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PRCTOERC Résumé de rapport

Project ID: 639253
Financé au titre de: H2020-EU.1.1.

Periodic Reporting for period 1 - PRCTOERC (Novel Regulatory Principles of Polycomb Repressive Complex 2)

Reporting period: 2015-06-01 à 2016-11-30

Summary of the context and overall objectives of the project

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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Work on the action commenced June 1, 2015. The start date of the Action was 6 months after the PI took up his first independent post at the hosting institution. Given the close timeline, the first few months of the Action were invested in preparatory work and completing the set up of the lab as well as initial generation of constructs for Work Package (WP) 1 with the help of the lab’s research assistant (funded through other sources).
A first PhD student started her work on WP1 in September 2015. After an initial training phase, we made good progress on the aims of WP1.1-WP1.3. Her work on WP1 is largely moving as planned, however, alternative approaches for aspects of WP1.1 and WP1.3 had to be developed due to issues with the original approaches. It was initially planned to employ methyl-and acetyl-lysine analogs for the generation of recombinant nucleosomes carrying specific modifications needed for WP1.1; however, results from on-going work in the laboratory indicated that histones carrying the proper modifications rather than analogs needed to be created by native chemical ligation in order for binding proteins to be identified. Setting up the required techniques incurred some delays, but the approach is now set up and has proven superior to the lysine analog approach. Moreover, it was initially planned to use EED-/- cell lines to study the role of PRC2 in promoter-enhancer interactions. However, recent work in the field suggested that these cell lines have undergone significant adaptive processes since their generation, so that we resorted to generating a distinct cell line that achieves knockout of the PRC2 complex by CRISPR-based approaches. These cell lines are currently being characterised for differentiation defects, and assays on chromatin conformation will be initiated shortly.
Preparatory work has been undertaken to address the aims of Work Package 2, which is expected to gain significant traction in due course with the addition of further researchers to the team. A second PhD student started his work in February 2016. His work on WP3 has so far focused on WP3.3. For this technically challenging aim, we are optimising the approach originally proposed to purify CpG islands and to characterise their proteome. He has generated constructs and expressed bait proteins to purify CpG islands and is exploring approaches to generate chromatin preparations suitable for CpG island purification and analysis of their proteome. In addition to experiments based on the approach originally outlined in the Action, alternative approaches are currently being explored as well in order to identify the most promising approach to purify CpG islands from endogenous chromatin.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

So far, this initial phase of the project has been mostly characterised by preparatory work and generation of essential tools. These are expected to be successfully employed in the second reporting period. We expect our work to reveal important novel facets of PRC2 regulation and its interplay with other chromatin modifying enzymes. 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.