Studies in Saccharomyces cerevisiae
Although the MLL gene was cloned twenty years ago, the first molecular function for an MLL homologue was defined by our laboratory when we demonstrated that yeast Set1 (the MLL homologue) is a component of a large complex, COMPASS, that methylates histones in the early transcribed region of the genes. Subsequent work by us and others demonstrated that MLL is part of a homologous complex in higher organisms and the components of its complex behave similarly from yeast to human.
To better define the molecular machinery required for proper histone methylation by COMPASS in yeast and the MLL complex in human, we devised a global functional proteomic screen. We call this screen the Global Proteomic analysis in S. cerevisiae (GPS). In GPS, we test extracts of each of the non-essential yeast gene deletion mutants in different mating types (~15,000 strains) for defects in modifications of histones by Western blotting. Employing an antibody specific to histone H3 methylated on its fourth lysine, GPS revealed that the ubiquitination of lysine 123 of histone H2B by Rad6 (the E2 conjugating enzyme) is required for histone methylation by COMPASS. We have taken advantage of GPS and have been able to put together a molecular pathway of factors required for proper histone methylation by COMPASS. This includes a role for Bre1 as Rad6’s E3 ligase and the elongation factor, the Paf1 complex, and the Bur1/Bur2 kinase for the proper regulation of H2B monoubiquitination.
We are employing GPS in yeast to better define the molecular machinery required for COMPASS's function and have also applied this screen to other posttranslational modifications of histones such as H3K36 methylation, H3K79 methylation and H3K56 acetylation. There is no doubt that the application of GPS will be extremely informative in defining such molecular pathways.