title: Functional analysis of Bre1p, an E3 ligase for histone H2B ubiquitylation, in regulation of RNA polymerase II association with active genes and transcription in vivo.
abstract: H2B ubiquitylation is carried out by Bre1p, an E3 ligase, along with an E2 conjugase, Rad6p. H2B ubiquitylation has been previously implicated in promoting the association of RNA polymerase II with the coding sequence of the active GAL1 gene, and hence transcriptional elongation. Intriguingly, we find here that the association of RNA polymerase II with the active GAL1 coding sequence is not decreased in Δbre1, although it is required for H2B ubiquitylation. In contrast, the loss of Rad6p significantly impairs the association of RNA polymerase II with GAL1. Likewise, the point mutation of lysine 123 (ubiquitylation site) to arginine of H2B (H2B-K123R) also lowers the association of RNA polymerase II with GAL1, consistent with the role of H2B ubiquitylation in promoting RNA polymerase II association. Surprisingly, unlike the Δrad6 and H2B-K123R strains, complete deletion of BRE1 does not impair the association of RNA polymerase II with GAL1. However, deletion of the RING domain of Bre1p (that is essential for H2B ubiquitylation) impairs RNA polymerase II association with GAL1. These results imply that a non-RING domain of Bre1p counteracts the stimulatory role of the RING domain in regulating the association of RNA polymerase II with GAL1, and hence RNA polymerase II occupancy is not impaired in Δbre1. Consistently, GAL1 transcription is impaired in the absence of the RING domain of Bre1p, but not in Δbre1. Similar results are also obtained at other genes. Collectively, our results implicate both the stimulatory and repressive roles of Bre1p in regulation of RNA polymerase II association with active genes (and hence transcription) in vivo.
authors: Department of Biochemistry and Molecular Biology, Southern Illinois University, School of Medicine, Carbondale, Illinois 62901.
authors: Rwik
authors: R
authors: Sen
authors: Department of Biochemistry and Molecular Biology, Southern Illinois University, School of Medicine, Carbondale, Illinois 62901.
authors: Shweta
authors: S
authors: Lahudkar
authors: Department of Biochemistry and Molecular Biology, Southern Illinois University, School of Medicine, Carbondale, Illinois 62901.
authors: Geetha
authors: G
authors: Durairaj
authors: Department of Biochemistry and Molecular Biology, Southern Illinois University, School of Medicine, Carbondale, Illinois 62901. Electronic address: sbhaumik@siumed.edu.
authors: Sukesh R
authors: SR
authors: Bhaumik
mesh_terms: D047369:Chromatin Immunoprecipitation; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006657:Histones / Q000737:chemistry*; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D025801:Ubiquitin / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism
chemical_list: C472168:Bre1 protein, S cerevisiae; D006657:Histones; D009687:Nuclear Proteins; C097698:PAF1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D012319:RNA Polymerase II