PRC1在胚胎干细胞生长和分化中识别和抑制靶基因的分子机制

Polycomb Repressive Complexes 1 and 2 (PRC1, PRC2) are epigenetic regulatory complexes that cause transcriptional repression of target genes through modifying chromatin. The PRC1/2-mediated gene silencing mechanism is required for the maintenance of the core properties of embryonic stems cells (ESC), pluripotency and self renewal. The spectra of target genes for PRC complexes are dynamically changing along with cell differentiation during developmental processes. One of the key questions in the field is how PRCs selectively recognize and switch targets in multiple cell types and under various physiological or pathological conditions. To address this question, our research has been focused on CBX protein(s), one of the core components in PRC1 that is responsible for the chromatin targeting of the complex. We generated and analyzed a number of ES cell lines with the expression of Cbx genes manipulated. Our preliminary data have provided us several lines of valuable information. 1) The interaction between CBX7 and ncRNA is structurally complicated and possibly prevalent in cells; 2) Cbx homologs and their transcription splicing variants expressed in ESC may have distinct functions. Combining previously published data and our new evidence, we propose that both the diversity of CBX proteins and their interaction with ncRNA contribute to the targeting specificity of PRC1. In this study, we will employ approaches in biochemistry, molecular and cellular biology, together with genome-wide analyses to identify and verify the ncRNA species that specifically interact with PRC1. We will further elucidate the structural foundation of these interactions and their role in selectively targeting PRC1 to chromatin. In addition, we plan to characterize main features of PRC1 and analyze its function in maintaining ESC pluripotency and regulating differentiation in established or newly generated ESC lines with manipulated Cbx expression. The data will help us make functional comparisons among Cbx homologs and their transcription splicing variants. Overall, our study will help elucidate the targeting and working mechanisms of PRC1.

多梳抑制复合体（PRC1或2）通过重塑染色质构象在表观遗传上抑制靶基因表达，从而定义和维持机体内多种细胞类型的特异表征和功能。CBX蛋白是参与PRC1识别靶基因的核心成员。我们对Cbx基因敲除或稳定表达外源Cbx基因及突变体的胚胎干细胞（ESC）株系的鉴定发现：CBX与非编码RNA的结合存在未知的复杂性和普遍性；Cbx同源基因及转录剪切体功能有所差异。我们推测CBX的多样性及其与非编码RNA的结合直接参与调节PRC1对靶基因的识别和抑制。本课题以小鼠ESC及其分化体系为模型，分离鉴定与PRC1特异性结合的非编码RNA分子，澄清作用的结构基础及非编码RNA调节PRC1靶向募集的分子机理；在表达不同CBX的遗传背景下剖析CBX的多样形式与PRC1特异性识别靶基因的相互关联。阐明PRC1随细胞分化动态识别靶基因的分子机理对揭示其如何在多种细胞类型和生理、病理状态下发挥特异性调控作用至关重要。

多梳抑制复合体（PRC1和PRC2）通过重塑染色质构象在表观遗传上抑制靶基因表达，从而定义和维持包括胚胎干细胞（ESC）在内的多种细胞类型的特异表征和功能。CBX蛋白是参与PRC1识别靶基因的核心成员。我们在本项目研究中发现：1）CBX蛋白与非编码RNA（ncRNA）在小鼠ESC中的结合相对广谱、特异性较差且其结合RNA的区段包含CD结构域以及中间区段，去除其介导RNA互作的中间区段不影响CBX蛋白的整体亚细胞核定位；2）ESC中表达多种CBX同源蛋白且首次证实CBX蛋白彼此间存在直接的相互作用，介导该相互作用的区段在其未知功能的中间区段；3）检测到小鼠ESC中表达多种CBX7的可变剪切体，不同的剪切体具有不同的组织及细胞类型特异表达谱，我们从小鼠脾脏中成功克隆到一个未知功能的CBX7剪切体，并制备抗体检测到了其蛋白产物，该蛋白具有CBX7蛋白的N端CD结构域，能够与染色质相结合，但不能结合RING1B因此不能形成完整的PRC1复合体，在调控PRC1稳态中发挥重要作用。本课题的研究一定程度上揭示了CBX蛋白作为PRC1的核心组分发挥功能的分子机制；CBX同源蛋白间的互作为其协同形成覆盖靶基因位点的PRC1多分子聚合物以及在分化过程中更替作用提供可能的分子基础；组织特异性CBX蛋白亚型的鉴定丰富了人们对CBX蛋白调控机制的认知。本研究有助于阐明PRC1随细胞分化和既定发育程序动态识别并抑制靶基因的分子机理以及揭示其如何在多种细胞类型和生理、病理状态下发挥特异性调控作用。本项目的部分成果已经发表，其余结果正在整理发表中。