Regulation of PRC2 by protein and RNA interactions during differentiation

摘要 在发育过程中，单个人类基因组产生数百种分化的细胞类型， 保持他们独特的身份。Polycomb组的蛋白质和复合物化学修饰染色质 并且是在不适当的细胞类型中维持谱系特异性基因的抑制所必需的。 Polycomb抑制复合物2（PRC 2）解释了细胞核的转录和表观遗传状态 并使组蛋白H3在赖氨酸27处三甲基化（H3 K27 me 3），从而施加基于染色质的沉默。自我- H3 K27 me 3的繁殖特性解释了这些沉默染色质结构域的表观遗传维持， 建立，并因此维持适当的细胞身份;然而，不同的基因， 在不同的谱系中，PRC 2沉默，这表明在发育的关键时刻，PRC 2必须是 能够选择新的基因进行抑制。 这项提案的目标是破译控制新PRC 2基因建立的分子逻辑， 在发育过程中沉默染色质结构域。具体来说，我们将测试两个PRC 2 PRC2.1和PRC2.2在发育过程中沉默不同的基因，这是由于它们的不同 辅助蛋白亚基和相互作用的RNA。我们将以两个具体目标来检验这一假设。 在目标1中，我们将利用最先进的诱导蛋白降解系统来辨别PRC2.1的作用。 和PRC2.2在早期发展的两个关键步骤，从基础到引发的多能性的过渡， 对神经谱系的承诺。我们将诱导辅助亚基的降解， 复杂的类型，并分析分子和功能表型在不同的时间点，在指导 胚胎干细胞向神经元祖细胞的分化。蛋白质降解的可逆性 系统将允许我们在分化期间或分化之后恢复复合物，并确定分化的确切时刻。 其功能所需。 在目标2中，我们将继续我们最近发表的工作，确定了多种RNA结合蛋白表面 在PRC2.1和PRC2.2上，并使用此信息设计功能分离RNA结合 变种人利用这些突变体，我们将鉴定与PRC2.1和PRC2.2的不同亚基结合的RNA。 接下来，我们将利用诱导蛋白降解，然后用RNA结合突变体进行拯救，以确定 RNA相互作用如何促进PRC2.1和PRC2.2在染色质上的募集和功能。 拟议的研究将提供有关创建新沉默的分子机制的见解 PRC 2在分化过程中的染色质区域，对我们理解表观遗传学具有广泛的意义。 在正常发育过程中的过程和疾病中的失调。

ABSTRACT During development, a single human genome gives rise to hundreds of differentiated cell types that must maintain their distinct identities. Proteins and complexes of the Polycomb group modify chromatin chemically and physically and are required to maintain repression of lineage-specific genes in inappropriate cell types. The Polycomb repressive complex 2 (PRC2) interprets the transcriptional and epigenetic state of the nucleus and trimethylates histone H3 at lysine 27 (H3K27me3), which imposes chromatin-based silencing. The self- propagating nature of H3K27me3 explains the epigenetic maintenance of these silent chromatin domains once established, and therefore the maintenance of appropriate cell identities; however, different genes are silenced by PRC2 in different lineages, suggesting that at critical junctures in development, PRC2 must be able to select new genes to be repressed. The goal of this proposal is to decipher the molecular logic that controls the establishment of new PRC2- silenced chromatin domains during development. Specifically, we will test the hypothesis that the two PRC2 complex types, PRC2.1 and PRC2.2 silence different genes during development due to their distinct accessory protein subunits and interacting RNAs. We will test this hypothesis with two specific aims. In Aim 1, we will utilize a state-of-the art inducible protein degradation system to discern the roles of PRC2.1 and PRC2.2 at two critical steps of early development, the transition from ground to primed pluripotency and the commitment to the neural lineage. We will induce degradation of accessory subunits that define the two complex types and analyze molecular and functional phenotypes at different time points during the directed differentiation of embryonic stem cells into neuronal progenitors. The reversibility of the protein degradation system will allow us to restore the complexes during or after differentiation and determine the exact moment at which their function is required. In Aim 2, we will follow up on our recently published work that identified multiple RNA-binding protein surfaces on both PRC2.1 and PRC2.2 and use this information to design separation-of-function RNA-binding mutants. With these mutants, we will identify RNAs bound to the different subunits of PRC2.1 and PRC2.2. Next, we will utilize inducible protein degradation followed by rescue with RNA-binding mutants to determine how RNA interactions contribute to PRC2.1 and PRC2.2 recruitment and function on chromatin. The proposed studies will provide insight on the molecular mechanisms that underpin the creation of new silent chromatin regions by PRC2 during differentiation, with broad implications for our understanding of epigenetic processes during normal development and their dysregulation in disease.