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

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.

H2B monoubiquitination (H2Bub1) is a central histone modification in many cellular processes ranging from transcription to DNA repair. H2Bub1 is deposited by a complex consisting of an E2 conjugating enzyme and an E3 ligase, which have been identified as Rad6 and Bre1 in S. cerevisiae. The human homolog is the RNF20/40 complex and in S. pombe this complex is represented by the proteins Brl1/Brl2 and the Rhp6 E2 ligase, named H2B ubiquitin ligase complex (HULC). Recently, a role for acetylation of Brl1 has been identified in protection of euchromatic gene transcription from heterochromatization by the RNA interference pathway. This suggests regulation of HULC by posttranslational modification. The aim of this proposal is to elucidate the molecular mechanism that underpins this regulation by studying the biochemistry and structure of the HULC complex. Furthermore, this proposal will target the structure of intact HULC in the free and nucleosome-bound state by cryo-EM and X-ray crystallography. This will provide unprecedented structural information for this highly conserved family of protein complexes.

This project aligns with a number of aspects of the BBSRC's Strategic Plan. The discovery program proposed here targets a central complex of eukaryotic transcription and will therefore lead to high impact publications, which contribute to maintaining the UK's position as a global leader in bioscience. The outcomes of the project contribute specifically to Strategic Research Priority 3 - Bioscience for Health - by driving advances in fundamental bioscience, which underpin pharmaceutical and healthcare industries.

1. Societal benefit: Supporting academic research excellence. By supporting academic research excellence in the central theme of transcription and chromatin this proposal benefits the UK science base, contributing to the maintenance of the UK's world leading role in the biosciences. As detailed in the academic beneficiaries section, this research is at the interface of several large biological research domains, and will therefore be widely recognized.

2. Economic impact: Stimulating industrial innovation. This study will support and stimulate new industrial innovation. With its connection to cancer and by delivering structural results at near-atomic resolution this proposal generates data that can be directly employed for development and optimization of bioactive molecules. New therapeutic agents and research reagents potentially being developed as a consequence of the proposed research may provide great commercial return.

3. Providing a scientifically well-trained professional workforce. This proposal will provide training in a wide range of disciplines (structural biology, biochemistry, genomics, fission yeast) and will equip a PDRA and a PhD student with publications, know-how, professional network and leadership experience that will allow them to move to the next level in their career.

4. Education of the general public. The PDRA and PI will engage in public outreach activities in order to inform the public about the results of the basic research pursued in this proposal. Outreach efforts will be part of the proposed research because it is an important aspect of basic science to communicate in understandable language about the advances and benefits that are produced by the proposed research.