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，这些细胞类型必须 保持他们独特的身份。多梳基团的蛋白质和络合物对染色质的化学修饰 在生理上，并需要在不适当的细胞类型中保持对谱系特异性基因的抑制。 多梳抑制复合体2(PRC2)解释核的转录和表观遗传状态 和三甲基化组蛋白H3的27位赖氨酸(H3K27me3)，这强加了染色质为基础的沉默。自我- H3K27me3的繁殖特性一次解释了这些沉默的染色质结构域的表观遗传维持 从而维持适当的细胞特性；然而，不同的基因 在不同的谱系中被PRC2沉默，这表明在发育的关键时刻，PRC2必须 能够选择要抑制的新基因。 这项建议的目标是破译控制新的PRC2建立的分子逻辑- 在发育过程中抑制染色质结构域。具体地说，我们将测试两个PRC2的假设 复杂类型的PRC2.1和PRC2.2由于其不同的基因类型在发育过程中使不同的基因沉默 辅助蛋白亚基和相互作用的RNA。我们将通过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可诱导蛋白质降解系统来识别PRC2.1的作用 和PRC2.2处于早期发展的两个关键阶段，从基础到启动的多能性过渡和 对神经谱系的承诺。我们将诱导定义这两个辅助亚基的降解 复杂类型和分析不同时间点的分子和功能表型 胚胎干细胞向神经前体细胞的分化。蛋白质降解的可逆性 系统将允许我们在分化期间或之后恢复复合体，并确定准确的时刻 它们的功能是必需的。 在目标2中，我们将继续我们最近发表的确定多个RNA结合蛋白质表面的工作 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不同亚基结合的RNA。 接下来，我们将利用可诱导的蛋白质降解，然后用RNA结合突变体进行救援来确定 RNA相互作用如何促进PRC2.1和PRC2.2在染色质上的招募和功能。 拟议中的研究将提供对新沉默形成的分子机制的洞察 PRC2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 正常发育过程及其在疾病中的失调。

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.