 mutants that were unable to cooperate with HPV16 E6 to immortalize NHFKs. Although E2F-1 was unable by itself to cooperate with E6, it did, in conjunction with E6, complement a p24GLY mutant of E7 that is defective for immortalization and binding of pRB and pRB-related proteins. By contrast, E2F-1 was unable to complement two other E7 mutants, p2PRO and p31/32ARG/PRO, which are also defective in the immortalization assay, although their proteins display wild-type binding of pRB in vitro. Since the binding of E7 to pRB results in disruption of pRB-E2F interaction and release of transcriptionally active E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E3F activity are important but not sufficient for E7-induced keratinocyte immortalization.
Adenovirus infection leads to E1A-dependent activation of the transcription factor E2F. E2F has recently been identified in complexes with cellular proteins such as the retinoblastoma protein (pRB) and the two pRB family members p107 and p130. E1A dissociates E2F from these cellular proteins, and another viral protein, E4 (ORF6/7), can bind to E2F. The binding of E4 to E2F induces the formation of a stable DNA-binding complex containing the two proteins, and stimulation of the adenovirus E2 early promoter can occur. Recent studies have shown that E2F is the combined activity of several proteins, and we demonstrate here that heterodimerization of two of these proteins, E2F-1 and DP-1, is required for stable binding to E4. This complex is formed independently of DNA binding and requires the C-terminal 20 amino acids of E4. Furthermore, the binding is dependent on a region of E2F-1 between amino acids 284 and 358. This region of E2F-1 is conserved in E2F-2 and E2F-3, and deletion of this region drastically reduces the transcriptional activity of the molecule without affecting DP-1 binding, suggesting that this region of the E2F transcription factors is involved in regulating their activity. Our experiments also demonstrate that pRB binding to the E2F-1/DP-1 heterodimer prevents the formation of an E2F-1/DP-1/E4 complex.
By performing DNase I footprint analysis, we had identified three distinct protein binding sequences (MT1, MT2, and MT3) located on the mouse thymidine kinase (TK) upstream promoter (Dou, Q.-P., Fridovich-Keil, J. L., and Pardee, A.B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1157-1161). Here we report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes. (iii) Formation of both these DNA-protein complexes were cell cycle-dependent: a G0/G1 phase-specific complex (E2F.G0/G1) was replaced by an S phase-specific complex(es) (E2F.S), whereas "free" E2F increased after the G1/S transition. (iv) Pulse inhibition of protein synthesis with cycloheximide interchanged these complexes with similar kinetics. (v) When MT2-shifted E2F.G0/G1, E2F.S, and free E2F were eluted and analyzed by Western blot assay using a specific antiserum to human E2F-1, two forms of murine E2F (62 and 66 kDa) were observed from all three complexes. The compositions of these MT2-bound complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex, a candidate repressor, from the MT2 site in late G1 may be essential for S phase-dependent transcription of the mouse TK gene.
Neutral endopeptidase (NEP) inhibitors have been shown to strengthen the effects of endogenous atrial natriuretic peptide (ANP). It has been well documented that angiotensin I-converting enzyme (ACE) inhibitors act beneficially in chronic congestive heart failure (CHF). In the present study, renal and hormonal effects of SCH 34826, an orally active NEP inhibitor, were studied in a coronary-ligation model of experimental CHF in the rat. The effects were compared to those of captopril. The drugs were also administered in combination. In anaesthetized rats with CHF, SCH 34826 (90 mg kg-1 sc) elevated plasma ANP from 382 +/- 85 to 658 +/- 120 ng l-1 compared with vehicle (p = 0.002). In sham-operated control rats, plasma ANP also increased slightly from 52 +/- 6 to 70 +/- 9 ng l-1 (p = 0.05). Plasma renin activity did not change in either group. When given orally for 36 h (90 mg kg-1 b.i.d.), SCH 34826 enhanced natriuresis in controls but not in rats with CHF. Captopril (0.2 mg ml-1 in drinking water) enhanced natriuresis in CHF rats and both natriuresis and kaliuresis in controls. When SCH 34826 and captopril were combined, natriuresis was potentiated in control rats as compared with captopril alone; in rats with CHF, however, a brisk kaliuresis was seen. The excretion of cyclic guanosine monophosphate was enhanced in CHF rats by 52% during treatment with SCH 34826 but not with captopril or combination of the two drugs. Moreover, captopril suppressed aldosterone excretion both in CHF rats and controls when administered alone but not when combined with SCH 34826.(ABSTRACT TRUNCATED AT 250 WORDS)
Recent experiments have demonstrated that the Polycomb group (PcG) gene EZH2 is highly expressed in metastatic prostate cancer and in lymphomas. EZH2 is a component of the PRC2 histone methyltransferase complex, which also contains EED and SUZ12 and is required for the silencing of HOX gene expression during embryonic development. Here we demonstrate that both EZH2 and EED are essential for the proliferation of both transformed and non-transformed human cells. In addition, the pRB-E2F pathway tightly regulates their expression and, consistent with this, we find that EZH2 is highly expressed in a large set of human tumors. These results raise the question whether EZH2 is a marker of proliferation or if it is actually contributing to tumor formation. Significantly, we propose that EZH2 is a bona fide oncogene, since we find that ectopic expression of EZH2 is capable of providing a proliferative advantage to primary cells and, in addition, its gene locus is specifically amplified in several primary tumors.
NPAT is an in vivo substrate of cyclin E-Cdk2 kinase and is thought to play a critical role in coordinated transcriptional activation of histone genes during the G(1)/S-phase transition and in S-phase entry in mammalian cells. Here we show that NPAT transcription is up-regulated at the G(1)/S-phase boundary in growth-stimulated cells and that the NPAT promoter responds to activation by E2F proteins. We demonstrate that endogenous E2F proteins interact with the promoter of the NPAT gene in vivo and that induced expression of E2F1 stimulates NPAT mRNA expression, supporting the idea that the expression of NPAT is regulated by E2F. Consistently, we find that the E2F sites in the NPAT promoter are required for its activation during the G(1)/S-phase transition. Moreover, we show that the expression of NPAT accelerates S-phase entry in cells released from quiescence. The inhibition of NPAT expression by small interfering RNA duplexes impedes cell cycle progression and histone gene expression in tissue culture cells. Thus, NPAT is an important E2F target that is required for cell cycle progression in mammalian cells. As NPAT is involved in the regulation of S-phase-specific histone gene transcription, our findings indicate that NPAT links E2F to the activation of S-phase-specific histone gene transcription.
