Structure and regulation of the H2B E3 ligase complex in the promotion of euchromatic domains

转录的精细调节是每个细胞功能的基础，在真核生物中，转录发生在基因组的背景下，基因组将组蛋白包裹在核小体中形成染色质。基因组分离成大范围的区室提供了确定活跃转录区（常染色质）和转录沉默区（异染色质）的全局调控模式。每个隔室的特征是组蛋白翻译后修饰的特定模式，包括乙酰化、甲基化、泛素化、磷酸化等等。例如，乙酰化与常染色质相关，而乙酰化的缺失是异染色质的特征。组蛋白H2B的泛素化是与活性转录密切相关的标记之一，特别是通过染色质模板促进转录。裂变酵母S. pombe具有与植物和动物的染色质高度相似的染色质机制，为研究真核基因组如何组织成正染色质和异染色质结构域的分子机制提供了一个有价值的系统。先前的研究已经确定，pombe中负责泛素化的酶，即H2B泛素连接酶复合物HULC本身也受到乙酰化的化学修饰，并且HULC的乙酰化保护基因免受异染色质形成的影响。该提案希望确定HULC如何作为一个分子机器工作，以及它的乙酰化如何有助于其活性及其与染色质和转录机制的相互作用。作为人类的HULC， RNF20/RNF40在转录调控和双链DNA断裂修复中起着至关重要的作用。因此，该提案也将针对人类酶复合物的结构和机制。低温电子显微镜的最新发展使研究复杂的分子靶标（如HULC）成为可能，申请人将使用这一尖端技术和广泛的结构和生化工具来发现HULC如何在分子水平上起作用。研究计划的结果将在裂变酵母和人类细胞中进行功能验证。

The fine-tuned regulation of transcription is fundamental to the functioning of every cell, and in eukaryotes transcription occurs in the context of a genome that is wrapped around histone proteins into nucleosomes to form chromatin. Separation of the genome into large scale compartments provides global patterns of regulation that determine actively transcribed (euchromatin) and transcriptionally silent regions (heterochromatin). Characteristic of each compartment are specific patterns of post-translational modifications on the histone proteins which include acetylation, methylation, ubiquitination, phosphorylation and many more. For example acetylation correlates with euchromatin while the absence of acetylation is characteristic of heterochromatin. Ubiquitination of histone H2B is one of the marks that is strongly associated with active transcription and is in particular involved in facilitating transcription through chromatin templates.The fission yeast S. pombe features a chromatin machinery that is highly similar to chromatin in plants and animals and provides a valuable system to study molecular mechanisms that determine how eukaryotic genomes are organized into euchromatic and heterochromatic domains. Previous research has established that the enzyme responsible for ubiqutination, the H2B ubiquitin ligase complex HULC in S. pombe is itself subject to chemical modifications by acetylation and that the acetylation of HULC protects genes from becoming subject to heterochromatin formation. This proposal wants to determine how HULC works as a molecular machine and how its acetylation contributes to its activity and its interactions with chromatin and the transcription machinery. The human equivalent of HULC, the RNF20/RNF40 plays a crucial role in regulation of transcription as well as in the double strand DNA break repair. The proposal will therefore also target the structure and mechanism of the human enzyme complex. Recent developments in cryo-electron microscopy make investigation of complex molecular targets such as HULC possible and the applicants will use this cutting-edge technology and a wide range of structural and biochemical tools to discover how HULC works at the molecular level. Results from the research proposal will be followed up by functional validation in fission yeast and human cells.