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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 do 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 are 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.
Either PRC1 or PRC2 can be classified as multiple different types of complexes, each includes different protein subunits of different properties. Some types of PRC1 can trigger the recruitment of certain PRC1 complexes and vice versa. Yet, very little is known about how these processes are taking place at the molecular level and, especially, how these complexes discriminate target- from non-target genes. Subunits of both the mammalian PRC1 and PRC2 include various paralogous proteins and isoforms, although little is known to what extent they are redundant or allow for functional diversification.
It is now clear that polycomb‐mediated epigenetic repression involves protein accessory factors, DNA sequence elements, nucleosomes carrying specific epigenetic marks and RNAs, including ncRNA and mRNA. It is far from being understood 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. 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. Our previous work into this researches them including identifying how PRC2 is interacting with RNA (Zhang et al. 2019) and the molecular basis for the regulation of PRC2 by its accessory subunit PALI1 (Zhang et al. 2021).
How are chromatin-modifying factors regulated by lncRNAs and mRNA 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 is associated with hundreds to thousands of RNA transcripts, including lncRNAs and mRNA, in both mouse and human cells. Some evidence implies that PRC1 is also subjected to RNA-mediated regulation, although the extent of this phenomenon and the molecular mechanism has not been studied in depth. 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 site within the regulatory centre of PRC2 (Zhang et al. 2019). From a technical point of view, while working on this project we also develop tools for the identification of protein-RNA interactions using crosslinking mass spectrometry (Gail et al. 2020). We aim to understand how RNA regulates PRCs at the molecular level.
How do polycomb-repressive complexes are dysregulated in diseases?
The involvement of polycomb-repressive complexes in cancer and congenital disorders has been widely observed. In healthy cells and during normal development, PRCs repress oncogenes and genes coding for cell-type-specific transcription factors. Accordingly, PRCs are among the most frequently mutated epigenetic modifiers in various types of cancers. Several cancer-associated mutations in PRCs already identified as drivers, while most other mutations are waiting to be explored mechanistically. This is an active area of research with a strong potential for targeted therapy, given the recent success in the introduction of PRC2 (EZH2) inhibitors for clinical usage in cancer.
There is now ample information about the composition of PRCs in different pathologies, including the expression level of their different subunits and the mutations they are harbouring. Yet, less is known about how the function of PRCs is dysregulated as a result of mutations in their subunits, post-translational modifications, fusions with other protein factors that take place in some types of tumours, or the overexpression or under-representation of certain subunits or their different isoforms. Understanding the molecular mechanism for the dysregulation of PRCs in diseases would foster the development of strategies for molecular diagnostics, targeted therapies and drug development. We are developing tools to understand the molecular mechanism dictating the regulation and dysregulation of PRCs. To overcome the complexity of PRCs and the various mechanisms for their dysregulation in diseases, this project is relaid on a combination of mammalian cell culture systems complemented with model organisms, synthetic biology approaches and in vitro studies using reconstituted chromatin and reconstituted holo-PRCs and other chromatin-modifying complexes.