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

点击翻译按钮获取中文摘要

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.