Biophysical interrogation of the RNA-mediated mechanisms regulating Polycomb Repressive Complex 2 activity

对调节 Polycomb 抑制复合物 2 活性的 RNA 介导机制的生物物理研究

基本信息

批准号：
10535223
负责人：
Wayne Oliver Hemphill
金额：
$ 6.72万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10535223
关键词：
Binding Biophysics Cell Differentiation process Chromatin Complex DNA Data Deposition Development Dissociation Embryo Embryonic Development Enzymes Epigenetic Process Fluorescence Polarization G-Quartets Gene Expression Gene Expression Regulation Genes Genomics Goals Hand Histone H3 Kinetics Knowledge Length Literature Lysine Malignant Neoplasms Mediating Modeling Nature Nucleic Acids Nucleosomes Play Poly(A)+ RNA Polycomb Polynucleotides Prevalence Proteins Proxy RNA RNA Binding Recruitment Activity Regulation Research Role Site Specificity Structure Testing Three Prime Repair Exonuclease 1 Transfer RNA Work base biophysical properties design epigenetic silencing experimental study flexibility genomic locus histone methyltransferase in vitro activity in vivo interest methyl group novel therapeutics nucleic acid binding protein recruit spatial relationship tumor progression

项目摘要

PROJECT SUMMARY Polycomb repressive complex 2 (PRC2) is a histone methyltransferase (HMTase) that sequentially deposits three methyl groups onto lysine 27 of histone H3 (H3K27me1/2/3), and its activity is crucial for epigenetic silencing during development and cancer. How PRC2 is targeted to genetic loci is of considerable interest, given its critical function and abundance of target genes. It’s well accepted that PRC2 interacts broadly with RNA, and that these interactions regulate PRC2’s localization at genomic sites destined for epigenetic silencing. However, the details about the nature and mechanism(s) of PRC2’s regulation by RNA remain quite controversial. While some studies have proposed a role for RNA in PRC2’s recruitment to chromatin, more have suggested roles in PRC2 eviction from chromatin and/or inhibition of PRC2 activity. Notably, a recent work has demonstrated that the PRC2-RNA interaction is critical in vivo for maintaining H3K27me3 levels and chromatin occupancy at PRC2 target genes. Thus, while there’s evidence RNA plays a role in evicting PRC2 from chromatin under certain conditions, it also seems it must facilitate chromatin binding and H3K27me3 deposition in at least some circumstances. To date, the PRC2 literature lacks direct evidence for a mechanism that can reconcile these prior data and explain RNA-mediated chromatin-binding and HMTase activity by PRC2. My preliminary data unexpectedly imply PRC2 has the intrinsic capacity to transfer directly between polynucleotides, and this phenomenon could be broadly relevant to RNA-mediated regulation of chromatin-modifying enzymes. Based on these and other data, I hypothesize a ‘hand-off’ model where PRC2 can be directly transferred between nascent RNA and spatially proximal chromatin to facilitate H3K27me3 deposition or eviction from chromatin. Studies proposed herein will quantify PRC2’s binding and competition kinetics for RNA and DNA/nucleosomes, determine the polynucleotide species PRC2 could be transferred between, characterize the prevalence and biophysical requisites of this phenomenon in other proteins, and interrogate the effect of RNA-nucleosome spatial relationships on PRC2’s HMTase activity. Findings from these studies will expand our understanding of how chromatin-modifying enzymes are regulated, which could have profound implications in our understanding of cell differentiation, embryonic development, and cancer.

项目概要 Polycomb 抑制复合物 2 (PRC2) 是一种组蛋白甲基转移酶 (HMTase)，可连续沉积三个甲基位于组蛋白 H3 (H3K27me1/2/3) 的赖氨酸 27 上，其活性对于表观遗传沉默至关重要。鉴于 PRC2 的关键功能和作用，它如何靶向遗传位点引起了人们的极大兴趣。人们普遍认为 PRC2 与 RNA 广泛相互作用，并且这些相互作用调节。 PRC2 在基因组位点的定位注定会发生表观遗传沉默，但有关其性质和细节的细节尚不清楚。 RNA 调节 PRC2 的机制仍然存在很大争议，尽管一些研究提出了 RNA 的作用。 RNA 在 PRC2 招募染色质中发挥作用，更多研究表明在 PRC2 从染色质驱逐和/或抑制中发挥作用值得注意的是，最近的一项工作表明 PRC2-RNA 相互作用在体内至关重要。维持 PRC2 靶基因上的 H3K27me3 水平和染色质占据，因此，虽然有证据表明 RNA 发挥着重要作用。在某些条件下将 PRC2 从染色质中驱逐的作用，似乎它也必须促进染色质结合和至少在某些情况下 H3K27me3 沉积，迄今为止，PRC2 文献缺乏机制的直接证据。可以协调这些先前的数据并解释 PRC2 介导的 RNA 介导的染色质结合和 HMTase 活性。初步数据出乎意料地表明 PRC2 具有在多核苷酸之间直接转移的内在能力，并且这基于这些，这种现象可能与 RNA 介导的染色质修饰酶的调节广泛相关。和其他数据，我领导了一个“交接”模型，其中 PRC2 可以直接在新生 RNA 和空间上邻近的染色质以促进 H3K27me3 沉积或从染色质中驱逐本文提出的研究将。量化 PRC2 对 RNA 和 DNA/核小体的结合和竞争动力学，确定多核苷酸种类 PRC2 可以在其他区域之间转移，表征这种现象的普遍性和生物物理要求蛋白质，并探讨 RNA-核小体空间关系对 PRC2 的 HMTase 活性的影响。这些研究将扩大我们对染色质修饰酶如何调节的理解，这可能会对我们对细胞分化、胚胎发育和癌症的理解具有广泛的意义。