Role of the pRB family in Quiescence and Differentiation

描述(申请人提供)：过去二十年的研究表明，视网膜母细胞瘤肿瘤抑制因子(PRb)和两种相关蛋白(p107和p130；统称为Pocket蛋白)在调节细胞周期中发挥重要作用，并且pRb已被证明是在很大一部分人类肿瘤中突变的典型肿瘤抑制因子。此外，pRb已被证明在几种组织的分化中起着关键作用，包括肌肉和骨骼。我们在前一个资助期间的工作，在我们的父母拨款申请的主持下，突出了每个口袋蛋白在响应生长停滞信号以及在促进和维持肌肉分化方面的独特作用。此外，我们首次成功地从增殖和分化的肌肉细胞中纯化了pRb复合体。在这项资助中，我们提出了以下目的，以进一步剖析口袋蛋白参与细胞周期退出和分化的机制。(1)我们将确定增殖和分化细胞中PRB复合体的特征，并研究耗尽相关蛋白对基因表达和分化的影响；(2)我们将确定培养细胞和肌肉组织中PRB复合体的靶标，并研究PRB复合体在靶基因染色质修饰中的作用；以及(3)我们将研究是否与AIMS 1和2中发现的机制类似，也与可逆生长停滞相关，并确定是否存在区分可逆和不可逆细胞周期退出的机制。在目前对原始亲本应用的修订中，我们建议扩大我们对与成肌分化相关的染色质修饰的研究，特别是那些依赖于pRb蛋白家族的变化。我们将结合新的、最先进的芯片测序方法，以一种公正的、全基因组的方式检查这些修改。这些研究将加强我们对调控的理解，调控对于可逆性和永久性地退出细胞周期和分化都是必不可少的。此外，他们还将阐明抑制物、共抑制物和染色质修饰之间的关键相互作用，因为它们发生在与发育相关的环境中。 与公共卫生相关：在某些情况下，当细胞不能正确分化时，癌症就会发生。分化与退出细胞周期有关，而视网膜母细胞瘤(retinoblastoma，PRB)抑癌基因在控制细胞生长停滞和分化中起着关键作用。这项建议试图了解pRb调节基因表达和控制永久停止分裂和终末分化的决定的潜在机制。

DESCRIPTION (provided by applicant): Research during the past two decades has suggested that the retinoblastoma tumor suppressor (pRB) and two related proteins (p107 and p130; collectively referred to as pocket proteins) play a fundamental role in regulating the cell cycle, and pRB has been shown to be a prototypical tumor suppressor mutated in a large portion of human tumors. In addition, pRB has been shown to play a pivotal role in differentiation of several tissues, including muscle and bone. Our work during the previous funding period, under the auspices of our parent grant application, has highlighted unique roles for each of the pocket proteins in responding to growth arrest cues and in both promoting and maintaining differentiation of muscle. In addition, we have succeeded for the first time in purifying pRB complexes from proliferating and differentiated muscle cells. In this grant, we propose the following aims to further dissect the mechanisms underlying pocket protein involvement in cell cycle exit and differentiation. (1) We will characterize pRB complexes in proliferating and differentiated cells and examine the impact of depleting associated proteins on gene expression and differentiation; (2) we will identify targets of the pRB complexes in cultured cells and in muscle tissue and examine the role of pRB complexes in modification of target gene chromatin; and (3) we will examine whether mechanisms analogous to those discovered in Aims 1 and 2 pertain to reversible growth arrest as well and determine if there are mechanisms that distinguish reversible and irreversible cell cycle exit. In the current Revision to the original parent application, we propose broadening our investigation of chromatin modifications associated with myogenic differentiation, focusing in particular on those changes that are dependent on the pRB family of proteins. We will incorporate the new, state-of-the-art ChIP-sequencing approach to examine these modifications in an unbiased, genome-wide manner. These studies will enhance our understanding of regulatory controls that are essential for both reversible and permanent withdrawal from the cell cycle and differentiation. In addition, they will elucidate critical interactions between repressors, co-repressors, and chromatin modifications as they occur in a developmentally relevant setting. PUBLIC HEALTH RELEVANCE: Cancer results in some cases when cells fail to properly differentiate. Differentiation is coupled to exit from the cell cycle, and the retinoblastoma (pRB) tumor suppressor plays a pivotal role in controlling growth arrest and differentiation. This proposal seeks to understand the underlying mechanisms whereby pRB regulates gene expression and controls the decision to permanently stop dividing and to terminally differentiate.