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: 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
• 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

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 多梳抑制复合物2（PRC 2）是一种组蛋白甲基转移酶（HMTase），其顺序地沉积三个 在组蛋白H3（H3 K27 me 1/2/3）的赖氨酸27上，它的活性对于表观遗传沉默至关重要。 发展和癌症。考虑到PRC 2的关键功能和作用，PRC 2如何靶向遗传基因座引起了相当大的兴趣。 丰富的靶基因。众所周知，PRC 2与RNA广泛相互作用，并且这些相互作用调节 PRC 2定位于注定用于表观遗传沉默的基因组位点。然而，关于性质和 PRC 2通过RNA调节的机制仍然存在相当大的争议。虽然一些研究提出了一个作用， RNA参与PRC 2向染色质的募集，更多的研究表明PRC 2从染色质中驱逐和/或抑制作用 PRC 2活动值得注意的是，最近的工作已经证明PRC 2-RNA相互作用在体内对于 维持H3 K27 me 3水平和PRC 2靶基因处的染色质占据。因此，尽管有证据表明RNA在 在某些条件下从染色质中驱逐PRC 2的作用，似乎它也必须促进染色质结合， H3 K27 me 3沉积。到目前为止，PRC 2文献缺乏直接证据表明 这可以调和这些先前的数据，并解释RNA介导的染色质结合和HMTase活性的PRC 2。我 初步数据出乎意料地暗示PRC 2具有在多核苷酸之间直接转移的内在能力，并且这 这种现象可能与RNA介导的染色质修饰酶的调节广泛相关。基于这些 和其他数据，我假设了一个“交接”模型，其中PRC 2可以直接在新生RNA和 在空间上接近染色质以促进H3 K27 me 3沉积或从染色质驱逐。本文提出的研究将 定量PRC 2对RNA和DNA/核小体的结合和竞争动力学， PRC 2可以在其他人之间转移，表征这种现象的流行和生物物理学特征， 蛋白质，并询问RNA-核小体空间关系对PRC 2的HMTase活性的影响。的结果 这些研究将扩大我们对染色质修饰酶如何调节的理解， 对我们理解细胞分化、胚胎发育和癌症有着深远的影响。