Chemical Strategies to Investigate Gene Regulation by Histone SUMOylation

描述(由申请人提供)：这项研究的长期目标是通过用小泛素样修饰物(SUMO)蛋白修饰组蛋白来获得对染色质调节的分子理解。组蛋白被一系列化学基团翻译后修饰(PTM)存在于所有真核生物中。大量工作表明，组蛋白PTM的动态调控和特定PTM之间的生化关系是DNA转录、修复和复制等关键过程的基础。一种戏剧性的PTM，组蛋白赖氨酸侧链与蛋白质SUMO的结合(称为SUMO作用)广泛存在，从酵母到人类，与转录沉默和DNA双链断裂修复有关。环境或遗传因素对这些关键过程的失调与许多人类疾病有关，例如血癌、脑癌、乳腺癌和肾癌等等。因此，阐明组蛋白SUMO化调控转录和基因修复的分子机制是设计合理的人类急性疾病治疗策略的关键第一步。直到最近，由于无法从培养细胞或酶方法中获得足够数量的均一组蛋白来进行体外研究，SuMO化染色质的生物物理和生化特性一直受到限制。因此，对组蛋白SUMO化影响人染色质结构和功能的直接和/或间接机制基本上一无所知。为了解决我们知识中的这一重大差距，我们的目标是将合成有机化学、生物化学、分子和细胞生物学的工具结合起来。我们的基于化学生物学的方法包括合成用于生化和生物物理研究的特定部位的SUMO化组蛋白，以及产生抗体来研究SUMO化组蛋白在基因组范围内的发生。通过采用体外和细胞内研究组蛋白SUMO化的方法，我们将全面了解这种修饰在正常生长和疾病进展中的机制作用。这一建议的具体研究目标是：(1)阐明SUMO化对染色质结构和稳定性的直接影响。(2)阐明组蛋白SUMO化与基因活化组蛋白修饰之间的生化关系；(3)研究人染色质活跃区和静止区的组蛋白SUMO化状态。这些目标的成功完成将导致对相扑介导的染色质结构和功能变化的分子理解。识别涉及SUMO化的组蛋白调控通路中新的蛋白质-蛋白质相互作用可能为组蛋白修饰失调引起的疾病的治疗发明提供新的靶点。最后，本提案中描述的方法具有广泛的适用性，并将作为研究相扑调节过程的变革性工具，在其他关键信号蛋白的背景下，如转录因子、DNA和组蛋白修饰酶。

DESCRIPTION (provided by applicant): The long-term goal of the research in this proposal is to gain a molecular understanding of chromatin regulation by histone modification with the small ubiquitin-like modifier (SUMO) protein. The post-translational modification (PTM) of histone proteins by a range of chemical groups is observed in all eukaryotes. An extensive body of work has established that the dynamic regulation of histone PTMs and the biochemical relationships between specific PTMs underlie critical processes such as DNA transcription, repair, and replication. One dramatic PTM, the conjugation of histone lysine side-chains with the protein SUMO (termed SUMOylation) occurs widely, from yeast to humans, and is implicated in transcriptional silencing and DNA double-strand break repair. The dysregulation of these critical processes by environmental or genetic factors is linked to many human diseases such as cancers of the blood, brain, breast, and kidneys, to name a few. Therefore elucidating the molecular mechanisms by which histone SUMOylation regulates transcription and gene repair are essential first steps toward devising rational therapeutic strategies for acute human diseases. The biophysical and biochemical characterization of SUMOylated chromatin has until recently been limited by the inability to obtain sufficient quantities of homogeneously SUMOylated histones for in vitro studies, either from cultured cells or by enzymatic means. Hence, essentially nothing is known about the direct and/or indirect mechanisms by which histone SUMOylation influences the structure and function of human chromatin. In order to address this significant gap in our knowledge, we aim to combine the tools of synthetic organic chemistry, biochemistry, molecular and cell biology. Our chemical biology-based approach involves synthesizing site-specifically SUMOylated histones for biochemical and biophysical studies, as well as generating antibodies to investigate the genome-wide occurrence of SUMOylated histones. By adopting methods to study histone SUMOylation both in vitro and in cells, we will gain a comprehensive understanding of the mechanistic roles for this modification in normal growth and in disease progression. The specific research objectives of this proposal are: (1) To elucidate the direct effects of SUMOylation on chromatin structure and stability. (2) To elucidate the biochemical relationship between histone SUMOylation and gene-activating histone modifications, and (3) To investigate the histone SUMOylation state of active and silent regions of human chromatin. The successful completion of these aims will lead to a molecular understanding of SUMO-mediated changes in chromatin structure and function. Identifying new protein- protein interactions in regulatory pathways involving SUMOylated histones may provide new targets for therapeutic invention in diseases arising from the dysregulation of histone modifications. Finally, the methodologies described in this proposal are broadly applicable and will serve as transformative tools for studying SUMO-mediated processes in the context of other key signaling proteins, such as transcription factors, DNA- and histone-modifying enzymes.