We report here the characterization of a mutant mouse line with a specific gene trap event in the Mdm4 locus. Absence of Mdm4 expression results in embryonic lethality (10.5 days postcoitum [dpc]), which was rescued by transferring the Mdm4 mutation into a Trp53-null background. Mutant embryos were characterized by overall growth deficiency, anemia, improper neural tube closure, and dilation of lateral ventricles. In situ analysis demonstrated increased levels of p21(CIP1/Waf1) and lower levels of Cyclin E and proliferating cell nuclear antigen expression. Consistent with lack of 5-bromo-2'-deoxyuridine incorporation, these data suggest a block of mutant embryo cells in the G(1) phase of the cell cycle. Accordingly, Mdm4-deficient mouse embryonic fibroblasts manifested a greatly reduced proliferative capacity in culture. Moreover, extensive p53-dependent cell death was specifically detected in the developing central nervous system of the Mdm4 mutant embryos. These findings unambiguously assign a critical role for Mdm4 as a negative regulator of p53 and suggest that Mdm4 could contribute to neoplasias retaining wild-type Trp53. Finally, we provide evidence indicating that Mdm4 plays no role on cell proliferation or cell cycle control that is distinct from its ability to modulate p53 function.
Mad2 is a key component of the spindle checkpoint, a device that controls the fidelity of chromosome segregation in mitosis. The ability of Mad2 to form oligomers in vitro has been correlated with its ability to block the cell cycle upon injection into Xenopus embryos. Here we show that Mad2 forms incompatible complexes with Mad1 and Cdc20, neither of which requires Mad2 oligomerization. A monomeric point mutant of Mad2 can sustain a cell cycle arrest of comparable strength to that of the wild-type protein. We show that the interaction of Mad2 with Mad1 is crucial for the localization of Mad2 to kinetochores, where Mad2 interacts with Cdc20. We propose a model that features the kinetochore as a 'folding factory' for the formation of a Mad2-Cdc20 complex endowed with inhibitory activity on the anaphase promoting complex.
p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5-6-fold upregulation of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced cell death, Apaf1 protein levels were increased. We addressed whether p53 directly regulates Apaf1 transcription via the two p53 consensus binding sites in the Apaf1 promoter. Electrophoretic mobility shift assays demonstrated p53-DNA binding activity at both p53 consensus binding sequences in extracts obtained from neurons undergoing p53-induced cell death, but not in healthy control cultures or when p53 or the p53 binding sites were inactivated by mutation. In transient transfections in a neuronal cell line with p53 and Apaf1 promoter-luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites. The importance of Apaf1 as a p53 target gene in neuronal cell death was evaluated by examining p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury.
Loss of function of the retinoblastoma protein, pRB, leads to lack of differentiation, hyperproliferation and apoptosis. Inactivation of pRB results in deregulated E2F activity, which in turn induces entry to S-phase and apoptosis. Induction of apoptosis by either the loss of pRB or the deregulation of E2F activity occurs via both p53-dependent and p53-independent mechanisms. The mechanism by which E2F induces apoptosis is still unclear. Here we show that E2F1 directly regulates the expression of Apaf-1, the gene for apoptosis protease-activating factor 1. These results provide a direct link between the deregulation of the pRB pathway and apoptosis. Furthermore, because the pRB pathway is functionally inactivated in most cancers, the identification of Apaf-1 as a transcriptional target for E2F might explain the increased sensitivity of tumour cells to chemotherapy. We also show that, independently of the pRB pathway, Apaf-1 is a direct transcriptional target of p53, suggesting that p53 might sensitize cells to apoptosis by increasing Apaf-1 levels.
The retinoblastoma protein (pRB) and its two relatives, p107 and p130, regulate development and cell proliferation in part by inhibiting the activity of E2F-regulated promoters. We have used high-density oligonucleotide arrays to identify genes in which expression changed in response to activation of E2F1, E2F2, and E2F3. We show that the E2Fs control the expression of several genes that are involved in cell proliferation. We also show that the E2Fs regulate a number of genes involved in apoptosis, differentiation, and development. These results provide possible genetic explanations to the variety of phenotypes observed as a consequence of a deregulated pRB/E2F pathway.
In animal cells the progression of the cell cycle through G(1)/S transition and S phase is under the control of the pRB/E2F regulatory pathway. The E2F transcription factors are key activators of genes coding for several regulatory proteins and for enzymes involved in nucleotide and DNA synthesis. In this report we have detected the presence of E2F-like DNA binding activities in carrot nuclear extracts, and we have isolated a carrot cDNA (DcE2F) encoding a plant E2F homologue. The DcE2F gene is expressed in proliferating cells and is induced during the G(1)/S transition of the cell cycle. Supershift experiments using anti-DcE2F antiserum have confirmed that the DcE2F protein is a component of the carrot E2F-like nuclear activities. DNA binding assays have demonstrated that the DcE2F protein can recognize a canonical E2F cis-element in association with a mammalian DP protein. Furthermore, transactivation assays have revealed that DcE2F is a functional transcription factor that can transactivate, together with a DP partner, an E2F-responsive reporter gene in both plant and mammalian cells.
Ever since its discovery, the RB-1 gene and the corresponding protein, pRB, have been a focal point of cancer research. The isolation of E2F transcription factors provided the key to our current understanding of RB-1 function in the regulation of the cell cycle and in tumor suppression. It is becoming more and more evident that the regulatory circuits governing the cell cycle are very complex and highly interlinked. Certain aspects of RB-1 function, for instance its role in differentiation, cannot be easily explained by the current models of pRB-E2F interaction. One reason is that pRB has targets different from E2F, molecules like MyoD for instance. Another reason may be that we have not completely understood the full complexity of E2F function, itself. In this review, we will try to illuminate the role of E2F in pRB- and p53-mediated tumor suppression pathways with particular emphasis on the aspect of E2F-mediated transcriptional regulation. We conclude that E2F can mediate transcriptional activation as well as transcriptional repression of E2F target genes. The net effect of E2F on the transcriptional activity of a particular gene may be the result of as yet poorly understood protein-protein interactions of E2F with other components of the transcriptional machinery, as well as it may reflect the readout of the different ways of regulating E2F activity, itself. We will discuss the relevance of a thorough understanding of E2F function for cancer therapy.
The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have functions similar to those of E2F proteins in higher eukaryotes, by regulating the timed expression of genes implicated in cell cycle progression and DNA synthesis. The CDC6 gene is a target for MBF and SBF-regulated transcription. S. cerevisiae Cdc6p induces the formation of the prereplication complex and is essential for initiation of DNA replication. Interestingly, the Cdc6p homolog in Schizosaccharomyces pombe, Cdc18p, is regulated by DSC1, the S. pombe homolog of MBF. By cloning the promoter for the human homolog of Cdc6p and Cdc18p, we demonstrate here that the cell cycle-regulated transcription of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells enter S phase. Our data also demonstrate that the human CDC6 protein (hCDC6) is essential and limiting for DNA synthesis, since microinjection of an anti-CDC6 rabbit antiserum blocks DNA synthesis and CDC6 cooperates with cyclin E to induce entry into S phase in cotransfection experiments. Furthermore, E2F is sufficient to induce expression of the endogenous CDC6 gene even in the absence of de novo protein synthesis. In conclusion, our results provide a direct link between regulated progression through G1 controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication.
