ROLE OF HISTONE H4 N-TERMINAL TAIL IN TRANSCRIPTION REGULATION

组蛋白 H4 N 末端尾部在转录调控中的作用

• 批准号: 7894600
• 负责人: WOOJIN AN
• 金额: $ 30.89万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7894600
• 关键词: Acetylation; Address; Architecture; Attenuated; Biochemical; Biological Assay; Cell physiology; Cells; Chemicals; Chromatin; Chromosomes; Coupled; Development; Disease; Environment; Epigenetic Process; Event; Factor Analysis; Future; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Goals; Histone Acetylation; Histone H4; Human; In Vitro; Lead; Light; Lysine; Maintenance; Malignant Neoplasms; Mediating; Modification; Molecular; Mutate; Mutation; N-terminal; Organ; Pattern; Peptides; Play; Process; Property; Protocols documentation; RNA Interference; Recombinant Proteins; Recombinants; Regulation; Relative (related person); Role; Signal Transduction; Site; Specificity; Switch Genes; System; TP53 gene; Tail; Therapeutic Agents; Tissues; Transcriptional Activation; Transcriptional Regulation; Tumor Suppression; base; cancer cell; cell growth; chromatin remodeling; design; established cell line; gene repression; histone modification; human CCDC6 protein; human disease; in vivo; insight; mutant; novel; public health relevance; reconstitution; research study

DESCRIPTION (provided by applicant): Acetylation of histone H4 N-terminal tail plays a crucial role in gene activation in chromatin environments; and alterations in this epigenetic process can lead to various diseases. Our long-term objective is to understand how H4 acetylation regulates p53-dependent transcription to attenuate cancer development. To investigate these aspects, we have developed new protocols to interrogate the cellular functions of H4 acetylation as a signal that would facilitate the recruitment/retention of chromatin regulatory machinery at the site of gene transcription. Importantly, our development of recombinant chromatin transcription systems using wild type or mutated recombinant H4 proteins has allowed us to evaluate the contribution of each specific H4 acetylation event in p53-dependent transcription from chromatin. Our preliminary results from transcription analysis, coupled with chromatin acetylation assays, have indicated that acetylation of only a small subset of lysines in H4 tail is critical for transcriptional activation. To gain mechanistic insight into the function of H4 acetylation, we have established cell lines that stably express H4 tail domains for biochemical purification of acetylated H4 tail-associated factors. Importantly, our functional analysis revealed that the acetylated tail-associated factors could significantly enhance p53-dependent transcription. In light of specific interactions between acetylated H4 tails and regulatory factors, we also have generated cell-permeable H4 tail mimics bearing specific acetylation marks to screen for the most active H4 acetylation events in cellular transcription. Based on these results, our specific aims are (i) to investigate the specificity of H4 acetylation events at the level of p53-dependent transcription by checking inhibitory effects of mutations of H4 lysine substrates in vitro as well as by characterizing repressive actions of acetylated H4 tail mimics in vivo, (ii) to detail the molecular mechanism underlying H4 acetylation-mediated activation of p53-dependent transcription by isolating and characterizing acetylated H4 tail-associated factors that could counteract chromatin-induced repression of transcription, and (iii) to define the functional core of the H4 tail-associated factors by reconstituting the key regulatory components transcriptionally equivalent to the entire factors. Specific H4 tail-associated factors identified as playing crucial roles in p53-dependent transcription will be assigned as a target for the future design of therapeutic agents to block aberrant p53 regulation related to many human cancers. PUBLIC HEALTH RELEVANCE Our proposal describes multiple approaches to comprehensively understand how a specific chemical modification, acetylation of histone H4 protein within chromosomes, switches genes on in human cells. Since the regulation of this gene activation process is important in the control of cell growth and the establishment and maintenance of tissues and organs, the proposed studies should significantly contribute to our understanding of the molecular basis of many human diseases that are associated with uncontrolled gene expression.

描述（申请人提供）：组蛋白H4 n端尾部乙酰化在染色质环境下的基因激活中起着至关重要的作用；这种表观遗传过程的改变会导致各种疾病。我们的长期目标是了解H4乙酰化如何调节p53依赖性转录以减轻癌症的发展。为了研究这些方面，我们开发了新的方案来询问H4乙酰化作为一种信号的细胞功能，该信号将促进基因转录位点染色质调节机制的招募/保留。重要的是，我们使用野生型或突变的重组H4蛋白开发的重组染色质转录系统使我们能够评估每个特定的H4乙酰化事件在染色质p53依赖性转录中的贡献。转录分析和染色质乙酰化实验的初步结果表明，H4尾部一小部分赖氨酸的乙酰化对转录激活至关重要。为了深入了解H4乙酰化的作用机制，我们建立了稳定表达H4尾部结构域的细胞系，对乙酰化的H4尾部相关因子进行生化纯化。重要的是，我们的功能分析显示，乙酰化的尾部相关因子可以显著增强p53依赖性转录。鉴于乙酰化的H4尾部与调节因子之间的特异性相互作用，我们还生成了带有特定乙酰化标记的细胞可渗透H4尾部模拟物，以筛选细胞转录中最活跃的H4乙酰化事件。基于这些结果，我们的具体目标是：(i)通过检查体外H4赖氨酸底物突变的抑制作用以及体内乙酰化H4尾部模拟物的抑制作用，在p53依赖转录水平上研究H4乙酰化事件的特异性；（ii）通过分离和表征可以抵消染色质诱导的转录抑制的乙酰化H4尾部相关因子，详细阐述H4乙酰化介导的p53依赖性转录激活的分子机制；（iii）通过重组转录等效于整个因子的关键调控组分，确定H4尾部相关因子的功能核心。特定的H4尾相关因子在p53依赖转录中起着至关重要的作用，将被指定为未来设计治疗药物的靶标，以阻断与许多人类癌症相关的p53异常调节。我们的提案描述了多种方法，以全面了解染色体内组蛋白H4蛋白乙酰化的特定化学修饰如何在人类细胞中开启基因。由于该基因激活过程的调控对细胞生长的控制以及组织和器官的建立和维持具有重要意义，因此所提出的研究将显著有助于我们了解许多与基因表达失控相关的人类疾病的分子基础。