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.

Project Narrative The Polycomb Repressive Complexes 1 and 2 (PRC1&PRC2) are conserved epigenetic regulators that are required for proper development whose dysregulation is linked to abnormal embryogenesis, various cancers, and neurodevelopmental disorders. While we are beginning to understand the mechanisms behind how PRCs maintain silencing at developmentally regulated genes, we do not know how they establish silencing at previously active genes during differentiation. I will employ a novel dual-degron line to acutely degrade each complex to obtain mechanistic insight into the interplay of PRC1 and PRC2 during polycomb establishment as well as upstream interactions that regulate polycomb localization.