Previous studies have shown that the cell cycle-regulated E2F transcription factor is subjected to both positive and negative control by phosphorylation. Here we show that in transient transfection experiments, adenovirus E1A activation of the viral E2 promoter is abrogated by coexpression of the viral E4 open reading frame 4 (E4-ORF4) protein. This effect does not to require the retinoblastoma protein that previously has been shown to regulate E2F activity. The inhibitory activity of E4-ORF4 appears to be specific because E4-ORF4 had little effect on, for example, E4-ORF6/7 transactivation of the E2 promoter. We further show that the repressive effect of E4-ORF4 on E2 transcription works mainly through the E2F DNA-binding sites in the E2 promoter. In agreement with this, we find that E4-ORF4 inhibits E2F-1/DP-1-mediated transactivation. We also show that E4-ORF4 inhibits E2 mRNA expression during virus growth. E4-ORF4 has previously been shown to bind to and activate the cellular protein phosphatase 2A. The inhibitory effect of E4-ORF4 was relieved by okadaic acid, which inhibits protein phosphatase 2A activity, suggesting that E4-ORF4 represses E2 transcription by inducing transcription factor dephosphorylation. Interestingly, E4-ORF4 did not inhibit the transactivation capacity of a Gal4-E2F hybrid protein. Instead, E4-ORF4 expression appears to result in reduced stability of E2F/DNA complexes.
The transcription factor E2F is present in independent complexes with the product of the retinoblastoma susceptibility gene, pRB, and a related gene product, p107, in association with the cyclin A-cdk2 or the cyclin E-cdk2 kinase complex. pRB and p107 can negatively regulate E2F activity, since overexpression of pRB or p107 in cells lacking a functional pRB leads to the repression of E2F activity. The products of the adenovirus E1A gene can disrupt E2F complexes and result in free and presumably active E2F transcription factor. The regions of E1A required for this function are also essential for binding to a number of cellular proteins, including pRB and p107. Through the use of a number of glutathione S-transferase fusion proteins representing different regions of E1A, as well as in vivo expression of E1A proteins containing deletions of either conserved region 1 (CR1) or CR2, we find that CR2 of E1A can form stable complexes with E2F. E1A proteins containing both CR1 and CR2 also associate with E2F, although the presence of these proteins results in the release of free E2F from its complexes. In vitro reconstitution experiments indicate that E1A-E2F interactions are not direct and that pRB can serve to facilitate these interactions. Complexes containing E1A, p107, cyclin A, and E2F were identified in vivo, which indicates that E1A may associate with E2F through either p107 or pRB. Peptide competition experiments demonstrate that the pRB-binding domain of the human E2F-1 protein can compete with the CR1 but not CR2 domain of E1A for binding to pRB. These results indicate that E1A CR1 and E2F-1 may bind to the same or overlapping sites on pRB and that E1A CR2 binds to an independent region. On the basis of our results, we propose a two-step model for the release of E2F from pRB and p107 cellular proteins.
The binding of viral oncogenes to cellular proteins is thought to modulate the activities of these cellular targets. The p107 protein is targeted by many viral proteins, including adenovirus E1A, simian virus 40 large T antigen, and human papillomavirus type 16 E7 protein. A panel of monoclonal antibodies against p107 was raised and used to identify cellular proteins that interact with the p107 protein in vivo. p107-associated proteins included cyclin A, cyclin E, and cdk2. In addition, p107 was found to associate with 62- to 65- and 50-kDa phosphoproteins in ML-1 cells, a human myeloid leukemia cell line. The 62- to 65-kDa proteins have many of the properties of the transcription factor E2F but were distinguished from pRB-associated E2F-1 by both immunologic and biochemical properties.
E2F is a transcription factor that helps regulate the expression of a number of genes that are important in cell proliferation. Recently, several laboratories have isolated a cDNA clone that encodes an E2F-like protein, known as E2F-1. Subsequent characterization of this protein showed that it had the properties of E2F, but it was difficult to account for all of the suggested E2F activities through the function of this one protein. Using low-stringency hybridization, we have isolated cDNA clones that encode two additional E2F-like proteins, called E2F-2 and E2F-3. The chromosomal locations of the genes for E2F-2 and E2F-3 were mapped to 1p36 and 6q22, respectfully, confirming their independence from E2F-1. However, the E2F-2 and E2F-3 proteins are closely related to E2F-1. Both E2F-2 and E2F-3 bound to wild-type but not mutant E2F recognition sites, and they bound specifically to the retinoblastoma protein in vivo. Finally, E2F-2 and E2F-3 were able to activate transcription of E2F-responsive genes in a manner that was dependent upon the presence of at least one functional E2F binding site. These observations suggest that the E2F activities described previously result from the combined action of a family of proteins.
The E2F transcription factor has been implicated in the regulation of genes whose products are involved in cell proliferation. Two proteins have recently been identified with E2F-like properties. One of these proteins, E2F-1, has been shown to mediate E2F-dependent trans-activation and to bind the hypophosphorylated form of the retinoblastoma protein (pRB). The other protein, murine DP-1, was purified from an E2F DNA-affinity column, and it was subsequently shown to bind the consensus E2F DNA-binding site. To study a possible interaction between E2F-1 and DP-1, we have now isolated a cDNA for the human homolog of DP-1. Human DP-1 and E2F-1 associate both in vivo and in vitro, and this interaction leads to enhanced binding to E2F DNA-binding sites. The association of E2F-1 and DP-1 leads to cooperative activation of an E2F-responsive promoter. Finally, we demonstrate that E2F-1 and DP-1 association is required for stable interaction with pRB in vivo and that trans-activation by E2F-1/DP-1 heterodimers is inhibited by pRB. We suggest that "E2F" is the activity that is formed when an E2F-1-related protein and a DP-1-related protein dimerize.
Loss of a functional retinoblastoma tumor suppressor gene product, pRB, is a key step in the development of many human tumors. pRB is a negative regulator of cell proliferation and appears to participate in control of entry into the S phase of the cell cycle. The recent demonstration that pRB binds to transcription factor E2F has provided a model for the mechanism of pRB-mediated growth regulation. Since adenovirus E1A proteins dissociate the pRB-E2F complexes and stimulate E2F-dependent transcription, it has been suggested that pRB inhibits E2F transactivation. Although some evidence for this hypothesis has been provided, it has not been possible to determine the mechanism of pRB-mediated inhibition of E2F transactivation. In this study, we constructed mutants of E2F-1 that do not bind to pRB yet retain the ability to transactivate the adenovirus E2 promoter through E2F DNA-binding sites. We demonstrated that transactivation mediated by the wild-type E2F-1 protein was inhibited by overexpression of wild-type pRB but not by a naturally occurring mutant of pRB. Transactivation mediated by mutants of E2F-1 which do not bind to pRB was not affected by overexpression of wild-type pRB. Furthermore, when the E2F-1 transactivation domain was fused to the GAL4 DNA-binding domain, pRB inhibited GAL4-E2F-1 transactivation through GAL4 sites. Expression of pRB did not inhibit transactivation mediated by GAL4-E2F-1 mutant constructs which were devoid of pRB binding. In conclusion, these data demonstrate that pRB inhibits E2F-dependent transactivation by direct protein-protein interaction.
