Dr Christian Zierhut
Group Leader: Genome Stability and Innate Immunity
OrcID: 0000-0003-3501-603X
Phone: +44 20 7153 5256
Email: [email protected]
Also on: https://www.zierhutlab.org
Location: Chelsea
OrcID: 0000-0003-3501-603X
Phone: +44 20 7153 5256
Email: [email protected]
Also on: https://www.zierhutlab.org
Location: ChelseaBiography
Dr Christian Zierhut obtained his undergraduate degree from the University of Vienna in Austria, where he studied meiotic DNA repair with Prof Franz Klein at the Department of Genetics (later incorporated into the Max Perutz Labs).
For his PhD, he worked with Dr John Diffley at the Clare Hall Laboratories of the Cancer Research UK London Research Institute (now part of the Francis Crick Institute). Here, Christian studied how double-stranded DNA breaks are processed and activate DNA damage response mechanisms within cells.
Christian then went to New York, where he joined Prof Hironori Funabiki’s laboratory at Rockefeller University to carry out postdoctoral work. Christian first revealed that the packaging of DNA into chromatin is essential for the assembly of the nuclear envelope and the mitotic spindle.
Subsequently, he found that during normal cell growth, chromatin is also crucial for helping the intracellular innate immune system distinguish between self-DNA and the DNA of invading pathogens. At the same time, Christian’s work revealed that innate immune signalling from self-DNA, which can happen under stressed conditions, may be important for certain types of chemotherapy, such as treatment with taxanes.
Christian joined the Cancer Biology Division of the ICR in 2020 to continue his work on immune responses to self-DNA, and on their relevance for cancer.
Related pages
Types of Publications
Journal articles
Nucleosomes are the fundamental unit of chromatin, but analysis of transcription-independent nucleosome functions has been complicated by the gene-expression changes resulting from histone manipulation. Here we solve this dilemma by developing Xenopus laevis egg extracts deficient for nucleosome formation and by analyzing the proteomic landscape and behavior of nucleosomal chromatin and nucleosome-free DNA. We show that although nucleosome-free DNA can recruit nuclear-envelope membranes, nucleosomes are required for spindle assembly and for formation of the lamina and of nuclear pore complexes (NPCs). We show that, in addition to the Ran G-nucleotide exchange factor RCC1, ELYS, the initiator of NPC formation, fails to associate with naked DNA but directly binds histone H2A-H2B dimers and nucleosomes. Tethering ELYS and RCC1 to DNA bypasses the requirement for nucleosomes in NPC formation in a synergistic manner. Thus, the minimal essential function of nucleosomes in NPC formation is to recruit RCC1 and ELYS.
Chromosomes are not only carriers of the genetic material, but also actively regulate the assembly of complex intracellular architectures. During mitosis, chromosome-induced microtubule polymerisation ensures spindle assembly in cells without centrosomes and plays a supportive role in centrosome-containing cells. Chromosomal signals also mediate post-mitotic nuclear envelope (NE) re-formation. Recent studies using novel approaches to manipulate histones in oocytes, where functions can be analysed in the absence of transcription, have established that nucleosomes, but not DNA alone, mediate the chromosomal regulation of spindle assembly and NE formation. Both processes require the generation of RanGTP by RCC1 recruited to nucleosomes but nucleosomes also acquire cell cycle stage specific regulators, Aurora B in mitosis and ELYS, the initiator of nuclear pore complex assembly, at mitotic exit. Here, we review the mechanisms by which nucleosomes control assembly and functions of the spindle and the NE, and discuss their implications for genome maintenance.
During DNA double-strand break (DSB) repair, the ring-shaped Ku70/80 complex becomes trapped on DNA and needs to be actively extracted, but it has remained unclear what provides the required energy. By means of reconstitution of DSB repair on beads, we demonstrate here that DNA-locked Ku rings are released by the AAA-ATPase p97. To achieve this, p97 requires ATP hydrolysis, cooperates with the Ufd1-Npl4 ubiquitin-adaptor complex, and specifically targets Ku80 that is modified by K48-linked ubiquitin chains. In U2OS cells, chemical inhibition of p97 or siRNA-mediated depletion of p97 or its adapters impairs Ku80 removal after non-homologous end joining of DSBs. Moreover, this inhibition attenuates early steps in homologous recombination, consistent with p97-driven Ku release also affecting repair pathway choice. Thus, our data answer a central question regarding regulation of Ku in DSB repair and illustrate the ability of p97 to segregate even tightly bound protein complexes for release from DNA.
