Regulatory Mechanisms for the Pol II Associated Histone Methyltransferase Set2

Nucleosomes are building blocks of the eukaryotic chromatin which package genomic DNA with histones. The modification patterns of histones constitute an important signaling pathway for various nuclear processes. H3K36 methylation is catalyzed by the histone methyltranserase Set2 during transcription elongation. This important histone mark is ubiquitously presented in all organisms from yeast to mammals. In this study, we set out to investigate the molecular mechanisms by which the Set2 activity is precisely regulated during dynamic transcription cycle. In the first part of the study we discovered a novel role of the Set2 SRI domain, which is responsible for the binding of Set2 to elongating RNA polymerase II. We show that SRI also binds to DNA which determines the substrate specificity of Set2. In addition, we identified a novel auto-inhibitory role for the middle region of Set2 in regulating catalytic activity of Set2. Remarkably, mutations at this region cause hyperactivities, which in turn lead to synthetic phenotype with an essential histone chaperone FACT. Our data suggests that a temporal control for dynamic chromatin regulation is needed during transcription elongation process. In the second part, we investigated the molecular mechanism by which elongating Pol II regulated the Set2 activity beyond its initial recruitment. Surprisingly, we found the excessive amount of phosphorylated serine residues on Pol II CTD inhibited Set2 activity in vitro. Subsequent biophysical examination revealed that the additional phosphorylated CTD repeats collaterally occupied the surface of SRI where SRI contacts DNA. Finally, we determined that the minimal recognition unit of Set2 on fully phosphorylated CTD tail is three heptad repeats, and demonstrated that this minimal unit is sufficient for the Set2 recruitment without disrupting its catalytic activity. Since Pol II CTD utilizes repeating sequence as a scaffold for multiple factors, our results implicate that an organized spatial arrangement of these factors along CTD is necessary for accommodating their individual functions. In the last part of this work, we examined the state-specific functions of H3K36 methylation. By manipulating the catalytic domain of Set2, we obtained two mutants that can catalyze specific methyl-states of H3K36 both in vitro and in vivo. Genetic studies showed that cells carrying these two mutants displayed distinct phenotypes in several functional pathways, including histone chaperone, CTD proline isomerization and double-strand DNA repair. Our results suggest individual methyl-state of H3K36 plays non-redundant biological roles in cells. In summary, we discovered multiple mechanisms by which Set2 is dynamically regulated by elongating Pol II. Setd2, the human homolog of yeast Set2, has been shown recently to be one of the most important tumor suppressors among chromatin regulators. Given the highly conserved nature of this histone methyltransferase family, we believe that our mechanistic studies here may shed lights on the roles of Setd2 in tumorigenesis.