ROLE OF HISTONE H4 N-TERMINAL TAIL IN TRANSCRIPTION REGULATION

描述（由申请人提供）：组蛋白H4 N-末端尾部的乙酰化在染色质环境中的基因激活中起着至关重要的作用;并且该表观遗传过程的改变可导致各种疾病。我们的长期目标是了解H4乙酰化如何调节p53依赖性转录以减弱癌症发展。为了研究这些方面，我们已经开发了新的协议来询问H4乙酰化的细胞功能，作为一个信号，这将有助于在基因转录的位点上的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蛋白的重组染色质转录系统的开发使我们能够评估每个特定的H4乙酰化事件在染色质的p53依赖性转录中的贡献。我们的初步结果，从转录分析，加上染色质乙酰化测定，表明乙酰化只有一小部分的赖氨酸在H4尾是转录激活的关键。为了深入了解H4乙酰化的功能，我们建立了稳定表达H4尾结构域的细胞系，用于乙酰化H4尾相关因子的生化纯化。重要的是，我们的功能分析表明，乙酰化的尾部相关因子可以显着增强p53依赖的转录。鉴于乙酰化的H4尾和调节因子之间的特异性相互作用，我们还产生了具有特异性乙酰化标记的细胞可渗透的H4尾模拟物，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通过表征乙酰化H4尾部模拟物的体内抑制作用，研究H4乙酰化事件在p53依赖性转录水平上的特异性，（ii）通过分离和表征乙酰化的H4尾相关因子来详细说明H4乙酰化介导的p53依赖性转录激活的分子机制，所述乙酰化的H4尾相关因子可以抵消染色质-诱导转录抑制，以及（iii）通过重建转录上相当于整个因子的关键调节组分来定义H4尾部相关因子的功能核心。被鉴定为在p53依赖性转录中起关键作用的特定H4尾相关因子将被指定为未来设计治疗剂的靶点，以阻断与许多人类癌症相关的异常p53调节。我们的提案描述了多种方法，以全面了解特定的化学修饰，染色体内组蛋白H4蛋白的乙酰化，如何在人类细胞中打开基因。由于这种基因激活过程的调控在控制细胞生长和组织器官的建立和维持中非常重要，因此拟议的研究应有助于我们了解与基因表达不受控制相关的许多人类疾病的分子基础。

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.