Chromatin recruitment of Rad6 determines H3K4me3 in Saccharomyces cerevisiae

Project abstract : The trimethylation of histone H3 lysine 4 (H3K4me3) is a crucial factor in defining the promoter regions of active genes in all eukaryotes ranging from Saccharomyces cerevisiae (yeast) to humans. In budding yeast, this trimethylation process facilitated by the Set1 complex results in H3K4me3 requiring a prior mono-ubiquitination at the histone H2BK123 residue (H2Bub) by E2 enzyme Rad6 and E3 enzyme Bre1. A previous in vitro study suggested that ubiquitinated H2B directly facilitates H3K4me3. However, even low levels of global H2Bub is sufficient for the required H3K4me3 in yeast cells, thereby indicating that other factors resulting in the H2Bub-dependent H3K4me3 remain unknown. This study revealed the high level of correlation of H3K4me3 with chromatin recruitment of Rad6 at the genome-wide level. Rad6 is confirmd to interact and co-localize with Swd2/Cps35, a key factor for the H2Bub-dependent H3K4me3 in genes with high levels of H3K4me3 and intronic genes rather than non-intronic genes. This study therefore provides a mechanistic insight of the H2Bub-Rad6- Swd2/Cps35-H3K4me3 axis and its potential role in RNA biogenesis. Overall design: To define the global binding sites of Rad6 and to identify whether chromatin recruitment of Rad6 is affected by catalytic activity or phosphorylation status of Rad6, chromatin immunoprecipitation (ChIP) sequencing against 9myc antibody is performed with yeast strains endogenously tagged with 9myc at Rad6 locus in WT, Rad6 C88A catalytically dead mutant, Rad6S120A phosphorylation site mutant background. To confirm if H2B ubiquitination contributes to chromatin recruitment of Rad6, ChIP with 9myc antibody is performed to endogenously expressed Rad6-9myc at H2B WT and H2B K123R strain. 9myc ChIPseq is carried out with no tag WT strain as control. To define the chromatin binding sites of Swd2 and to compare with chromatin binding of Rad6 and H3K4me3, ChIP against 3HA tag of endogenously tagged Swd2-3HA WT strain is performed.