Vannini Group

The below outlines what the lab is currently focussing on.

Yeast RNA Polymerase III architecture:

We recently obtained the cryo-EM structure of yeast Pol III pre-initiation complex comprising TFIIIB, Pol III and the promoter sequence (Abascal-Palacios et al., Nature, 2018). Using state-of-the-art cryo-electron microscopy (cryo-EM) we obtained reconstructions of Pol III PIC and demonstrated the function of TFIIIB in the rearrangement of Pol III-specific subunits C34 and C37. Our study rationalised the mechanisms leading to DNA strand separation and template-strand loading into the active site and shed light into the general mechanism of gene transcription initiation. Likewise, using structural and biochemical approaches we have analysed the role of the TFIIIC transcription factor in the initial recruitment of TFIIIB to DNA promoters.

The stable association of these transcription factors to the DNA provides a platform for the further recruitment of RNA Pol III. Additionally, these assemblies are relevant for other cellular processes such the correct positioning of nucleosomes close to RNA Pol III-transcribed genes or the specific integration of Ty3 retro-transposons upstream of the transcription start site (TSS). These and other processes depend in the direct interaction of specific factors with components of TFIIIB and TFIIIC machineries.

RNA Polymerase III at type 3 promotors:

We previously demonstrated a redox control of Pol III transcription at the type 3 promoters providing a mechanistic link between Pol III deregulation and cancer (Gouge et al, 2015, Cell). We are now seeking to decipher how the promoter architecture contributes to human Pol III transcription at the type 3 promoters using structural and in vitro techniques.

TFIIIC in genome organisation:

In addition to acting as a transcription factor, TFIIIC has a potential role in genome organisation, with chromatin capture experiments localising extra TFIIIC DNA binding sites at the boundary regions of topological associating DNA domains (TADs). TFIIIC is enriched with and thought to directly interact with known genome structural protein, Condensin II at these sites. To further investigate this interaction, we are characterising human Condensin complexes using integrative approaches.

Eukaryotic transcription relies on three different RNA polymerases: RNA polymerase I (Pol) transcribes ribosomal RNA, RNA Polymerase II synthesizes messenger RNAs and RNA polymerase III produces short and non-translated RNAs, including the entire pool of tRNAs, which are essential for cell growth.

For a long time, it was assumed that only Pol II was regulated whereas Pol I and Pol III, being devoted to house-keeping genes, did not require such control. However, growing evidence show that RNA Polymerase III transcription is tightly regulated. Deregulation of its recruitment has been linked to neurodegenerative diseases and cancer.

RNA Pol III is recruited at only 3 types of promoters. While the type 1 and 2 are conserved from yeast to human, the type 3 promoters are found solely in higher eukaryotes. At the type 1 and 2, TFIIIC binds the promoter sequence to recruit TFIIIB that places the polymerase at the transcriptional start site. In humans, several tumour suppressors proteins and oncogenes interact directly with the transcription factor TFIIIB and, as a consequence, modulate RNA polymerase III occupancy at target genes. During carcinogenesis, this layer of regulation is lost, resulting in an augmented RNA polymerase III transcriptional output. Our research is aimed at mechanistically understanding the role of RNA polymerase III deregulation in cancer.

It is becoming increasingly clear that Pol III (and its associated factors) play a paramount role into genome structure and organisation. These extratranscriptional roles are effected through interactions with transposon machineries, SMC complexes and specific chromatin remodellers; we are aiming to obtain a detailed mechanistic understanding of these fundamental processes.