Role of Polycomblike1-histone interaction in PRC2 activities

描述(申请人提供)：多梳样蛋白1(PCL1)是多梳状抑制复合体2(PRC2)的一种成分，是发育期间和谱系承诺后基因抑制的主要调节因子。PRC2亚单位的失调会导致分化缺陷，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)，产生翻译后修饰(PTM)，通常与转录抑制有关。Pcl1含有PTMS潜在阅读器的保守组合：N-末端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这两个模块都是组蛋白PTMS的阅读器，Tudor结构域识别H3K36me3，这是一个活跃的转录标志。然而，Pcl1通过这两个阅读器结构域招募PRC2进行翻译后修饰染色质的分子机制以及这些相互作用如何调节复合体的活性尚未阐明。我们假设，Pcl1、Tudor和PhD结构域与组蛋白的结合标志着微调的PRC2靶向并调节了PRC2甲基转移酶的活性，以响应当地的表观遗传格局。本项目的具体目的是：(1)阐明Pcl1都铎的染色质识别的分子机制；(2)确定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射线结晶学来确定Pcl1Tudor和PHD Finger及其各自的组蛋白配体的原子分辨结构。我们还将在EMSA检测中使用改进的核小体核心颗粒。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-染色质结合对PrC2活性的生物学意义，我们将产生Pcl1的功能缺失突变体，并在功能实验中对它们进行检测，包括蛋白质印迹分析、甲基转移酶分析、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，并为表观遗传异常如何导致发育障碍和癌症提供洞察力。

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.