Cell Cycle Regulation of Histone Gene Expression

描述（申请人提供）：进入S期是调节哺乳动物细胞增殖的关键事件。组蛋白基因表达在G1/S期的受控激活对于新生DNA的染色质包装是必不可少的。我们的项目已经确定HiNF-P是组蛋白基因的主要转录调节因子，是细胞周期蛋白E/CDK 2/p220 NPAT通路的终点分子，HiNF-P缺乏会损害细胞周期通过S期的进展。HiNF-P的发现和表征代表了我们项目最重要的成就之一，拟议的研究将建立在这一重大成果的基础上，为细胞周期控制开辟一个新的维度。我们的中心假设是，细胞周期蛋白E/CDK 2/p220 NPAT/ HiNF-P基因调控级联是一个主要的细胞周期途径，其功能是实现组蛋白H4生物合成的能力，以支持DNA包装为染色质。为了解决这一假设，我们将结合联合收割机生化，分子，细胞和体内遗传策略，以确定如何HiNF-P/p220 NPAT途径支持S期激活和进展。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，其控制HiNF-P/p220 NPAT复合物的活性，以及调节信号在亚核位点（“组蛋白基因座体”）的原位整合，其介导组蛋白基因转录和mRNA加工（特异性目的1）。然后，我们将研究HiNF-P缺陷如何影响正常和肿瘤细胞的细胞周期进程，并解决HiNF-P相关机制参与调节细胞增殖（具体目标2）。为了建立HiNF-P途径的体内相关性，我们将研究HiNF-P在胚胎发生和出生后生长期间是否对体内正常发育重要（具体目标3）。提出的三个目标旨在提供一个完整的理解细胞周期蛋白E/CDK 2/p220 NPAT/HiNF-P途径的分子，细胞和生物学的贡献，在正常和肿瘤细胞的细胞增殖。公共卫生相关性：细胞增殖受一系列复杂且相互依赖的生化事件调节，这些事件涉及基因表达中的细胞周期阶段特异性修饰。组蛋白基因的S期特异性表达与DNA复制在时间和功能上都是耦合的。通过HiNF-P和p220 NPAT对组蛋白基因转录的细胞周期依赖性调节提供了在G1/S相变诱导组蛋白蛋白合成的初始限速步骤。我们正在研究的HiNF-P依赖性机制代表了参与细胞周期控制的全新途径。我们的研究将深入了解控制S期进展能力的机制，并可能产生选择性治疗疾病的新靶点，特别是生长控制受到影响的癌症。

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.