The cellular protein p107 shares many structural and biochemical features with the retinoblastoma gene product, pRB. We have isolated a full-length cDNA for human p107 and have used this clone to study the function of p107. We show that, like pRB, p107 is a potent inhibitor of E2F-mediated trans-activation, and overexpression of p107 can inhibit proliferation in certain cell types, arresting sensitive cells in G1. Several experiments, however, showed that growth inhibition by pRB and p107 did not occur through the same mechanism. First, in the cervical carcinoma cell line C33A, p107 was able to block cell proliferation, whereas pRB could not, even though both proteins were potent inhibitors of E2F-mediated transcription in this cell line. Second, growth arrest by pRB and p107 was rescued differentially by various cell cycle regulators. Third, some mutants of p107 that cannot associate with adenovirus E1A were still able to inhibit cell proliferation, whereas analogous mutants in pRB are known to be unable to block cell growth. Together, these results suggest a biological role of p107 that is related, but not identical, to that of pRB.
The retinoblastoma protein, pRB, appears to play a key role in coordinating the regulation of cell cycle position and transcriptional events. pRB undergoes specific cell-cycle-dependent phosphorylation, being underphosphorylated in G1 and heavily phosphorylated in S, G2, and M. The underphosphorylated form is able to interact with the E2F transcription factor. Recently, we have cloned a cDNA for E2F-1. By using this clone and a series of non-pRB binding mutants, we have been able to show that the binding of pRB to E2F-1 causes inhibition of E2F-mediated transactivation. pRB's inhibition of E2F-mediated transcription would be lost by mutation in the retinoblastoma gene in human tumours, by pRB's interaction with DNA tumour virus oncoproteins, or by phosphorylation during the cell cycle.
The retinoblastoma protein (pRB) is one of the best-studied tumour suppressor gene products. Its loss during the genesis of many human tumours, its inactivation by several DNA tumour virus oncoproteins, and its ability to inhibit cell growth when introduced into dividing cells all suggest that pRB negatively regulates some aspect of normal cell growth. The discovery that pRB associates with transcription factors such as E2F has provided the first model for pRB function. In this review, we discuss how pRB may regulate cell growth by repressing transcription of genes essential for cell proliferation.
The epidermal growth factor (EGF) receptor (EGFR) and the erbB-2 gene product, gp185erbB-2, exhibit distinct abilities to stimulate mitogenesis in different target cells. By using chimeric molecules between these two receptors, we have previously shown that their intracellular juxtamembrane regions are responsible for this specificity. Here we describe a genetically engineered EGFR mutant containing a threonine for arginine substitution at position 662 in the EGFR juxtamembrane domain, corresponding to threonine 694 in gp185erbB-2. This mutant, designated EGFRThr662, displayed affinity for EGF binding and catalytic properties that were indistinguishable from those of the wild type EGFR. However, EGFRThr662 behaved much as gp185erbB-2 in a number of bioassays which readily distinguish between the mitogenic effects of EGFR and gp185erbB-2. Moreover, significant differences were detected in the pattern of intracellular proteins phosphorylated on tyrosine in vivo by EGFR and EGFRThr662 in response to EGF. Thus, small differences in the primary sequence of two closely related receptors have dramatic effects on their ability to couple with mitogenic pathways.
The retinoblastoma gene product, pRB, regulates cell proliferation by binding to and inhibiting the activity of key growth promoting proteins. Several cellular proteins have been shown to bind directly to pRB and the genes encoding a number of them have been isolated. The protein product of one of these genes is the transcription factor E2F. We have now isolated cDNA clones that contain the full-length coding sequence of two other proteins, RBP1 and RBP2, cloned originally by their interaction with pRB. The products of the RBP1 and RBP2 genes are ubiquitously expressed, large (200 kDa for RBP1 and 195 kDa for RBP2) nuclear phosphoproteins with structural motifs that suggest a role in transcriptional regulation. In addition we have been able to identify complexes of pRB and RBP1 in vivo that are dissociated in the presence of purified human papillomavirus E7 protein.
Autophosphorylation of gp185erbB-2 in vivo is confined to its carboxy terminus and is required for optimal erbB-2 transforming activity under conditions of receptor overexpression. It remains unresolved, however, to what extent autophosphorylation regulates erbB-2 mitogenic signaling in normal cells, nor is the biochemical basis for such a regulatory function known. To address these issues, we utilized a chimeric molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) fused to the transmembrane and intracellular domains of the erbB-2 product. In this EGFR/erbB-2 chimera, erbB-2 kinase activity is regulated by EGF binding. An EGFR/erbB-2 mutant bearing multiple Tyr----Phe substitutions at erbB-2 autophosphorylation sites (EGFR/erbB-2 5P) displayed markedly reduced phosphotyrosine content following EGF stimulation in comparison with the non-mutated chimera. When expressed in NR6 cells, the EGFR/erbB-2 5P mutant was unable to deliver a sizeable mitogenic signal when activated by EGF at physiological levels. In intact cells, the 5P mutant was still able to stimulate phosphorylation of the gamma isozyme of phospholipase C (PLC-gamma), a prototype erbB-2 substrate, although with a delayed time course, indicating that the 5P mutation decreased the affinity of the erbB-2 kinase for this substrate. This conclusion was further supported by the inability of the 5P mutant to associate with PLC-gamma in co-immunoprecipitation experiments. We infer that a major role of autophosphorylation is to increase the affinity of the erbB-2 kinase for its cellular substrates, so that, under physiological conditions, autophosphorylation is absolutely required for erbB-2 mitogenic signaling.
The role of epidermal growth factor (EGF) receptor autophosphorylation sites in the regulation of receptor functions has been studied using cells transfected with mutant EGF receptors. Simultaneous point mutation of 4 tyrosines (Y1068, Y1086, Y1148, Y1173) to phenylalanine, as well as removal of these sites by truncation of the carboxyl-terminal 123 amino acid residues, resulted in reduced receptor phosphorylation of an in vivo specific substrate phospholipase C-gamma 1 to less than 50% compared to the wild-type receptor. The internalization rate constant Ke was also significantly lower in these mutants (0.15/min) compared to cells transfected with wild-type receptor (0.27/min). Additional mutation of tyrosine 992 to phenylalanine in the truncated receptor mutant (Dc-123F) further decreased the receptor internalization rate to a minimal level (ke = 0.07-0.10/min), equivalent to the ke measured for cells expressing kinase-negative receptor (A721). Moreover, tyrosine kinase activity of the Dc-123F receptor toward phospholipase C-gamma 1, compared to wild-type receptor, was reduced by 90%. Taken together, these results show that EGF receptor lacking five autophosphorylation sites functions similar to a kinase-negative receptor. Mutation of tyrosine residue Y992 alone in the context of full length EGF receptor, however, did not affect receptor internalization or kinase activity toward phospholipase C-gamma 1. These data indicate that tyrosine 992 is critical for substrate phosphorylation and internalization only in the context of the truncated receptor, and that minor autophosphorylation sites, such as Y992, may act as compensatory regulatory sties in the absence of the major EGF receptor autophosphorylation sites.
