Role of Polycomblike1-histone interaction in PRC2 activities

描述（由申请人提供）：Polycomb样蛋白1 （Pcl1）是Polycomb抑制复合物2 （PRC2）的一个组成部分，PRC2是发育期间和谱系承诺后基因抑制的主要调节因子。PRC2亚基的失调导致分化缺陷，并已在多种类型的癌症中观察到，包括乳腺癌和前列腺癌。PRC2三甲基化Lysine27上的组蛋白H3 (H3K27me3)，产生通常与转录抑制相关的翻译后修饰（PTM）。Pcl1包含一个保守的ptm潜在读者组合：一个n端Tudor结构域和一个植物同源结构域（PHD）手指，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两个模块都是组蛋白ptm的读取器，Tudor结构域识别H3K36me3，这是主动转录的标志。然而，Pcl1通过这两个读取器结构域将PRC2招募到翻译后修饰的染色质上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对PRC2靶向进行微调，并调节PRC2甲基转移酶活性，以响应局部表观遗传景观。本项目的具体目的是：(1)阐明Pcl1 Tudor的染色质识别分子机制；(2)明确Pcl1 PHD手指靶向染色质的分子基础。我们将使用核磁共振和x射线晶体学来确定Pcl1Tudor和PHD手指及其各自的组蛋白配体的原子分辨率结构。我们还将在EMSA检测中使用修饰的核小体核心颗粒。组蛋白的特异性和结合亲和力将通过核磁共振、色氨酸荧光和ITC来表征。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的功能缺失突变体，并在功能实验中对其进行检测，包括western blot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功能的分子机制，并为表观遗传畸变如何导致发育障碍和癌症提供见解。

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.