Repressive Heterochromatin Establishment by Polycomb Complexes

Project Summary The goal of this proposal is to dissect the distinct molecular roles of Polycomb Repressive Complexes 1 and 2 (PRC1&PRC2) and upstream factors in establishing silencing at developmentally regulated genes. The PRCs are conserved chromatin modifiers that represses transcription via chemical and structural alterations of chromatin architecture. PRC1 mutations

cause neurodevelopmental disorders characterized by microcephaly, intellectual disabilities, and dysmorphic body features, while mutations in PRC2 are the main cause of Weaver syndrome making it crucial to gain understanding about polycomb repression during early development. Despite ubiquitous expression, PRC1 and PRC2 can target distinct sets of genes

for silencing in different cell lineages, resulting in cell-type specific expression. Though there are decades of research on Polycomb protein function, a key unanswered question is how polycomb repressive complexes select different target genes as cells differentiate into different cell types. While we understand the interplay of PRC1 and PRC2 during the maintenance of

polycomb silencing, we do not understand the order of events in the establishment of new polycomb domains at de novo silenced genes. Others in the field have shown that correct PRC2 localization can be re-established de novo in mouse embryonic stem cells (mESCs) after ablation, indicating that there is information

upstream of PRC2 dictating localization. Additionally, my preliminary data from a genome wide knock out screen implicates several factors of interest in regulating polycomb establishment including RNA binding protein ERH. I will investigate the regulatory role of PRC1 and these additional factors identified by the screen in PRC2 localization during the establishment of

polycomb domains in mESCs with the following specific aims. In Aim 1, I will utilize acute protein degradation to investigate PRC1’s ability to re-establish correct localization on chromatin after depletion. Using a similar strategy with an orthogonal dual-degron line, I will also determine if PRC1 is necessary for correct PRC2 localization during

establishment. In Aim 2, I will investigate the functional roles of candidates I identified in my genome wide KO screen during PRC2’s re-establishment on chromatin in mESCs, leveraging my lab’s expertise in designing acute protein degradation lines. The experiments in this proposal will elucidate the

order of events and factors involved in the establishment of silent polycomb domains. To achieve these aims, I have developed with my sponsor a rigorous and comprehensive training program with three primary goals: 1) become an expert in epigenomics methods, 2) sharpen my scientific communication skills to broad audiences, 3) increase proficiency in

bioinformatics and data analysis. I am confident that my choice of sponsor combined with my diverse training background and the collaborative nature of my training environment will enable me to achieve my goals and the proposed research plan simultaneously.