Role of the pRB family in Quiescence and Differentiation

描述（由申请人提供）：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，统称口袋蛋白）在调节细胞周期中起着重要作用，pRB已被证明是在大部分人类肿瘤中发生突变的典型肿瘤抑制因子。此外，pRB已被证明在包括肌肉和骨在内的几种组织的分化中发挥关键作用。在之前的资助期内，我们在母体资助申请的支持下，强调了每个口袋蛋白在响应生长停滞信号以及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复合物。在这项资助中，我们提出了以下目标，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)我们将表征增殖和分化细胞中的pRB复合物，并研究消耗相关蛋白对基因表达和分化的影响；(2)我们将在培养细胞和肌肉组织中确定pRB复合物的靶点，并研究pRB复合物在修饰靶基因染色质中的作用；(3)我们将研究与目标1和目标2中发现的机制类似的机制是否也适用于可逆生长停滞，并确定是否存在区分可逆和不可逆细胞周期退出的机制。在当前对原始亲本应用程序的修订中，我们建议扩大与肌源性分化相关的染色质修饰的研究，特别关注那些依赖于pRB蛋白家族的变化。我们将采用新的、最先进的芯片测序方法，以无偏倚的全基因组方式检查这些修饰。这些研究将增强我们对调控控制的理解，这些调控控制对于细胞周期和分化的可逆和永久退出都是必不可少的。此外，他们将阐明在与发育相关的环境中，阻遏子、共阻遏子和染色质修饰之间的关键相互作用。

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.