Role of Polycomblike1-histone interaction in PRC2 activities

Polycomblike1-组蛋白相互作用在 PRC2 活性中的作用

• 批准号: 8782121
• 负责人: Jovylyn Gatchalian
• 金额: $ 2.97万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8782121
• 关键词: Affinity; Binding; Biological; Biological Assay; Calorimetry; Cells; Chromatin; Complex; DNA; DNA Binding; Defect; Development; Disease; Electrophoretic Mobility Shift Assay; Embryo; Epigenetic Process; Fingers; Fluorescence; Gene Expression Regulation; Genetic Transcription; Germ; Germ Cells; Histone H3; Histones; Lead; Ligands; Light; Malignant Neoplasms; Malignant neoplasm of prostate; Maps; Measures; Mediating; Methylation; Methyltransferase; Modeling; Molecular; Mus; Mutate; N-terminal; Nucleosome Core Particle; Peptides; Plants; Polycomb; Post-Translational Protein Processing; Proteins; Reader; Recruitment Activity; Regulator Genes; Repression; Research; Resolution; Role; Signal Transduction; Site-Directed Mutagenesis; Specificity; Structure; Testing; Titrations; Transcriptional Regulation; Tryptophan; Western Blotting; X-Ray Crystallography; base; cancer type; chromatin immunoprecipitation; developmental disease; gene repression; histone methyltransferase; homeodomain; in vivo; insight; loss of function; male; malignant breast neoplasm; mutant; public health relevance; research study; response

DESCRIPTION (provided by applicant): Polycomblike protein 1 (Pcl1) is a component of the Polycomb Repressive Complex2 (PRC2), a master regulator of gene repression during development and after lineage commitment. Dysregulation of PRC2 subunits results in differentiation defects and has been observed in multiple types of cancer, including breast and prostate cancers. PRC2 tri-methylates histone H3 on Lysine27 (H3K27me3), generating a posttranslational modification (PTM) generally associated with transcriptional repression. Pcl1 contains a conserved combination of potential readers of PTMs: an N-terminal Tudor domain and a plant homeodomain (PHD) finger, however, the biological roles of these modules are not well understood. Preliminary studies show that both modules, Tudor and PHD, act as readers of histone PTMs and the Tudor domain recognizes H3K36me3, a mark of active transcription. However, the molecular mechanisms by which Pcl1 recruits PRC2 to post-translationally modified chromatin via these two reader domains as well as how these interactions may modulate the complex's activity have yet to be elucidated. We hypothesize that binding of the Pcl1 Tudor and PHD domains to histone marks fine tunes PRC2 targeting and modulates PRC2 methyltransferase activity in response to the local epigenetic landscape. The specific aims of this project are: (1) To elucidate the molecular mechanism of chromatin recognition of Pcl1 Tudor and (2) To define the molecular basis of Pcl1 PHD finger targeting to chromatin. We will use NMR and X-ray crystallography to determine the atomic-resolution structures of Pcl1Tudor and PHD finger with their respective histone ligands. We will also utilize modified nucleosome core particles in EMSA assays. Histone specificities and binding affinities will be characterized by NMR, tryptophan fluorescence, and ITC. To define the biological significance of the Pcl1-chromatin association for PRC2 activity we will generate loss-of-function mutants of Pcl1 and examine them in functional experiments, including western blot analysis, methyltransferase assays, chromatin immunoprecipitation (ChIP) and quantitative PCR. Our studies will aid in our understanding of the molecular mechanisms that underlie Pcl1 function and provide insight into how epigenetic aberrations can lead to developmental disorders and cancer.

描述（由申请人提供）：Polycomblike 蛋白 1 (Pcl1) 是 Polycomb Repressive Complex2 (PRC2) 的组成部分，PRC2 是发育期间和谱系定型后基因抑制的主要调节因子。 PRC2 亚基的失调会导致分化缺陷，并且已在多种类型的癌症中观察到，包括乳腺癌和前列腺癌。 PRC2 将赖氨酸 27 (H3K27me3) 上的组蛋白 H3 三甲基化，产生通常与转录抑制相关的翻译后修饰 (PTM)。 Pcl1 包含 PTM 潜在阅读器的保守组合：N 端 Tudor 结构域和植物同源结构域 (PHD) 指，然而，这些模块的生物学作用尚不清楚。初步研究表明，Tudor 和 PHD 这两个模块都充当组蛋白 PTM 的读取器，并且 Tudor 结构域可识别 H3K36me3（活性转录标记）。然而，Pcl1 通过这两个阅读器结构域将 PRC2 募集到翻译后修饰的染色质的分子机制以及这些相互作用如何调节复合物的活性尚未阐明。我们假设 Pcl1 Tudor 和 PHD 结构域与组蛋白的结合标志着微调 PRC2 靶向并调节 PRC2 甲基转移酶活性以响应局部表观遗传景观。该项目的具体目标是：（1）阐明Pcl1 Tudor染色质识别的分子机制；（2）定义Pcl1 PHD手指靶向染色质的分子基础。我们将使用 NMR 和 X 射线晶体学来确定 Pcl1Tudor 和 PHD Finger 及其各自组蛋白配体的原子分辨率结构。我们还将在 EMSA 测定中利用修饰的核小体核心颗粒。组蛋白特异性和结合亲和力将通过 NMR、色氨酸荧光和 ITC 进行表征。为了确定 Pcl1-染色质关联对 PRC2 活性的生物学意义，我们将生成 Pcl1 功能缺失突变体，并在功能实验中检查它们，包括蛋白质印迹分析、甲基转移酶测定、染色质免疫沉淀 (ChIP) 和定量 PCR。我们的研究将有助于我们理解 Pcl1 功能背后的分子机制，并深入了解表观遗传畸变如何导致发育障碍和癌症。