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Novel Regulatory Principles of Polycomb Repressive Complex 2

Periodic Reporting for period 4 - PRCTOERC (Novel Regulatory Principles of Polycomb Repressive Complex 2)

Reporting period: 2019-12-01 to 2021-11-30

Posttranslational modifications of histone proteins have emerged as central regulators of gene expression. Through the factors that install, interpret, and erase them, histone marks control access to the genome, establishing chromatin environments that either support or counteract transcription. The histone methyltransferase Polycomb repressive complex 2 (PRC2) is crucially involved in gene repression all throughout development and adulthood, and it is often misregulated in cancer. Despite significant advances in the field, key aspects of PRC2 function remain largely elusive. The overarching goal of this project is to enhance our understanding of how PRC2 is regulated and how it controls the expression of developmental genes in embryonic stem cells. To this end, my research team and I will analyse how PRC2 cooperates with other histone modifiers and chromatin organisers at enhancers to achieve poising of developmental genes (Work Package 1). These studies will enable us to appreciate how the pivotal PRC2 module interfaces with other players in a complex system of chromatin regulators, contributing to a much-needed integrated view of chromatin regulation. We will further unravel how generation of the recently discovered asymmetric nucleosomes, in which the two copies of histone H3 are disparately modified (Voigt et al., Cell, 2012), is controlled by PRC2-intrinsic catalytic properties and through interactions with other chromatin modifiers (Work Package 2). This will ultimately allow modulating asymmetry in vivo, providing unprecedented means to assess its impact on PRC2 function and chromatin structure. Lastly, we aim to re-evaluate the issue of PRC2 recruitment to its target loci by employing a systems biology-informed quantitative approach (Work Package 3). Together, the aims of this ambitious project will significantly advance our understanding of PRC2 and its role in the establishment of chromatin states, which are crucial to embryonic stem cell physiology and deregulated in cancer.
After an initial setup phase, we have made good progress on several work packages of the Action. We have established methodology to perform nucleosome pulldowns with site-specifically modified histones and are currently setting up pulldown experiments that will determine how asymmetric nucleosome modification found at enhancers controls recruitment of binding proteins to these sites. In addition, we have used such nucleosome templates to perform enzymatic assays with modifying enzymes that are active at enhancers, uncovering novel interactions between these activities. These are currently being characterized further both in vitro and in vivo. To complement these studies, we are setting up an approach to determine the enhancer proteome and have made significant progress on that.

PRC2 has been shown to be involved in interactions between both enhancers and bivalent promoters in embryonic stem cells (ESCs). We have set up ESC cell lines harboring several mutants of PRC2 in order to reveal the mechanistic details of PRC2's role in these processes. This work indicates novel roles for PRC2, which are currently being characterized further.

In order to clarify the action of CpG islands in controlling expression of developmental genes in ESCs, we have been setting up an approach to purify these genomic elements and determine the proteome of these sites. This ambitious goal has so far not been met, but methodologies have been established that will make such purifications feasible within the course of the Action. To complement these approaches, we have devised assays to determine the impact of CpG islands on chromatin state and gene expression in ESCs using. The required cell lines have been generated and their characterization is underway.
Over this initial phase of the project, we have established several novel methodologies to elucidate the mechanisms of chromatin modifying enzymes. We have set up native chemical ligation-based approaches to generate recombinant chromatin templates carrying specific histone modifications. These are being used to determine binding proteins for chromatin states found at enhancers. Moreover, these recombinant chromatin templates allow to study how certain histone modifications affect the activity of other histone modifying enzymes. Using these approaches, we have already uncovered novel interactions and feedback loops between histone modifications that so far have been considered as being independent in their placement on chromatin. We expect our work to uncover further such interactions, providing mechanistic insight into how chromatin states are set up at important regulatory sites in mammalian genomes. This work might ultimately pave the way to novel treatment strategies for conditions that involve deregulation of epigenetic activities, such as certain types of cancer.

We also established CRISPR-based approaches to study the function of PRC2 and its histone modification H3K27me3. With so-called CRISPRi tools to site-specifically add or remove histone marks we are determining the effect of this modification on the action of enhancers during neuronal differentiation. Moreover, we are studying novel roles of PRC2 in chromatin organization. We expect our work to reveal novel interactions of PRC2 with other chromatin modifiers as well as further insight into its recruitment to specific sites on chromatin. This work will be pivotal to understanding the role of PRC2 in embryonic stem cell differentiation, informing strategies for regenerative medicine as well as for the treatment of cancer.