Cell Cycle Regulation of Histone Gene Expression

描述(申请人提供)：进入S期是调控哺乳动物细胞增殖的关键事件。组蛋白基因表达在G1/S期的受控激活是新生DNA染色质包装所必需的。本课题组已经确定HiNF-P是组蛋白基因的主要转录调控因子，是细胞周期蛋白E/CDK2/p220NPAT通路的终点分子，并且HiNF-P缺乏通过S期损害细胞周期进程。HiNF-P的发现和鉴定代表了我们计划最重要的成就之一，拟议的研究将以这一主要结果为基础，为细胞周期控制开辟一个新的维度。我们的中心假设是，Cyclin E/CDK2/p220NPAT/HiNF-P基因调控级联是主要的细胞周期途径，其功能是实现组蛋白H4的生物合成，以支持DNA作为染色质的包装。为了解决这一假设，我们将结合生化、分子、细胞和体内遗传策略来定义HiNF-P/p220NPAT通路如何支持S阶段的激活和进展。首先，我们将表征蛋白质-蛋白质相互作用结构域和翻译后修饰，这些修饰控制HiNF-P/p220NPAT复合体的活性，以及介导组蛋白基因转录和mRNA加工的亚核部位(组蛋白基因体)调控信号的原位整合(特定目标1)。然后，我们将研究HiNF-P缺乏如何影响正常细胞和肿瘤细胞的细胞周期进程，并解决涉及调控细胞增殖的HiNF-P相关机制(特定目标2)。为了确定HiNF-P途径在体内的相关性，我们将研究HiNF-P在胚胎发育和出生后生长过程中是否对体内正常发育重要(特定目标3)。这三个被提议的目的旨在提供一个完整的了解在正常和肿瘤细胞中细胞周期蛋白E/CDK2/p220NPAT/HiNF-P通路对细胞增殖的分子、细胞和生物学作用。与公共健康相关：细胞增殖受到一系列复杂且相互依赖的生化事件的调控，这些生化事件涉及细胞周期阶段基因表达的特定修饰。组蛋白基因的S期特异性表达在时间上和功能上都与DNA复制相耦合。组蛋白基因转录依赖于细胞周期的调节组蛋白基因转录通过HiNF-P和p220NPAT提供了在G1/S相变中诱导组蛋白合成的最初限速步骤。我们正在研究的HiNF-P依赖机制代表了参与细胞周期控制的基本新途径。我们的研究将为S阶段进展的能力控制机制提供洞察力，并可能为选择性治疗疾病，尤其是癌症提供新的靶点，在这些疾病中，生长控制受到影响。

DESCRIPTION (provided by applicant): Entry into S phase is a key event for regulation of mammalian cell proliferation. The controlled activation of histone gene expression at the G1/S phase transition is essential for chromatin packaging of nascent DNA. Our program has identified HiNF-P as the principal transcriptional regulator of histone genes, the end-point molecule for the cyclin E/ CDK2/ p220NPAT pathway, and HiNF-P deficiency impairs cell cycle progression through S phase. The discovery and characterization of HiNF-P represents one of the most important accomplishments of our program and the proposed studies will build on this major result to open a new dimension to cell cycle control. Our central hypothesis is that the cyclin E/ CDK2/ p220NPAT/ HiNF-P gene regulatory cascade is a principal cell cycle pathway that functions to achieve competency for histone H4 biosynthesis to support packaging of DNA as chromatin. To address this hypothesis, we will combine biochemical, molecular, cellular and in vivo genetic strategies to define how the HiNF-P/p220NPAT pathway supports S phase activation and progression. First, we will characterize protein-protein interaction domains and post-translational modifications that control the activity of the HiNF-P/p220NPAT complex, as well as the in situ integration of regulatory signals at subnuclear sites ('Histone Locus Bodies') that mediate histone gene transcription and mRNA processing (Specific Aim 1). We will then examine how HiNF-P deficiency affects cell cycle progression in normal and tumor cells and address HiNF-P related mechanisms involved in regulating cell proliferation (Specific Aim 2). To establish the in vivo relevance of the HiNF-P pathway, we will investigate whether HiNF-P is important for normal development in vivo during embryogenesis and post-natal growth (Specific Aim 3). The three proposed aims are designed to provide an integrated understanding of the molecular, cellular, and biological contributions of the cyclin E/CDK2/p220NPAT/HiNF-P pathway to cell proliferation in normal and tumor cells. PUBLIC HEALTH RELEVANCE: Cell proliferation is regulated by a complex and interdependent series of biochemical events involving cell cycle-stage specific modifications in gene expression. The S-phase specific expression of histone genes is both temporally and functionally coupled with DNA replication. Cell cycle dependent modulation of histone gene transcription via HiNF-P and p220NPAT provides the initial rate-limiting step in the induction of histone protein synthesis at the G1/S phase transition. The HiNF-P dependent mechanisms we are investigating represent fundamentally novel pathways involved in cell cycle control. Our studies will provide insight into mechanisms that control competency for S phase progression and may yield novel targets for selective treatment of diseases, particularly cancer, in which growth control is compromised.