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How genes are turned off and then maintained in a repressed state throughout countless cell divisions? During embryonic development, or in disease, large sets of lineage-specific genes become transcriptionally inactive and spatially compacted. The repressed-state of these genes is then maintained, allowing for the maintenance of cell identity. In all multicellular organisms, these processes are dependent on the histone modifier polycomb repressive complexes (PRCs). The enzymatic activity and the recruitment of PRCs to chromatin are regulated by sub-stoichiometric protein co-factors — accessory subunits. Mechanistic studies into how PRCs are regulated by their accessory subunits are challenging, given their redundancy in vivo. Proteomic studies indicate that different types of PRCs do not interact externally to the context of chromatin, but biochemical and cell-based assays imply for cooperation between them. How chromatin-bound histone modifiers work together to facilitate gene repression? This fundamental question remained open, challenged by redundancy between subunits of modifier complexes in vivo and difficulties in the purification and studies of fully assembled holo-enzymatic complexes in vitro.
The Davidovich lab aims to discover how different chromatin-modifying complexes work together and how they utilise their various accessory subunits to maintain the repressed state of genes. We seek to understand, down to atomic resolution, how the functions of polycomb-group proteins function are modulated by their environment and their various binding partners. To do this, we are combining next-generation sequencing‐based techniques with molecular biology and biochemical approaches, in vitro and in vivo, for coherent functional study. We are further studying the structural basis for the function of chromatin-modifying complexes at low and high resolution using structural biology approaches, including X-ray crystallography, high-resolution cryo‐electron microscopy (cryo‐EM), electron cryotomography and crosslinking mass-spectrometry (XL-MS and RBDmap).
How Polycomb-mediated epigenetic repression takes place?
Most polycomb-group proteins assembled into two major types of enzymatic complexes: polycomb repressive complexes 1 and 2 (PRC1 and PRC2, respectively). PRC2 is a histone methyltransferase that mono‐, di‐ and tri‐methylates lysine 27 of histone H3 (H3K27me1, H3K27me2 and H3K27me3, respectively). PRC1 is a ubiquitin ligase that monoubiquitylates lysine 119 of histone H2A (H2AK119Ub). These histone modifications provide an epigenetic mark of repressed chromatin. PRC1 is preferentially recruited to nucleosomes that have been methylated by PRC2. PRC2 can be recruited to nucleosomes that were ubiquitylated by PRC1. Transcription shut-off of PcG target genes is also sufficient for the recruitment of PRC2. Yet, very little is known about how these processes are taking place at the molecular level in a gene-specific manner and to what extent the various paralogous protein subunits of either PRC1 or PRC2 are redundant or allow for functional diversification.
Polycomb‐mediated epigenetic repression has been suggested to involve protein accessory factors, DNA sequence elements, nucleosomes carrying specific epigenetic marks and long non‐coding RNAs (lncRNAs). How these factors work together to modulate the function of PRCs throughout development and in cancer in order to maintain the repressed state of genes is far from being understood. We are using cutting-edge genomic and proteomic approaches in mammalian cells, in combination with structural biology, biochemistry and biophysics in order to determine the molecular mechanism of gene-repression by the polycomb machinery.
How are chromatin-modifying factors regulated by lncRNAs and RNA transcripts?
Multiple chromatin-modifying factors were shown to be recruited to chromatin by interactions with lncRNAs. Among these chromatin modifiers, PRC2 is one of the most studied. Previous studies indicated that PRC2 associated with hundreds to thousands of RNA transcripts in both mouse and human cells. We provided evidence for promiscuous RNA-binding by PRC2 in vitro and in vivo (Davidovich et al. 2013, Davidovich et al. 2015) and showed that it is attributed to interactions with an abundant motif of multiple G-tracts and G-quadruplex-forming sequences (Wang et al. 2017). From the protein side, we mapped an RNA-binding sire within the regulatory center of PRC2 (Zhang et al. 2019). We aim to understand how RNA regulates PRC2 at the molecular level.
How does epigenetic derepression take place during development and in cancer?
The involvement of polycomb-repressive complexes in cancer has been widely observed, suggesting a function for PRC2 and PRC1 as tumour‐suppressors. During cancer development and progression, the balance between polycomb‐mediated repression and derepression is altered and lead to transcriptional activation of oncogenes. Yet, derepression of polycomb-target genes is poorly understood at the molecular level.
We are developing tools to understand the molecular mechanism dictating the derepression of polycomb‐target genes in mammalian cells. This will be an important step toward understanding how loss-of-function mutations in genes coding polycomb-group proteins contribute to cancer and growth disorders. We will determine how the processes of repression and derepression are coordinated to regulate gene expression and to reshape repressed chromatin domains. This project is relaid on a combination of cell culture systems complemented with in vitro studies using reconstituted chromatin and reconstituted holo-PRCs and other chromatin-modifying complexes.