- Chromatin regulation
Protein ubiquitination has emerged as a critical mechanism involved in many different aspects of chromatin dynamics: controlling transcription factor activation, mediating DNA damage repair decisions, regulating the timing of DNA replication, and influencing silencing. In fact, the discovery of ubiquitin is rooted in chromatin; ubiquitin was discovered as a covalent modifier of histone H2A and H2B nearly 30 years ago. It was later found that ubiquitinated H2B and H2A are associated with transcriptionally active chromatin in higher eukaryotes, though the role of ubiquitin in this capacity wasn't clear until fairly recently. As it turns out, ubiquitination of histone H2B is required for methylation of histone H3 on 2 distinct residues, Lys4 and Lys79. It's suggested that the ubiquitin moiety on H2B may recruit proteasomal ATPases to prepare the chromatin for Set1p and Dot1p activity.
In Saccharomyces cerevisiae, chromatin-mediated silencing at telomeres and the HM loci is particularly sensitive to histone H3 methylation at Lys4 and Lys79, which appear to disrupt the ability of the silencing proteins Sir3 and Sir4 to bind to, or assemble along, the nucleosome fiber. From an original screen run in Dan Gottschling's lab, it was discovered that the ubiquitin protease Ubp10 (also known as Dot4) is an important regulator of silencing and binds directly to Sir4. With the association of Ubp10 with silent chromatin and the role of H2B ubiquitination in the silencing-antogonistic H3 Lys4 and Lys79 methylation, it seemed reasonable that one function for Ubp10 would be to regulate the levels of H2B ubiquitination to limit H3 Lys4 and Lys79 methylation in silent regions. In fact, that's exactly what we found. Ubp10 is preferentially localized to silent chromatin where its ubiquitin-protease activity removes ubiquitin from histone H2B and maintains low levels of H3 Lys4 and Lys79 methylation to allow optimal Sir protein binding to telomeres and global telomeric silencing.
Interestingly, Ubp10's chromatin function of deubiquitinating histone H2B doesn't appear to be limited to silent chromatin, but also appears to have a role in active chromatin. For example, deletion of any of the silencing SIR genes, which disrupts silencing, does not effect global H2B ubiquitination, whereas deletion of UBP10, which also disrupts silencing, does have an observable effect on global H2B ubiquitination. Thus the bulk of Ubp10 action appears to be in active chromatin. Furthermore, loss of silencing has no effect on genes located out in active chromatin, but eliminating Ubp10 function does result in significant changes in expression in genes located in active chromatin. Lastly, Ubp10 appears to have a synergistic role in terms of H2B ubiquitination with Ubp8, another ubiquitin protease that's been shown to remove ubiquitin from H2B during SAGA-mediated transcription. Loss of both Ubp10 and Ubp8 results in a synergistic increase in histone H2B ubiquitination, indicating that they partially overlap in their target chromatin regions. Loss of both Ubp10 and Ubp8 also results in a synergistic increase in the number of genes with altered expression, indicating that Ubp10 and Ubp8 may share a similar function in gene expression at some loci. Ubp10 and Ubp8 appear to be the only ubiquitin proteases that normally remove mono-ubiquitin from histone H2B and, though there are regions of the genome to which each are specifically targeted, both combine to regulate the global balance of H2B ubiquitination.
Future studies in the lab will focus on understanding Ubp10's role in removing ubiquitin from H2B located in active chromatin. Is this used as a way to regulate gene expression, like Ubp8's role in SAGA, or does it do something entirely different? It's also likely that Ubp10 has targets other than ubiquitinated histone H2B, and we'll be examining the scope of Ubp10 targets through global ubiquitin proteomics studies.
- The other projects
There are a variety of projects going on in the lab from devising global ubiquitin proteomic strategies that will allow us to elucidate the functions of ubiquitin-protein ligases and ubiquitin proteases, to understanding the roles ubiquitination and deubiquitination play in gene transcription and silencing, to uncovering the ways in which the cell destroys aberrant proteins in the nucleus for the purposes of protein quality control. Move mouse over pictures to learn more...