The Role of pRB and Co-repressors in Transcriptional Regulation

描述(申请人提供)：细胞分化与永久细胞周期出口密切协调，并依赖于永久细胞周期出口。肿瘤抑制因子pRb通过其抑制增殖的能力防止肿瘤的发生。很明显，pRb肿瘤家族由pRb及其相关蛋白p107和p130(也称为Pocket蛋白)组成，通过E2F转录因子招募到启动子。一旦被招募，口袋蛋白在细胞分化时发生的短暂生长停滞(静止)和永久生长停滞期间，协调基因表达与细胞周期进展和细胞周期退出的变化。Pocket蛋白在招募一批共抑制物，如Sin3-HDAC和组蛋白甲基转移酶，改变染色质和沉默基因的表达方面发挥了作用。然而，依赖于pRB的基因表达变化背后的机制还不是很清楚。破解这些机制很重要，因为它们可能有助于阐明与正常细胞生长和肿瘤抑制机制相关的转录调控。目前还不清楚是什么转录控制将细胞从生长停滞(G0/G1相变)中恢复过来，以及在因子募集和染色质修饰方面的差异如何决定了一过性生长停滞和永久性细胞周期退出。我们发现PRB和SIN3共抑制物在这两种情况下都起到了作用。在这项提案中，我们试图在前一个资助阶段的结果的基础上，从以下几个方面探索这些基本问题的答案：(1)我们将研究E2F、Pocket蛋白和SIN3亚型在肌肉细胞永久细胞周期退出过程中的因子招募、染色质修饰和核小体重塑中的作用。(2)确定Sin3A和Sin3B亚型是否能特异性调节靶基因转录和纯化内源性复合体。(3)我们将研究SIN3条件性基因敲除的表型，并试图揭示SIN3亚型在维持动物模型细胞周期停滞中的作用。

DESCRIPTION (provided by applicant): Cellular differentiation is tightly coordinated with, and dependent upon, permanent cell cycle exit. The tumor suppressor pRB prevents tumorigenesis by virtue of its ability to suppress proliferation. It is clear that the pRB tumor family, which is comprised of pRB and the related proteins, p107 and p130 (also known as pocket proteins), is recruited to promoters through the E2F transcription factor. Once recruited, pocket proteins coordinate changes in gene expression with cell cycle progression and cell cycle exit, during transient growth arrest (quiescence) and permanent growth arrest that occurs upon cellular differentiation. Pocket proteins play a role in recruiting a cadre of co-repressors, such as Sin3-HDAC and histone methyltransferases, that modify chromatin and silence gene expression. Yet the mechanisms underlying the pRB-dependent changes in gene expression are not well understood. It is important to unravel these mechanisms, since they are likely to shed light on transcriptional controls associated with normal cell growth and tumor suppressive mechanisms. It is also unclear what transcriptional controls distinguish cells progressing through the M/G1 phase transition from those observed in cells re-emerging from growth arrest (the G0/G1 phase transition) and how differences in factor recruitment and chromatin modifications dictate transient growth arrest versus permanent cell cycle exit. We have found that pRB and the Sin3 co-repressor play a role in both settings. In this proposal, we seek to build upon results of the previous funding period and explore answers to these fundamental issues in the following Aims: (1) We will examine the role of E2F, pocket proteins, and Sin3 isoforms in factor recruitment, chromatin modification, and nucleosome remodeling during permanent cell cycle exit in muscle cells. (2) We will determine whether Sin3A and Sin3B isoforms can specifically regulate target gene transcription and purify endogenous complexes. (3) We will investigate the phenotype of Sin3 conditional knock-outs and attempt to unravel a role for Sin3 isoforms in maintaining cell cycle arrest in an animal model.