Chemical Strategies to Investigate Gene Regulation by Histone SUMOylation

描述（由申请人提供）：本课题研究的长期目标是通过组蛋白修饰小泛素样修饰物（SUMO）蛋白来获得染色质调控的分子理解。在所有真核生物中都观察到一系列化学基团对组蛋白的翻译后修饰（PTM）。大量的研究表明，组蛋白PTMs的动态调控和特定PTMs之间的生化关系是DNA转录、修复和复制等关键过程的基础。一种引人注目的PTM，组蛋白赖氨酸侧链与SUMO蛋白的结合（称为SUMO化）广泛发生，从酵母到人类，并涉及转录沉默和DNA双链断裂修复。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，如血癌、脑癌、乳腺癌和肾癌，仅举几例。因此，阐明组蛋白summoylation调节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必不可少的第一步。由于无法从体外培养细胞或通过酶促方法获得足够数量的均匀的summoylated组蛋白，因此直到最近，summoylated染色质的生物物理和生化表征一直受到限制。因此，对于组蛋白summoylation影响人类染色质结构和功能的直接和/或间接机制，基本上一无所知。为了解决我们知识上的这一重大差距，我们的目标是结合合成有机化学，生物化学，分子和细胞生物学的工具。我们基于化学生物学的方法包括合成特定位点的summoylated组蛋白用于生化和生物物理研究，以及产生抗体来研究summoylated组蛋白的全基因组发生。通过在体外和细胞中研究组蛋白SUMOylation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。本课题的具体研究目标是：(1)阐明SUMOylation对染色质结构和稳定性的直接影响。(2)阐明组蛋白summoylation与基因激活组蛋白修饰之间的生化关系；(3)研究人染色质活性区和沉默区组蛋白summoylation状态。这些目标的成功完成将导致对sumo介导的染色质结构和功能变化的分子理解。在涉及SUMOylated组蛋白的调控途径中发现新的蛋白-蛋白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，本建议中描述的方法广泛适用，并将作为在其他关键信号蛋白（如转录因子、DNA和组蛋白修饰酶）的背景下研究sumo介导过程的变革性工具。

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.