Pathogenic and other cytoplasmic DNAs activate the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway to induce inflammation via transcriptional activation by IRF3 and nuclear factor κB (NF-κB), but the functional consequences of exposing cGAS to chromosomes upon mitotic nuclear envelope breakdown are unknown. Here, we show that nucleosomes competitively inhibit DNA-dependent cGAS activation and that the cGAS-STING pathway is not effectively activated during normal mitosis. However, during mitotic arrest, low level cGAS-dependent IRF3 phosphorylation slowly accumulates without triggering inflammation. Phosphorylated IRF3, independently of its DNA-binding domain, stimulates apoptosis through alleviating Bcl-xL-dependent suppression of mitochondrial outer membrane permeabilization. We propose that slow accumulation of phosphorylated IRF3, normally not sufficient for inducing inflammation, can trigger transcription-independent induction of apoptosis upon mitotic aberrations. Accordingly, expression of cGAS and IRF3 in cancer cells makes mouse xenograft tumors responsive to the anti-mitotic agent Taxol. The Cancer Genome Atlas (TCGA) datasets for non-small cell lung cancer patients also suggest an effect of cGAS expression on taxane response.
DNA double strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HR). HR requires nucleolytic degradation of 5' DNA ends to generate tracts of single-stranded DNA (ssDNA), which are also important for the activation of DNA damage checkpoints. Here we describe a quantitative analysis of DSB processing in the budding yeast Saccharomyces cerevisiae. We show that resection of an HO endonuclease-induced DSB is less extensive than previously estimated and provide evidence for significant instability of the 3' ssDNA tails. We show that both DSB resection and checkpoint activation are dose-dependent, especially during the G1 phase of the cell cycle. During G1, processing near the break is inhibited by competition with NHEJ, but extensive resection is regulated by an NHEJ-independent mechanism. DSB processing and checkpoint activation are more efficient in G2/M than in G1 phase, but are most efficient at breaks encountered by DNA replication forks during S phase. Our findings identify unexpected complexity of DSB processing and its regulation, and provide a framework for further mechanistic insights.
<h4>Background</h4>While double-strand break (DSB) repair is vital to the survival of cells during both meiosis and mitosis, the preferred mechanism of repair differs drastically between the two types of cell cycle. Thus, during meiosis, it is the homologous chromosome rather than the sister chromatid that is used as a repair template.<h4>Results</h4>Cells attempting to undergo meiosis in the absence of Mnd1 arrest in prophase I due to the activation of the Mec1 DNA-damage checkpoint accumulating hyperresected DSBs and aberrant synapsis. Sporulation of mnd1Delta strains can be restored by deleting RED1 or HOP1, which permits repair of DSBs by using the sister chromatid as a repair template. Mnd1 localizes to chromatin as foci independently of DSB formation, axial element (AE) formation, and synaptonemal complex (SC) formation and does not colocalize with Rad51. Mnd1 does not preferentially associate with hotspots of recombination.<h4>Conclusions</h4>Our results suggest that Mnd1 acts specifically to promote DSB repair by using the homologous chromosome as a repair template. The presence of Rec8, Red1, or Hop1 renders Mnd1 indispensable for DNA repair, presumably through the establishment of interhomolog (IH) bias. Localization studies suggest that Mnd1 carries out this function without being specifically recruited to the sites of DNA repair. We propose a model in which Mnd1 facilitates chromatin accessibility, which is required to allow strand invasion in meiotic chromatin.
A hallmark of mitosis is the appearance of high levels of histone phosphorylation, yet the roles of these modifications remain largely unknown. Here, we demonstrate that histone H3 phosphorylated at threonine 3 is directly recognized by an evolutionarily conserved binding pocket in the BIR domain of Survivin, which is a member of the chromosomal passenger complex (CPC). This binding mediates recruitment of the CPC to chromosomes and the resulting activation of its kinase subunit Aurora B. Consistently, modulation of the kinase activity of Haspin, which phosphorylates H3T3, leads to defects in the Aurora B-dependent processes of spindle assembly and inhibition of nuclear reformation. These findings establish a direct cellular role for mitotic histone H3T3 phosphorylation, which is read and translated by the CPC to ensure accurate cell division.
Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a major responder to the pathogenic DNA of viruses and bacteria. Upon DNA binding, cGAS becomes enzymatically active to generate the second messenger cGAMP, leading to activation of inflammatory genes, type I interferon production, autophagy, and cell death. Following genotoxic stress, cGAS can also respond to endogenous DNA, deriving from mitochondria, endogenous retroelements, and chromosomes to affect cellular signaling, secretion, and cell fate decisions. However, under unperturbed conditions, signaling from self-DNA is largely, but not completely, inhibited. Here we review how endogenous DNA is exposed to cGAS, how signaling is attenuated but activated under pathological conditions, and how low-level signaling under unperturbed conditions might prime antipathogenic responses.
