Project Summary The objective of this proposal is to determine the distinct molecular roles and temporal requirements of the two subtypes of the Polycomb Repressive Complex 2 (PRC2), PRC2.1 and PRC2.2, during differentiation. PRC2 is an essential chromatin modifier that trimethylates lysine 27 on histone H3 (H3K27me3), which leads to the formation of facultative heterochromatin, a structure that is refractory to transcription and prevents ectopic gene activation. The epigenetic function of PRC2 is essential for embryogenesis, and defects in this complex cause Weaver Syndrome, a congenital multi-system developmental disorder. Despite its ubiquitous expression during development, PRC2 is capable of targeting distinct sets of genes for silencing in different cell lineages, resulting in cell-type specific gene repression. However, the mechanisms regulating the targeting of PRC2 are poorly understood. PRC2 interacts with a variety of accessory subunits, resulting in the formation of two biochemically distinct subcomplexes—PRC2.1 and PRC2.2. Genetic knockouts of subunits comprising PRC2.1 and PRC2.2 in mice result in embryonic lethality at different times and with different phenotypes, demonstrating that the two PRC2 subcomplexes have non-overlapping essential functions in development. Unlike PRC2, the accessory subunits comprising PRC2.1 and PRC2.2 exhibit dynamic expression patterns during development, suggesting they may regulate distinct developmental stages. However, the molecular roles that PRC2.1 and PRC2.2 may play at different stages of development, as well as whether they target different genomic regions for silencing, remain unknown. I will investigate these questions using auxin- inducible degradation (AID) to acutely degrade subunits specific to PRC2.1 and PRC2.2 during the differentiation of mouse embryonic stem cells (mESCs). Because of its rapid kinetics, this approach will allow me to define the immediate molecular consequences of removing the selected subunit, avoiding confounding secondary and compensatory effects that have plagued previous genetic approaches. In Aim 1 I will combine AID with nascent RNA sequencing to determine the functions of PRC2.1 and PRC2.2 in regulating the transcriptional state of pluripotent mESCs. In Aim 2 I will use AID to selectively degrade PRC2.1 and PRC2.2 subunits at different times during the differentiation of mESCs to epiblast-like cells (EpiLCs) and neuronal progenitors (NPCs). I will measure resulting changes in gene expression, chromatin structure, and cell morphology to determine the functions of and temporal requirements for each PRC2 subcomplex at different developmental stages. Collectively, these Aims will shed mechanistic light on the distinct molecular roles of PRC2.1 and PRC2.2 in regulating pluripotency and differentiation.