title: The Set2 histone methyltransferase functions through the phosphorylated carboxyl-terminal domain of RNA polymerase II.
abstract: The histone methyltransferase Set2, which specifically methylates lysine 36 of histone H3, has been shown to repress transcription upon tethering to a heterologous promoter. However, the mechanism of targeting and the consequence of Set2-dependent methylation have yet to be demonstrated. We sought to identify the protein components associated with Set2 to gain some insights into the in vivo function of this protein. Mass spectrometry analysis of the Set2 complex, purified using a tandem affinity method, revealed that RNA polymerase II (pol II) is associated with Set2. Immunoblotting and immunoprecipitation using antibodies against subunits of pol II confirmed that the phosphorylated form of pol II is indeed an integral part of the Set2 complex. Gst-Set2 preferentially binds to CTD synthetic peptides phosphorylated at serine 2, and to a lesser extent, serine 5 phosphorylated peptides, but has no affinity for unphosphorylated CTD, suggesting that Set2 associates with the elongating form of the pol II. Furthermore, we show that set2Delta ppr2Delta double mutants (PPR2 encodes TFIIS, a transcription elongation factor) are synthetically hypersensitive to 6-azauracil, and that deletions in the CTD reduce in vivo levels of H3 lysine 36 methylation. Collectively, these results suggest that Set2 is involved in regulating transcription elongation through its direct contact with pol II.
authors.affiliation: Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802-4500, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.forename: LeAnn
authors.initials: L
authors.lastname: Howe
authors.forename: Scott
authors.initials: S
authors.lastname: Anderson
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.forename: Jerry L
authors.initials: JL
authors.lastname: Workman
mesh_terms: D000963:Antimetabolites / Q000494:pharmacology; D019175:DNA Methylation; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D017353:Gene Deletion; D006657:Histones / Q000378:metabolism; D015151:Immunoblotting; D008239:Lysine / Q000737:chemistry; D008780:Methyltransferases / Q000737:chemistry / Q000502:physiology*; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000502:physiology*; D014158:Transcription, Genetic; D014498:Uracil / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D000963:Antimetabolites; D005656:Fungal Proteins; D006657:Histones; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D014498:Uracil; D008780:Methyltransferases; C448996:Set2 protein, S cerevisiae; D012319:RNA Polymerase II; C100248:azauracil; D008239:Lysine