Molecular interactions of histone ubiquitylation enzymes with the nucleosome

描述(申请人提供)：组蛋白的翻译后修饰现在被确定为调节真核细胞中基因表达的重要机制。然而，对于组蛋白修饰酶如何在其核小体底物上发挥作用的结构和机制缺乏了解。这一缺陷限制了对丰富的遗传、基因组和生化数据的解释，并阻碍了针对与包括癌症在内的人类疾病相关的许多染色质酶的新疗法的开发。为了解决这一不足，我们建议研究PRC1多梳抑制复合体如何与核小体中组装的泛素和组蛋白H2A结合。在组蛋白H_2A中加入泛素具有重要的生物医学意义：组蛋白H_2A的单一泛素化在胚胎干细胞的维持中起重要作用，而BRCA1对组蛋白H_2A的单一泛素化可能介导该乳腺癌蛋白的肿瘤抑制功能。本提案主要针对以下两个具体目标：1.确定核小体上的Prc1泛素化模块的结构。我们建议将UbcH5c/Ring1/Bmi1的E2/E3泛素化络合物与核小体核心粒子形成络合物，并用X射线结晶学确定其原子结构。2.阐明核小体Prc1模块泛素化的机制。结晶学研究将得到生化研究的补充，以分析UbcH5c E2泛素结合和Ring1b/Bmi1 E3泛素连接酶蛋白在泛素化核小体H2A中的作用。

DESCRIPTION (provided by applicant): The post-translational modification of histone proteins is now established as an important mechanism for regulating gene expression in eukaryotic cells. However, a structural and mechanistic understanding of how the histone modification enzymes function on their nucleosome substrate is lacking. This shortcoming limits interpretation of the wealth of genetic, genomic and biochemical data available, and it hampers development of new therapeutics that target the many chromatin enzymes associated with human diseases including cancer. To address this deficiency, we propose to study how the PRC1 polycomb repressive complex conjugates ubiquitin to histone H2A assembled in nucleosomes. The addition of ubiquitin to histone H2A has significant biomedical consequences: mono-ubiquitylation of histone H2A by PRC1 plays a critical role in embryonic stem cell maintenance, while mono-ubiquitylation of histone H2A by BRCA1 may mediate this breast cancer protein's tumor suppressor function. This proposal focuses on the following two specific aims: 1. Determine structure of the PRC1 ubiquitylation module on the nucleosome. We propose to crystallize the E2/E3 ubiquitylation complex of UbcH5c/Ring1/Bmi1 in complex with the nucleosome core particle and to determine the atomic structure of the complex by X-ray crystallography. 2. Elucidate mechanism for PRC1 module ubiquitylation of nucleosomes. The crystallographic studies will be complemented by biochemical studies to analyze the roles of the UbcH5c E2 ubiquitin-conjugating and the Ring1b/Bmi1 E3 ubiquitin ligase proteins in ubiquitylating nucleosomal H2A.