The E2F transcription factors are key targets for the retinoblastoma protein, pRB. By inactivation of E2Fs, pRB prevents progression to the S phase. To test proliferative functions of E2F, we generated transgenic mice expressing human E2F-1 and/or human DP-1. When the hydroxymethyl glutaryl coenzyme A reductase promoter was used to express DP-1, overexpression occurred in a variety of tissues and did not confer phenotypic changes. In contrast, expression of E2F-1 from the same promoter was obtained only in testicles, in which E2F-1 overexpression caused atrophy and sterility through a process involving increased apoptosis in the germinal epithelium. This effect was potentiated by simultaneous overexpression of DP-1. Testicular atrophy as a result of overexpression of E2F-1 and DP-1 is independent of functional p53, since p53-nullizygous transgenic mice overexpressing E2F-1 and DP-1 also suffered testicular atrophy.
The family of E2F transcription factors have an essential role in mediating cell cycle progression, and recently, one of the E2F protein family, E2F-1, has been shown to participate in the induction of apoptosis. Cooperation between E2F and the p53 tumor suppressor protein in this apoptotic response had led to the suggestion that cell cycle progression induced by E2F-1 expression provides an apoptotic signal when placed in conflict with an arrest to cell cycle progression, such as provided by p53. We show here that although apoptosis is clearly enhanced by p53, E2F-1 can induce significant apoptosis in the absence of p53. Furthermore, this apoptotic function of E2F-1 is separable from the ability to accelerate entry into DNA synthesis. Analysis of E2F-1 mutants indicates that although DNA-binding is required, transcriptional transactivation is not necessary for the induction of apoptosis by E2F-1, suggesting that it may be mediated through alleviation of E2F-dependent transcriptional repression. These results indicate that E2F-1 can show independent cell cycle progression and apoptotic functions, consistent with its putative role as a tumor suppressor.
The E2F family of transcription factors regulate genes, whose products are essential for progression through the mammalian cell cycle. The transcriptional activity of the E2Fs is inhibited through the specific binding of the retinoblastoma protein, pRB, and the pRB homologs p107 and p130 to their transactivation domains. Seven members of the E2F transcription factor family have been isolated so far, and we were interested in investigating the possible contribution of the various E2Fs to cell cycle control. By presenting the results of the generation of cell lines with tetracycline-controlled expression of E2F-1 and E2F-4 and microinjection of expression plasmids for all members of the E2F family, we demonstrate here that the pRB-associated ED2Fs (E2F-1, E2F-2, and E2F-3) all induce S phase in quiescent rate fibroblasts when expressed alone. In contrast, the p107/p130-associated E2Fs require the coexpression of the heterodimeric partner DP-1 to promote S-phase entry and accelerate G1 progression. Furthermore, the pRB-associated E2Fs were all able to overcome a G1 arrest mediated by the p16INK4 tumor suppressor protein, and E2F-1 was shown to override a G1 block mediated by a neutralizing antibody to cyclin D1. The p16INK4-induced G1 arrest was not affected by expression of E2F-4, E2F-5, or DP-1 alone, but simulataneous expression of E2F-4 and DP-1 could overcome this block. Our results demonstrate that the generation of E2F activity is rate limiting for G1 progession, is sufficient to induce S-phase entry, and overcomes a p16-mediated G1 block, and since E12F-1, E2F-2, and E2F-3 are associated with pRB, they are the most likely downstream effectors in the p126-cyclin D-pRB pathway. Furthermore, our date suggest that the two subsets of E2Fs are regulated by distinct mechanisms and/or that they have distinct functions in cell cycle control. Since E2F-4 and E2F-5 cannot promote S-phase entry by themselves, our results may provide an explanation for the apparent lack of aberrations in p107 or p130 in human cancer.
The retinoblastoma protein (pRB) plays an important role in the control of cell proliferation, apparently by binding to and regulating cellular transcription factors such as E2F. Here we describe the characterization of a cDNA clone that encodes a protein with properties of E2F. This clone, RBP3, was identified by the ability of its gene product to interact with pRB. RBP3 bound to pRB both in vitro and in vivo, and this binding was competed by viral proteins known to disrupt pRB-E2F association. RBP3 bound to E2F recognition sequences in a sequence-specific manner. Furthermore, transient expression of RBP3 caused a 10-fold transactivation of the adenovirus E2 promoter, and this transactivation was dependent on the E2F recognition sequences. These properties suggest that RBP3 encodes E2F, or an E2F-like protein.
Mutant epidermal growth factor (EGF) receptors (obtained by substitution of one, two or three C-terminal autophosphorylable tyrosine residues with phenylalanine residues or by deletion of the C-terminal 19 amino acids, including the distal tyrosine) were expressed in mouse NIH-3T3 fibroblast clones at densities comparable (less than 25% difference) with those in control clones expressing the wild-type receptor. Total EGF-induced phosphorylation of the mutated receptors was not appreciably changed with respect to controls, whereas autophosphorylation at tyrosine residues was decreased, especially in the double and the triple mutants. In the latter mutant, expression of the EGF-receptor-activated lipolytic enzyme phospholipase C gamma was unchanged, whereas its tyrosine phosphorylation induced by the growth factor was lowered to approx. 25% of that in the controls. In all of the cell clones employed, the accumulation of inositol phosphates induced by treatment with fetal calf serum varied only slightly, whereas the same effect induced by EGF was consistently lowered in those lines expressing mutated receptors. This decrease was moderate for those receptors missing only the distal tyrosine (point and deletion mutants), intermediate in the dual mutants and almost complete in the triple mutants. Likewise, increases in intracellular Ca2+ concentrations [( Ca2+]i) induced by fibroblast growth factor were approximately the same in all of the clones, whereas those induced by EGF were decreased in the mutants, again in proportion to the loss of the phosphorylable C-terminal tyrosine residues. The same trend occurred with membrane hyperpolarization, an effect secondary to the increase in [Ca2+]i via the activation of Ca2(+)-dependent K+ channels. We conclude that C-terminal autophosphorylable tyrosine residues play a positive role in the regulation of transmembrane signalling at the EGF receptor. The stepwise decrease in signal generation observed in single, double and triple point mutants suggest that the role of phosphotyrosine residues is not in the participation in specific amino acid sequences, but rather in the introduction of strong negative charges at strategic sites of the receptor tail. As a consequence of autophosphorylation, the receptor could become competent for specific association with phospholipase C gamma, with ensuing activation by tyrosine phosphorylation followed by the chains of intracellular responses ultimately leading to DNA synthesis and cell duplication.
