Chemical strategies to investigate biochemical crosstalk in human chromatin

项目总结 染色质是一种巨大的核蛋白复合体，包装了大约30亿 有核细胞中遗传信息的碱基对。组蛋白与转录因子 (TFS)是与染色质相关的两个重要蛋白质家族，起着关键作用 在组织、保护和激活我们的基因方面的作用。其中一个保守的特征 组蛋白和转录因子在翻译后修饰(PTM)中起着关键作用 控制着它们的不同功能。在我们对这一问题的理解上仍然存在许多差距 低丰度的特定组蛋白和转铁蛋白PTM的机械作用 或者没有必要的分子生物和化学工具 机械学研究。这在研究监管方面尤其具有挑战性。 肿瘤抑制基因P53被我们体内的蛋白酶体机制迅速翻转 细胞。拟议的研究项目旨在克服 组蛋白和P53修饰的低丰度和异质性 结合化学和分子生物学工具来产生特定部位的 修饰的蛋白质。具体地说，总和化组蛋白H2B和H4以及甲基化的P53 将由新的蛋白质半合成技术产生。半合成的 蛋白质将接受一系列生物物理和生化分析，以便 阐明苏莫化和甲基化在染色质调节中的机制作用 结构和功能。生化分析的结果将在细胞内进一步验证- 基于研究得出分子机制的完整图景 组蛋白苏莫化和P53甲基化对基因的调控。的长期目标是 这个项目是为了识别细胞中新的生化关系，或称串扰。 染色质可以被控制来控制细胞的命运并选择性地 在治疗上针对人类疾病。

PROJECT SUMMARY Chromatin is a massive nucleoprotein complex that packages about three billion base pairs of genetic information in nucleated cells. Histones and transcription factors (TFs) are two important families of proteins associated with chromatin that play key roles in organizing, protecting and activating our genes. A conserved feature among histones and TFs is the critical role that post-translational modifications (PTMs) play in controlling their diverse functions. Many gaps remain in our understanding of the mechanistic roles for specific histone and TF PTMs that are either low in abundance or where the necessary molecular biological and chemical tools are unavailable for mechanistic studies. This is especially challenging when studying regulation of the tumor suppressor p53 that is rapidly turned over by the proteasomal machinery in our cells. The proposed research project seeks to overcome challenges arising from the low abundance and heterogeneity of histone and p53 modifications by applying a combination of chemical and molecular biological tools to generate site-specifically modified proteins. Specifically, sumoylated histones H2B and H4 and methylated p53 will be generated by new protein semisynthesis techniques. The semisynthetic proteins will be subjected to a range of biophysical and biochemical assays in order to elucidate the mechanistic roles for sumoylation and methylation in regulating chromatin structure and function. Results from biochemical assays will be further validated in cell- based studies to arrive at a complete picture of the molecular mechanisms underlying gene regulation by histone sumoylation and p53 methylation. The long-term goal of this project is to identify new biochemical relationships, or crosstalk, in cellular chromatin that may be controlled to engineer cellular fates and selectively therapeutically targeted in human diseases.