The Role of pRB and Co-repressors in Transcriptional Regulation

描述（由申请人提供）：细胞分化与永久细胞周期的退出紧密协调并依赖于此。肿瘤抑制因子pRB通过其抑制增殖的能力来防止肿瘤发生。很明显，由pRB及其相关蛋白p107和p130（也称为口袋蛋白）组成的pRB肿瘤家族通过E2F转录因子被募集到启动子中。一旦募集，口袋蛋白协调基因表达变化与细胞周期进程和细胞周期退出，在短暂生长停滞（静止）和永久生长停滞发生在细胞分化。口袋蛋白在招募一系列协同抑制因子方面发挥作用，如Sin3-HDAC和组蛋白甲基转移酶，它们可以修饰染色质并沉默基因表达。然而，prb依赖性基因表达变化的机制尚不清楚。揭示这些机制是很重要的，因为它们很可能揭示与正常细胞生长和肿瘤抑制机制相关的转录控制。目前还不清楚是什么转录控制将细胞从生长停滞（G0/G1）中重新出现的细胞与经过M/G1期转变的细胞区分出来，以及因子募集和染色质修饰的差异如何决定短暂的生长停滞与永久的细胞周期退出。我们发现pRB和Sin3共同抑制因子在这两种情况下都起作用。在本提案中，我们寻求建立在先前资助期的结果基础上，并在以下目标中探索这些基本问题的答案：(1)我们将研究E2F，口袋蛋白和Sin3亚型在肌肉细胞永久细胞周期退出过程中因子募集，染色质修饰和核小体重塑中的作用。(2)我们将确定Sin3A和Sin3B亚型是否能够特异性调控靶基因转录和纯化内源性复合物。(3)我们将在动物模型中研究Sin3条件敲除的表型，并试图揭示Sin3异构体在维持细胞周期阻滞中的作用。

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.