The epidermal growth factor receptor (EGF-R) C-terminus contains three conserved tyrosines (Y-1068, Y-1148, Y-1173) which are phosphorylated upon EGF activation. To clarify the functional role of these tyrosines, each has been mutated to phenylalanine and studied as single, double and triple mutants in the full length receptor. EGF-dependent transforming ability of the single point mutants is similar to that of the wild type, while that of double mutants is decreased and an even lower activity is present in the triple mutant. In each bioassay, including EGF-dependent focal transformation, growth in agar and growth in low serum, mutant receptors display a similar hierarchy of activity. The lower activity is intrinsic in the mutants since they are expressed at similar level as the wild type and bind EGF with similar affinity. Deletion mutants lacking the last 19 or 63 amino acids (Velu et al., 1989a) show a similar decline in biological activity when compared to the corresponding point mutants, although the reduction is more pronounced than with the point mutants. Deletion of the last 123 aa, which removes all three tyrosines (Dc123), results in a receptor that is almost inactive biologically. The EGF-R kinase activity is affected by tyrosine substitution since in vitro phosphorylation of exogenous substrates is reduced in the double and triple mutants. Autophosphorylation, in vivo and in vitro, is also reduced, but not totally abolished in the triple point mutant and Dc123 indicating the existence of other autophosphorylation sites. A new site of autophosphorylation is found in the Dc123 mutant. We conclude, therefore, that the tyrosines at the extreme C-terminus positively regulate the biological and transforming activity of the EGF-R, probably via autophosphorylation.
The C terminus of the epidermal growth factor receptor (EGF-R) contains three tyrosines (Y1068, Y1148, and Y1173) which correspond to the major autophosphorylation sites. To investigate the role of the tyrosines in internalization and down-regulation of the EGF-R, mutational analysis was performed with receptors in which 1, 2, or all 3 tyrosines were changed to phenylalanines. The triple point mutant EGF-R, expressed in NIH-3T3, exhibited low autophosphorylation in vivo, low biological and reduced kinase activities. Single and double point mutants were down-regulated, as well as wild type EGF-R in response to EGF showing a half-life of about 1 h. Degradation of the triple point mutant, however, was impaired and resulted in a half-life of 4 h in the presence of EGF. EGF-dependent down-regulation of surface receptors was decreased in the triple point mutant EGF-R as was internalization and degradation of EGF. The specific rate of internalization of the triple point mutant was reduced. By contrast, intracellular processing of ligand previously internalized at 20 degrees C was similar between wild type and mutant receptors. Taken together the data indicate that the delay in degradation observed in cells expressing the triple point mutant EGF-R can be attributed mainly to a slower removal from the cell surface. Our results show that in the full-length EGF-R all three C-terminal tyrosines are necessary for rapid internalization, suggesting that autophosphorylation is required for efficient EGF-dependent receptor endocytosis.
We have previously shown that the adenovirus E1A oncogene can reactivate the cell cycle in terminally differentiated cells. Current models imply that much or all of this E1A activity is mediated by the release of the E2F transcription factors from pocket-protein control. In contrast, we show here that overexpression of E2F-1, E2F-2 and E2F-4, or a chimeric E2F-4 tethered to a nuclear localization signal cannot reactivate postmitotic skeletal muscle cells (myotubes). This is not due to lack of transcriptional activity, as demonstrated on both a reporter construct and a number of endogenous target genes. Although cyclin E was strongly overexpressed in E2F-transduced myotubes, it lacked associated kinase activity, possibly explaining the inability of the myotubes to enter S phase and accumulate cyclin A. Although E2F is not sufficient to trigger DNA synthesis in myotubes, its activity is necessary even in the presence of E1A, as dominant-negative DP-1 mutants inhibit E1A-mediated cell cycle reentry. Our data show that, to reactivate myotubes, E1A must exert other functions, in addition to releasing E2F. They also establish mouse myotubes as an experimental system uniquely suited to study the most direct E2F functions in the absence of downstream cell cycle effects.
Cyclin-dependent kinases (CDKs) are essential for regulating key transitions in the cell cycle, including initiation of DNA replication, mitosis and prevention of re-replication. Here we demonstrate that mammalian CDC6, an essential regulator of initiation of DNA replication, is phosphorylated by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21(WAF1/SDI1) and E2F-1. The in vivo phosphorylation of CDC6 was dependent on three N-terminal CDK consensus sites, and the phosphorylation of these sites was shown to regulate the subcellular localization of CDC6. Consistent with this notion, we found that the subcellular localization of CDC6 is cell cycle regulated. In G1, CDC6 is nuclear and it relocalizes to the cytoplasm when Cyclin A/CDK2 is activated. In agreement with CDC6 phosphorylation being specifically mediated by Cyclin A/CDK2, we show that ectopic expression of Cyclin A, but not of Cyclin E, leads to rapid relocalization of CDC6 from the nucleus to the cytoplasm. Based on our data we suggest that the phosphorylation of CDC6 by Cyclin A/CDK2 is a negative regulatory event that could be implicated in preventing re-replication during S phase and G2.
PML is a nuclear protein with growth-suppressive properties originally identified in the context of the PML-retinoic acid receptor alpha (RAR alpha) fusion protein of acute promyelocytic leukemia. PML localizes within distinct nuclear structures, called nuclear bodies, which are disrupted by the expression of PML-RAR alpha. We report that PML colocalizes with the nonphosphorylated fraction of the retinoblastoma protein (pRB) within nuclear bodies and that pRB is delocalized by PML-RAR alpha expression. Both PML and PML-RAR alpha form complexes with the nonphosphorylated form of pRB in vivo, and they interact with the pocket region of pRB. The regions of PML and PML-RAR alpha involved in pRB binding differ; in fact, the B boxes and the C-terminal region of PML, the latter of which is not present in PML-RAR alpha, are essential for the formation of stable complexes with pRB. Functionally, PML abolishes activation of glucocorticoid receptor-regulated transcription by pRB, whereas PML-RAR alpha further increases it. Our results suggest that PML may be part of transcription-regulatory complexes and that the oncogenic potential of the PML-RAR alpha protein may derive from the alteration of PML-regulated transcription.
Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative disorders caused by an expansion of a polyglutamine tract. It is characterized by ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells. To understand the pathogenesis of SCA1, we examined the subcellular localization of wild-type human ataxin-1 (the protein encoded by the SCA1 gene) and mutant ataxin-1 in the Purkinje cells of transgenic mice. We found that ataxin-1 localizes to the nuclei of cerebellar Purkinje cells. Normal ataxin-1 localizes to several nuclear structures approximately 0.5 microm across, whereas the expanded ataxin-1 localizes to a single approximately 2-microm structure, before the onset of ataxia. Mutant ataxin-1 localizes to a single nuclear structure in affected neurons of SCA1 patients. Similarly, COS-1 cells transfected with wild-type or mutant ataxin-1 show a similar pattern of nuclear localization; with expanded ataxin-1 occurring in larger structures that are fewer in number than those of normal ataxin-1. Colocalization studies show that mutant ataxin-1 causes a specific redistribution of the nuclear matrix-associated domain containing promyelocytic leukaemia protein. Nuclear matrix preparations demonstrate that ataxin-1 associates with the nuclear matrix in Purkinje and COS cells. We therefore propose that a critical aspect of SCA1 pathogenesis involves the disruption of a nuclear matrix-associated domain.
