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

• 批准号: 10683162
• 负责人: Wayne Oliver Hemphill
• 金额: $ 6.95万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 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/10683162
• 关键词: Binding; Biological Assay; Biophysics; Cell Differentiation process; Chromatin; Complex; DNA; Data; Deposition; Development; Dissociation; Embryonic Development; Enzymes; Fluorescence Polarization; G-Quartets; Gene Expression; Gene Expression Regulation; Genes; Genomics; Goals; Histone H3; Kinetics; Knowledge; Length; Literature; Lysine; MS2 coat protein; Malignant Neoplasms; Mediating; Modeling; Nature; Nucleic Acids; Nucleosomes; 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; 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; permissiveness; recruit; spatial relationship; tumor progression

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.

项目摘要 Polycomb反射复合物2（PRC2）是一种组蛋白甲基转移酶（HMTase），依次沉积三个 在组蛋白H3的赖氨酸27（H3K27ME1/2/3）上甲基，其活性对于表观遗传沉默至关重要 发展与癌症。鉴于其关键功能和 靶基因的抽象。 PRC2与RNA广泛相互作用，这些相互作用已得到很好的接受，并且这些相互作用调节 PRC2在注定要进行表观遗传沉默的基因组部位的定位。但是，有关性质和的细节 PRC2通过RNA调节的机制仍然存在争议。虽然一些研究提出了 PRC2募集到染色质中的RNA，越来越多建议在染色质和/或抑制的PRC2驱逐中作用 Prc2活性。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靶基因的H3K27ME3水平和染色质占用率。那虽然有证据表明RNA播放 在某些条件下从染色质驱逐PRC2中的作用，似乎还必须促进染色质结合和 H3K27me3至少在某些情况下沉积。迄今为止，PRC2文献缺乏直接的机制证据 这可以调和这些先前的数据，并通过PRC2解释RNA介导的染色质结合和HMTase活性。我的 初步数据出乎意料地暗示PRC2具有直接在多核苷酸之间转移的内在能力，这 现象可能与RNA介导的调节酶的调节广泛有关。基于这些 和其他数据，我假设一个“交接”模型，其中Prc2可以直接转移到新生的RNA和 本文提出的研究将 量化PRC2的RNA和DNA/核小体的结合和竞争动力学，确定多核苷酸种类 可以在其他情况下转移PRC2，以表征这种现象的患病率和生物物理要求 蛋白质，并询问RNA核体空间关系对PRC2 HMTase活性的影响。来自 这些研究将扩展我们对如何调节染色质修饰酶的理解，这可能具有 在我们对细胞分化，胚胎发育和癌症的理解中的深刻含义。

项目成果

PRC2 direct transfer from G-quadruplex RNA to dsDNA has implications for RNA-binding chromatin modifiers.

• DOI: 10.1073/pnas.2220528120
• 发表时间: 2023-06-06
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Hemphill, Wayne O.;Fenske, Regan;Gooding, Anne R.;Cech, Thomas R.
• 通讯作者: Cech, Thomas R.

Multiple RNA- and DNA-binding proteins exhibit direct transfer of polynucleotides with implications for target-site search.

• DOI: 10.1073/pnas.2220537120
• 发表时间: 2023-06-27
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Hemphill, Wayne O.;Voong, Calvin K.;Fenske, Regan;Goodrich, James A.;Cech, Thomas R.
• 通讯作者: Cech, Thomas R.