The majority of eukaryotic chromosomal DNA exists in the form of nucleosomes, where ∼147 bp DNA wraps around histone hetero-octamers, composed of histone H3, H4, H2A, and H2B. Despite their obvious importance in DNA compaction and accessibility, studying their specific roles, such as regulation of mitotic progression, in a physiological environment is associated with critical caveats because of their major contributions in transcriptional control. Through establishing a method to deplete endogenous histones H3 and H4 from frog egg extracts and complementing their functions using recombinant nucleosome arrays, we are now able to analyze their roles in mitotic progression without affecting overall transcriptomic profiles. Here we summarize advancements learned from this system, illustrating that microtubule and nuclear envelope assembly can be regulated by two major nucleosome-bound protein complexes, RCC1-Ran and the chromosomal passenger complex (CPC) containing the mitotic protein kinase Aurora B. We also discuss roles of the CPC on the proteomic composition of mitotic chromatin. The CPC promotes dissociation of a variety of nucleosome remodelers and DNA repair pathway proteins, suggesting its role in suppressing DNA processing activities on mitotic chromosomes. We speculate that this suppression particularly on chromosomes under microtubule tension may be important to preserve genome integrity.
The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) senses invasion of pathogenic DNA and stimulates inflammatory signaling, autophagy, and apoptosis. Organization of host DNA into nucleosomes was proposed to limit cGAS autoinduction, but the underlying mechanism was unknown. Here, we report the structural basis for this inhibition. In the cryo-electron microscopy structure of the human cGAS-nucleosome core particle (NCP) complex, two cGAS monomers bridge two NCPs by binding the acidic patch of the histone H2A-H2B dimer and nucleosomal DNA. In this configuration, all three known cGAS DNA binding sites, required for cGAS activation, are repurposed or become inaccessible, and cGAS dimerization, another prerequisite for activation, is inhibited. Mutating key residues linking cGAS and the acidic patch alleviates nucleosomal inhibition. This study establishes a structural framework for why cGAS is silenced on chromatinized self-DNA.
The DNA sensor cyclic GMP-AMP synthase (cGAS) is critical in host antiviral immunity. Vaccinia virus (VACV) is a large cytoplasmic DNA virus that belongs to the poxvirus family. How vaccinia virus antagonizes the cGAS-mediated cytosolic DNA-sensing pathway is not well understood. In this study, we screened 80 vaccinia genes to identify potential viral inhibitors of the cGAS/Stimulator of interferon gene (STING) pathway. We discovered that vaccinia E5 is a virulence factor and a major inhibitor of cGAS. E5 is responsible for abolishing cGAMP production during vaccinia virus (Western Reserve strain) infection of dendritic cells. E5 localizes to the cytoplasm and nucleus of infected cells. Cytosolic E5 triggers ubiquitination of cGAS and proteasome-dependent degradation via interacting with cGAS. Deleting the E5R gene from the Modified vaccinia virus Ankara (MVA) genome strongly induces type I IFN production by dendritic cells (DCs) and promotes DC maturation, and thereby improves antigen-specific T cell responses.
Genome instability due to the loss of DNA repair factors can drive developmental defects, autoinflammatory disease, and cancer. Two major signaling pathways are activated by genome instability-DNA damage checkpoint signaling, leading to p53 activation, and innate immunity, largely driven by the DNA sensor cGAS. Like p53, cGAS is thought to drive cell death and senescence during genotoxic stress in addition to its canonical inflammatory functions, but its role during cellular differentiation and carcinogenesis is poorly understood. Furthermore, it is heavily debated whether the cGAS pathway primarily has tumor-suppressive or oncogenic functions. A study in this issue of Cancer Research used a hematopoietic lineage-specific knockout of the ribonucleotide repair gene Rnaseh2b to introduce genotoxic stress, resulting in severe hematopoiesis defects and increased incidence of hematologic cancers. These two effects were driven by and antagonized by p53, respectively. Surprisingly, despite increased innate immune signaling, the cGAS pathway did not seem to play a role in either process. These findings suggest that innate immune responses to genotoxic stress may be more subtle and context-specific than appreciated, indicating that a more detailed understanding of pathway activation and signaling consequences is needed. See related article by Dressel et al., p. 2858.
The major innate immune responder to the DNA of pathogens is the cyclic GMP-AMP (cGAMP) synthase (cGAS) - stimulator of interferon genes (STING) pathway. Most prominently, the outcome of cGAS signalling is the activation of inflammatory transcription through interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-kB). In addition, the cGAS-STING pathway can lead to the direct modulation of cellular processes independently of transcription, such as activation of autophagy. Under unperturbed conditions, several mechanisms are in place to prevent the activation of cGAS by self-DNA, chiefly its sequestration on chromatin, which interferes with binding to stimulatory DNA. However, under conditions of genotoxic stress and chromosomal instability, this inhibition breaks down, resulting in the activation of cGAS, which drives sterile inflammation, as well as cell fate and immune responses in cancer. Recently, several studies have suggested that cGAS, STING, or downstream pathway components can also regulate the DNA damage response, DNA damage checkpoint signalling, DNA repair and DNA replication. Here, I review these proposed mechanisms, and discuss some unanswered questions relating to them.