Studies in Saccharomyces cerevisiae
The MLL gene was cloned over twenty years ago, yet the first molecular function for a MLL homolog was defined by our laboratory when we demonstrated that yeast Set1 (the MLL homolog) is a component of a large complex, Set1/COMPASS, which methylates histone H3 on its fourth lysine (H3K4) in the early transcribed regions of genes. Subsequent work by us and other labs demonstrated that the COMPASS family is highly conserved from yeast to Drosophila to mammalian cells functioning as H3K4 methylases. Although there is only one COMPASS in yeast, there are at least three COMPASS family members in Drosophila (dSet1, trx and Trr) and six COMPASS family members in human cells (Set1A-B, MLL1-4) with non-redundant functions.
To better define the molecular machinery required for proper histone H3K4 methylation by Set/1COMPASS in yeast and the COMPASS family in Drosophila and human cells, we devised a global functional proteomic screen, which we call Global Proteomic analysis in S. cerevisiae (GPS). With GPS, we tested 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 monoubiquitination 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 a role for the Paf1 complex, the elongation factor, and the Bur1/Bur2 kinase for the proper regulation of H2B monoubiquitination. We now know that this H3K4 methylation machinery pathway is also highly conserved from yeast to Drosophila to human.
Given the extraordinary power of GPS in yeast, we are employing GPS to better define the molecular machinery required for COMPASS 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.