Project description:Histone methylation plays important roles in the regulation of chromatin dynamics and transcription. Steady state levels of histone lysine methylation are regulated by a balance between enzymes that catalyze either the addition or removal of methyl groups. Using an activity-based biochemical approach, we recently uncovered the JmjC domain as an evolutionarily conserved signature motif for histone demethylases. Furthermore, we demonstrated that Jhd1, a JmjC domain-containing protein in S. cerevisiae, is an H3K36-specific demethylase. Here we report further characterization of Jhd1. Similar to its mammalian homolog, Jhd1-catalyzed histone demethylation requires iron and alpha-ketoglutarate as cofactors. Mutation and deletion studies indicate that the JmjC domain and adjacent sequences are critical for Jhd1 enzymatic activity, while the N-terminal PHD domain is dispensable. Overexpression of JHD1 results in a global reduction of H3K36 methylation in vivo. Finally, chromatin immunoprecipitation coupled microarray (ChIP-chip) studies reveal subtle changes in the distribution of H3K36me2 upon overexpression or deletion of JHD1. Our studies establish Jhd1 as a histone demethylase in budding yeast and suggest that Jhd1 functions to maintain the fidelity of histone methylation patterns along transcription units. Keywords: ChIP-chip H3K36me2 ChIPs were performed on wild type, jhd1 knockout, and JHD1 overexpression yeast strains.
Project description:The transcription elongation factor Spt6 and the H3K36 methyltransferase Set2 are both required for H3K36 methylation and transcriptional fidelity in Saccharomyces cerevisiae. By selecting for suppressors of a transcriptional defect in an spt6 mutant, we have isolated dominant SET2 mutations (SET2sup mutations) in a region encoding a proposed autoinhibitory domain. The SET2sup mutations suppress the H3K36 methylation defect in the spt6 mutant, as well as in other mutants that impair H3K36 methylation. ChIP-seq studies demonstrate that the H3K36 methylation defect in the spt6 mutant, as well as its suppression by a SET2sup mutation, occur at a step following the recruitment of Set2 to chromatin. Other experiments show that a similar genetic relationship between Spt6 and Set2 exists in Schizosaccharomyces pombe. Taken together, our results suggest a conserved mechanism by which the Set2 autoinhibitory domain requires multiple interactions to ensure that H3K36 methylation occurs specifically on actively transcribed chromatin.
Project description:Histone methylation plays important roles in the regulation of chromatin dynamics and transcription. Steady state levels of histone lysine methylation are regulated by a balance between enzymes that catalyze either the addition or removal of methyl groups. Using an activity-based biochemical approach, we recently uncovered the JmjC domain as an evolutionarily conserved signature motif for histone demethylases. Furthermore, we demonstrated that Jhd1, a JmjC domain-containing protein in S. cerevisiae, is an H3K36-specific demethylase. Here we report further characterization of Jhd1. Similar to its mammalian homolog, Jhd1-catalyzed histone demethylation requires iron and alpha-ketoglutarate as cofactors. Mutation and deletion studies indicate that the JmjC domain and adjacent sequences are critical for Jhd1 enzymatic activity, while the N-terminal PHD domain is dispensable. Overexpression of JHD1 results in a global reduction of H3K36 methylation in vivo. Finally, chromatin immunoprecipitation coupled microarray (ChIP-chip) studies reveal subtle changes in the distribution of H3K36me2 upon overexpression or deletion of JHD1. Our studies establish Jhd1 as a histone demethylase in budding yeast and suggest that Jhd1 functions to maintain the fidelity of histone methylation patterns along transcription units. Keywords: ChIP-chip
Project description:Histone lysine methylation is a key epigenetic modification that regulates eukaryotic transcription. In Saccharomyces cerevisiae, it is controlled by a reduced but evolutionarily conserved suite of methyltransferase (Set1p, Set2p, Dot1p, and Set5p) and demethylase (Jhd1p, Jhd2p, Rph1p, and Gis1p) enzymes. Many of these enzymes are extensively phosphorylated in vivo; however, the functions of specific phosphosites are poorly understood. Here, we comprehensively investigate the phosphoregulation of the yeast histone methylation network by analysing 40 phosphosites on six enzymes through mutagenesis. A total of 82 genomically-edited S. cerevisiae strains were generated and screened for changes in native H3K4, H3K36, and H3K79 methylation levels, and for sensitivity to environmental stress conditions. This demonstrated the functional relevance of phosphosites on methyltransferase Set2p (S6, S8, S10, and T127) and demethylase Jhd1p (S44) in the regulation of H3K36 methylation in vivo, and in the coordination of specific stress response pathways in budding yeast. Proteomic analysis of SET2 mutants revealed that phosphorylation site mutations lead to significant downregulation of membrane-associated proteins and processes, consistent with changes brought about by SET2 deletion. This study represents the first systematic investigation into the phosphoregulation of an entire epigenetic network in any eukaryote, and our findings establish phosphorylation as an important regulator of histone lysine methylation in S. cerevisiae.