In cells of higher eukaryotes, cyclin D-dependent kinases Cdk4 and Cdk6 and, possibly, cyclin E-dependent Cdk2 positively regulate the G1- to S-phase transition, by phosphorylating the retinoblastoma protein (pRb), thereby releasing E2F transcription factors that control S-phase genes. Here we performed microinjection and transfection experiments using rat R12 fibroblasts, their derivatives conditionally overexpressing cyclins D1 or E, and human U-2-OS cells, to explore the action of G1 cyclins and the relationship of E2F and cyclin E in S-phase induction. We demonstrate that ectopic expression of cyclin E, but not cyclin D1, can override G1 arrest imposed by either the p16INK4a Cdk inhibitor specific for Cdk4 and Cdk6 or a novel phosphorylation-deficient mutant pRb. Several complementary approaches to assess E2F activation, including quantitative reporter assays in live cells, showed that the cyclin E-induced S phase and completion of the cell division cycle can occur in the absence of E2F-mediated transactivation. Together with the ability of cyclin E to overcome a G1 block induced by expression of dominant-negative mutant DP-1, a heterodimeric partner of E2Fs, these results provide evidence for a cyclin E-controlled S phase-promoting event in somatic cells downstream of or parallel to phosphorylation of pRb and independent of E2F activation. They furthermore indicate that a lack of E2F-mediated transactivation can be compensated by hyperactivation of this cyclin E-controlled event.
Cyclin E is necessary and rate limiting for the passage of mammalian cells through the G1 phase of the cell cycle. Control of cell cycle progression by cyclin E involves cdk2 kinase, which requires cyclin E for catalytic activity. Expression of cyclin E/cdk2 leads to an activation of cyclin A gene expression, as monitored by reporter gene constructs derived from the human cyclin A promoter. Promoter activation by cyclin E/cdk2 requires an E2F binding site in the cyclin A promoter. We show here that cyclin E/cdk2 kinase can directly bind to E2F/p107 complexes formed on the cyclin A promoter-derived E2F binding site, and this association is controlled by p27KIP1, most likely through direct protein-protein interaction. These observation suggest that cyclin E/cdk2 associates with E2F/p107 complexes in late G1 phase, once p27KIP1 has decreased below a critical threshold level. Since a kinase-negative mutant of cdk2 prevents promoter activation, it appears that transcriptional activation of the cyclin A gene requires an active cdk2 kinase tethered to its promoter region.
Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation is mediated by the transcription factor E2F, which binds to a specific site in the cyclin A promoter. It differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing cyclin E and p107 but not pRB. Ectopic expression of cyclin D1 triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4.
The urokinase-type plasminogen activator receptor (uPAR) focuses at the cell surface the activation of pro-uPA and, hence, the formation of plasmin, thus enhancing directional extracellular proteolysis. To characterize the transcriptional regulatory mechanisms that control receptor expression, we have cloned an uPAR DNA segment containing upstream regulatory sequences from both the human and murine genomes. We report that a proximal promoter, contained within 180 bp from the major transcription start sites of the human uPAR gene, drives basal transcription. This region lacks TATA and CAAT boxes and contains relatively GC-rich proximal sequences. A subregion of this sequence, highly conserved between human and murine genes, contains most of the promoter activity and is specifically bound by HeLa nuclear proteins, one of which belongs to the SP1 class.
The parB locus of plasmid R1, which mediates plasmid stability via postsegregational killing of plasmid-free cells, encodes two genes, hok and sok. The hok gene product is a potent cell-killing protein. The hok gene is regulated at the translational level by the sok gene-encoded repressor, a small anti-sense RNA complementary to the hok mRNA. The hok mRNA is extraordinarily stable, while the sok RNA decays rapidly. The mechanism of postsegregational killing is explained by the following model; the sok RNA molecule rapidly disappears in cells that have lost a parB-carrying plasmid, leading to translation of the stable hok mRNA. Consequently, the Hok protein is synthesized and killing of the plasmid-free cell follows.
Efficient treatment of acute myeloid leukemia (AML) patients remains a challenge despite recent therapeutic advances. Here, using a CRISPRi screen targeting chromatin factors, we identified the nucleosome-remodeling factor (NURF) subunit BPTF as an essential regulator of AML cell survival. We demonstrate that BPTF forms an alternative NURF chromatin remodeling complex with SMARCA5 and BAP18, which regulates the accessibility of a large set of insulator regions in leukemic cells. This ensures efficient CTCF binding and boundary formation between topologically associated domains that is essential for maintaining the leukemic transcriptional programs. We also demonstrate that the well-studied PHD2-BROMO chromatin reader domains of BPTF, while contributing to complex recruitment to chromatin, are dispensable for leukemic cell growth. Taken together, our results uncover how the alternative NURF complex contributes to leukemia and provide a rationale for its targeting in AML.
The human silencing hub (HUSH) complex is an epigenetic repressor complex whose role has emerged as an important guardian of genome integrity. It protects the genome from exogenous DNA invasion and regulates endogenous retroelements by recruiting histone methyltransferases catalyzing histone 3 lysine 9 trimethylation (H3K9me3) and additional proteins involved in chromatin compaction. In particular, its regulation of transcriptionally active LINE1 retroelements, by binding to and neutralizing LINE1 transcripts, has been well characterized. HUSH is required for mouse embryogenesis and is associated with disease, in particular cancer. Here we provide insights into the structural and biochemical features of the HUSH complex. Furthermore, we discuss the molecular mechanisms by which the HUSH complex is recruited to specific genomic regions and how it silences transcription. Finally, we discuss the role of HUSH complex members in mammalian development, antiretroviral immunity, and diseases such as cancer.
NSD2 is a histone methyltransferase predominantly catalyzing di-methylation of histone H3 on lysine K36. Increased NSD2 activity due to mutations or fusion-events affecting the gene encoding NSD2 is considered an oncogenic event and a driver in various cancers, including multiple myelomas carrying t(4;14) chromosomal translocations and acute lymphoblastic leukemia's expressing the hyperactive NSD2 mutant E1099 K. Using DNA-encoded libraries, we have identified small molecule ligands that selectively and potently bind to the PWWP1 domain of NSD2, inhibit NSD2 binding to H3K36me2-bearing nucleosomes, but do not inhibit the methyltransferase activity. The ligands were subsequently converted to selective VHL1-recruiting NSD2 degraders and by using one of the most efficacious degraders in cell lines, we show that it leads to NSD2 degradation, decrease in K3 K36me2 levels and inhibition of cell proliferation.
