Molecular interactions of histone ubiquitylation enzymes with the nucleosome

描述（由申请人提供）：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，缺乏对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的理解。这一缺点限制了对可用的大量遗传、基因组和生物化学数据的解释，并且阻碍了靶向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。为了解决这一缺陷，我们建议研究如何PRC 1 polycomb抑制复合物共轭泛素组蛋白H2 A组装在核小体。泛素添加到组蛋白H2 A具有显著的生物医学后果：PRC 1对组蛋白H2 A的单泛素化在胚胎干细胞维持中起关键作用，而BRCA 1对组蛋白H2 A的单泛素化可能介导这种乳腺癌蛋白的肿瘤抑制功能。这项建议着重于以下两个具体目标：1.确定核小体上PRC 1泛素化模块的结构。我们建议结晶的E2/E3泛素化复合物的UbcH 5c/Ring 1/Bmi 1在复杂的核小体核心颗粒，并确定通过X-射线晶体学的原子结构的复合物。2.阐明PRC 1模块在核小体泛素化的机制。晶体学研究将补充生化研究，以分析泛素化核小体H2 A中UbcH 5c E2泛素缀合和Ring 1b/Bmi 1 E3泛素连接酶蛋白的作用。

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.