Regulation of PRC2 by protein and RNA interactions during differentiation

分化过程中蛋白质和 RNA 相互作用对 PRC2 的调节

基本信息

批准号：
10031001
负责人：
Roberto Bonasio
金额：
$ 32.4万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-15 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10031001
关键词：
ART protein Acute Address Affect Auxins Binding Binding Sites Biochemical Biological Cell Differentiation process Cell Maintenance Cell Nucleus Cells Chemicals Chromatin Chromatin Remodeling Factor Complex Data Development Disease Embryo Embryonic Lethal Mutation Epigenetic Process Gatekeeping Gene Silencing Gene Targeting Genes Genetic Genetic Transcription Genome Goals Heterochromatin Histone H3 Histones Human Human Genome Investigation Knock-out Logic Lysine Maintenance Malignant Neoplasms Maps Mediating Membrane Proteins Molecular Mus Mutation Nature Pathway interactions Phenotype Play Polycomb Process Protein Subunits Proteins Publishing RNA RNA Binding RNA Sequences RNA-Binding Proteins RNA-Protein Interaction Regulation Repression Role Set protein Shapes Specific qualifier value Specificity System Techniques Testing Time Tissues Weaver Syndrome Work base cell type design developmental disease embryonic stem cell experimental study follow-up functional restoration in vivo insight interest mutant nerve stem cell new technology pluripotency prevent protein complex protein degradation recruit relating to nervous system

项目摘要

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.

抽象的在发育过程中，单个人类基因组会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。 Polycomb 组的蛋白质和复合物对染色质进行化学修饰在身体上和身体上，都需要在不适当的细胞类型中维持谱系特异性基因的抑制。 Polycomb 抑制复合物 2 (PRC2) 解释细胞核的转录和表观遗传状态并使组蛋白 H3 在赖氨酸 27 (H3K27me3) 处三甲基化，从而产生基于染色质的沉默。自我 H3K27me3 的繁殖性质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 PRC2在不同谱系中被沉默，这表明在发育的关键时刻，PRC2必须被抑制能够选择要抑制的新基因。该提案的目标是破译控制新 PRC2- 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，我们将检验两个 PRC2 的假设：复杂类型，PRC2.1 和 PRC2.2 在发育过程中沉默不同的基因，因为它们具有不同的特性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诱导蛋白降解系统来辨别 PRC2.1 的作用和 PRC2.2 在早期发育的两个关键步骤，即从地面多能性到初发多能性的转变和对神经谱系的承诺。我们将诱导定义两个的辅助亚基的降解复杂类型并分析定向期间不同时间点的分子和功能表型胚胎干细胞分化为神经祖细胞。蛋白质降解的可逆性系统将允许我们在分化过程中或分化后恢复复合物，并确定准确的时刻它们的功能是必需的。在目标 2 中，我们将跟进最近发表的鉴定多个 RNA 结合蛋白表面的工作在 PRC2.1 和 PRC2.2 上进行分析，并使用此信息来设计功能分离的 RNA 结合突变体。通过这些突变体，我们将鉴定与 PRC2.1 和 PRC2.2 不同亚基结合的 RNA。接下来，我们将利用诱导蛋白降解，然后用 RNA 结合突变体进行救援，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。拟议的研究将提供对支持创造新沉默的分子机制的见解。 PRC2 在分化过程中对染色质区域的影响，对我们对表观遗传的理解具有广泛的影响正常发育期间的过程及其在疾病中的失调。