Regulation of protein synthesis is a key factor in hematopoietic stem cell maintenance and differentiation. Rio-kinase 2 (RIOK2) is a ribosome biogenesis factor that has recently been described an important regulator of human blood cell development. Additionally, we have previously identified RIOK2 as a regulator of protein synthesis and a potential target for the treatment of acute myeloid leukemia (AML). However, its functional relevance in several organ systems, including normal hematopoiesis, is not well understood. Here, we investigate the consequences of RIOK2 loss on normal hematopoiesis using two different conditional knockout mouse models. Using competitive and non-competitive bone marrow transplantations, we demonstrate that RIOK2 is essential for the differentiation of hematopoietic stem and progenitor cells (HSPCs) as well as for the maintenance of fully differentiated blood cells in vivo as well as in vitro. Loss of RIOK2 leads to rapid death in full-body knockout mice as well as mice with RIOK2 loss specific to the hematopoietic system. Taken together, our results indicate that regulation of protein synthesis and ribosome biogenesis by RIOK2 is essential for the function of the hematopoietic system.
Poor survival and lack of treatment response in glioblastoma (GBM) is attributed to the persistence of glioma stem cells (GSCs). To identify novel therapeutic approaches, we performed CRISPR/Cas9 knockout screens and discovered TGFβ activated kinase (TAK1) as a selective survival factor in a significant fraction of GSCs. Loss of TAK1 kinase activity results in RIPK1-dependent apoptosis via Caspase-8/FADD complex activation, dependent on autocrine TNFα ligand production and constitutive TNFR signaling. We identify a transcriptional signature associated with immune activation and the mesenchymal GBM subtype to be a characteristic of cancer cells sensitive to TAK1 perturbation and employ this signature to accurately predict sensitivity to the TAK1 kinase inhibitor HS-276. In addition, exposure to pro-inflammatory cytokines IFNγ and TNFα can sensitize resistant GSCs to TAK1 inhibition. Our findings reveal dependency on TAK1 kinase activity as a novel vulnerability in immune-activated cancers, including mesenchymal GBMs that can be exploited therapeutically.
<h4>Significance</h4>Combined EZH2 and RAS pathway inhibitors kill KRAS-mutant colorectal cancer cells and promote durable tumor regression in vivo. These agents function by cooperatively suppressing the WNT pathway, driving differentiation, and epigenetically reprogramming cells to permit the induction of apoptotic signals, which then kill these more differentiated tumor cells.
DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies, although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein (TOPORS), which encodes a ubiquitin/SUMO E3 ligase, augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis, suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks, which undergo SUMOylation, followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. In TOPORS knockout cells, DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination, resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1, thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently, the ubiquitination inhibitor, TAK-243, and the SUMOylation inhibitor, TAK-981, show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks.
Epigenetic modifications, including posttranslational modifications of histones, are closely linked to transcriptional regulation. Trimethylated H3 lysine 4 (H3K4me3) is one of the most studied histone modifications owing to its enrichment at the start sites of transcription and its association with gene expression and processes determining cell fate, development, and disease. In this review, we focus on recent studies that have yielded insights into how levels and patterns of H3K4me3 are regulated, how H3K4me3 contributes to the regulation of specific phases of transcription such as RNA polymerase II initiation, pause-release, heterogeneity, and consistency. The conclusion from these studies is that H3K4me3 by itself regulates gene expression and its precise regulation is essential for normal development and preventing disease.
Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and has the highest rate of recurrence<sup>1</sup>. The predominant standard of care for advanced TNBC is systemic chemotherapy with or without immunotherapy; however, responses are typically short lived<sup>1,2</sup>. Thus, there is an urgent need to develop more effective treatments. Components of the PI3K pathway represent plausible therapeutic targets; more than 70% of TNBCs have alterations in PIK3CA, AKT1 or PTEN<sup>3-6</sup>. However, in contrast to hormone-receptor-positive tumours, it is still unclear whether or how triple-negative disease will respond to PI3K pathway inhibitors<sup>7</sup>. Here we describe a promising AKT-inhibitor-based therapeutic combination for TNBC. Specifically, we show that AKT inhibitors synergize with agents that suppress the histone methyltransferase EZH2 and promote robust tumour regression in multiple TNBC models in vivo. AKT and EZH2 inhibitors exert these effects by first cooperatively driving basal-like TNBC cells into a more differentiated, luminal-like state, which cannot be effectively induced by either agent alone. Once TNBCs are differentiated, these agents kill them by hijacking signals that normally drive mammary gland involution. Using a machine learning approach, we developed a classifier that can be used to predict sensitivity. Together, these findings identify a promising therapeutic strategy for this highly aggressive tumour type and illustrate how deregulated epigenetic enzymes can insulate tumours from oncogenic vulnerabilities. These studies also reveal how developmental tissue-specific cell death pathways may be co-opted for therapeutic benefit.
Book chapters
TET proteins are methylcytosine dioxygenases that interact directly with chromatin to shape the DNA methylation landscape. To increase the understanding of TET protein function in a specific cellular context, it is important to be able to map the interactions between TET proteins and DNA. This ChIP-seq protocol details our procedure to analyze TET2 bound DNA in disuccinimidyl glutarate (DSG) and formaldehyde-crosslinked chromatin but can also be adapted to study other TET enzymes.
Conferences
SUZ12 is a core component of the polycomb repressive complex 2 (PRC2) and is required for the differentiation of mouse embryonic stem cells (ESCs). PRC2 is associated with transcriptional repression via methylation of H3 Lys-27. We applied quantitative mass spectrometry to investigate the effects of Suz12 deficiency on H3.2 and H3.3 from mouse ESCs. Using high mass accuracy MS combined with CID or electron transfer dissociation (ETD) tandem mass spectrometry, we identified a total of 81 unique modified peptides from H3.2 and H3.3 and assigned 46 modifications at 22 different positions, including distinct coexisting modifications. In certain cases, high mass accuracy LTQ-Orbitrap MS/MS allowed precise localization of near isobaric coexisting PTMs such as trimethylation and acetylation within individual peptides. ETD MS/MS facilitated sequencing and annotation of phosphorylated histone peptides. The combined use of ETD and CID MS/MS increased the total number of identified modified peptides. Comparative quantitative analysis of histones from wild type and Suz12-deficient ESCs using stable isotope labeling with amino acids in cell culture and LC-MS/MS revealed a dramatic reduction of H3K27me2 and H3K27me3 and an increase of H3K27ac, thereby uncovering an antagonistic methyl/acetyl switch at H3K27. The reduction in H3K27 methylation and increase in H3K27 acetylation was accompanied by H3K36 acetylation and methylation. Estimation of the global isoform percentage of unmodified and modified histone peptides (amino acids 27-40) showed the relative distribution of distinct coexisting histone marks. Our study revealed limitations of antibody-based Western blotting methods for detection of coexisting protein modifications and demonstrated the utility of quantitative tandem mass spectrometry for detailed analysis of the dynamics of coexisting post-translational modifications in proteins.
The generation of different cell types from stem cells containing identical genetic information and their organization into tissues and organs during development is a highly complex process that requires defined transcriptional programs. Maintenance of such programs is epigenetically regulated and the factors involved in these processes are often essential for development. The activities required for cell-fate decisions are frequently deregulated in human tumors, and the elucidation of the molecular mechanisms that regulate these processes is therefore important for understanding both developmental processes and tumorigenesis.