Toward Understanding the Role of the Polycomb Complex in Skin Control

 描述（由申请人提供）：Polycomb复合物是一种主要的染色质阻遏物，控制干细胞身份、组织发育和肿瘤发生。它的活性分为两种多蛋白复合物，称为Polycomb抑制复合物（PRC）1和2。Polycomb介导的抑制的经典模型假定PRC复合物的严格分级招募，其中PRC 2招募PRC 1以建立染色质压缩和基因抑制。然而，我们最近的生物化学和遗传学数据对这一模型提出了质疑。首先，在对比的分层模型，我们发现，招聘的PRC 1发生独立的PRC 2。其次，我们通过切除每个复合物的必需亚基- EED（PRC 2）和Ring 1b（PRC 1），产生了表皮祖细胞中缺乏PRC 1/2复合物的小鼠。虽然缺乏EED或Ring 1b的小鼠没有明显的表皮改变，但双敲除EED/Ring 1b动物的表皮形成发生了显著变化，其特征在于基底层的结构改变，基底细胞与基底膜的附着丧失，以及基底/基底上细胞-细胞粘附缺陷。这也与模型相反，该模型指出，PRC 1和PRC 2的伴随损失应与PRC 1或PRC 2单独损失相同。这些发现为我们提供了一个独特的机会，证明Polycomb抑制的经典模型过于简单，至少对表皮来说是如此。这项资助的主要目标是了解PRC 1和PRC 2复合物如何调节表皮发育。由于Polycomb复合物的功能是控制基因表达，我们假设PRC 1和PRC 2调节对表皮发育至关重要的基因。为了检验这一假设，首先，我们将分析EED/Ring 1b无效表皮，以确定何时观察到第一个表型异常。其次，通过进行转录和生物化学研究，我们将确定PRC 1/PRC 2的EED/Ring 1b无效表皮细胞中激活的靶基因。这些分析之后将进行功能研究，以确定负责EED/Ring 1b无效表皮表型的基因。第三，最近在ES细胞中的研究已经鉴定了一种称为RYBP-PRC 1的非典型PRC 1复合物，其在没有PRC 2复合物的预先存在的结合的情况下被募集到染色质。我们的初步数据使我们假设RYBP-PRC 1复合物与PRC 2一起调节对表皮发育至关重要的基因。在这里，我们将进行生物化学，转录和功能丧失的研究，以确定RYBP-PRC 1复合物的作用，以及它如何与PRC 2合作，以控制表皮发育。总之，概述的研究将为Polycomb介导的基因控制机制提供新的见解，并揭示控制表皮发育的调节因子，表皮是我们生存所必需的组织。

 DESCRIPTION (provided by applicant): The Polycomb complex is a major chromatin repressor that controls stem cell identity, tissue development, and tumorigenesis. Its activity is divided between two multi-protein complexes called Polycomb repressive complex (PRC) 1 and 2. The classical model of Polycomb-mediated repression postulates a strict hierarchical recruitment of the PRC complexes, where PRC2 recruits PRC1 to establish chromatin compaction and gene repression. Our recent biochemical and genetic data, however, questioned this model. First, in contrast to the hierarchical model, we found that the recruitment of PRC1 occurs independently of PRC2. Second, we have generated mice that lack the PRC1/2 complexes in epidermal progenitors by ablating the essential subunits of each complex - EED (PRC2) and Ring1b (PRC1). While mice lacking solely EED or Ring1b had no obvious epidermal alterations, double knockout EED/Ring1b animals had striking changes in epidermal formation characterized by the altered architecture of the basal layer, a loss of basal cell attachment to the basement membrane, and defective basal/suprabasal cell-cell adhesion. This is again in contrast to the model, which states that concomitant loss of PRC1 and PRC2 should be identical to the loss of PRC1 or PRC2 alone. These findings provide us with the unique opportunity to demonstrate that the classical model of Polycomb repression is too simplistic, at least for the epidermis. The main goal of this grant is to understand how the PRC1 and PRC2 complexes regulate epidermal development. As the Polycomb complexes function to control gene expression, we hypothesize that PRC1 and PRC2 regulate genes that are critical for epidermal development. To test this hypothesis, first, we will analyze the EED/Ring1b-null epidermis to pinpoint when the first phenotypic abnormalities are observed. Second, by performing transcriptional and biochemical studies, we will identify PRC1/PRC2 target genes that are activated in the EED/Ring1b-null epidermal cells. These analyses will be followed by functional studies to determine the genes responsible for the EED/Ring1b-null epidermal phenotypes. Third, recent studies in ES cells have identified a non-canonical PRC1 complex called RYBP- PRC1 that is recruited to chromatin without the pre-existent binding of the PRC2 complex. Our preliminary data have led us to hypothesize that the RYBP-PRC1 complex functions with PRC2 to regulate genes that are critical for epidermal development. Here, we will perform biochemical, transcriptional, and loss-of-function studies to determine the role of the RYBP-PRC1 complex and how it cooperates with PRC2 to control epidermal development. In summary, the outlined studies will provide novel insights into the mechanisms of Polycomb-mediated gene control and uncover regulators controlling the development of the epidermis, a tissue that is essential for our survival.