title: The bromodomain of Gcn5 regulates site specificity of lysine acetylation on histone H3.
abstract: In yeast, the conserved histone acetyltransferase (HAT) Gcn5 associates with Ada2 and Ada3 to form the catalytic module of the ADA and SAGA transcriptional coactivator complexes. Gcn5 also contains an acetyl-lysine binding bromodomain that has been implicated in regulating nucleosomal acetylation in vitro, as well as at gene promoters in cells. However, the contribution of the Gcn5 bromodomain in regulating site specificity of HAT activity remains unclear. Here, we used a combined acid-urea gel and quantitative mass spectrometry approach to compare the HAT activity of wild-type and Gcn5 bromodomain-mutant ADA subcomplexes (Gcn5-Ada2-Ada3). Wild-type ADA subcomplex acetylated H3 lysines with the following specificity; H3K14 > H3K23 > H3K9 ≈ H3K18 > H3K27 > H3K36. However, when the Gcn5 bromodomain was defective in acetyl-lysine binding, the ADA subcomplex demonstrated altered site-specific acetylation on free and nucleosomal H3, with H3K18ac being the most severely diminished. H3K18ac was also severely diminished on H3K14R, but not H3K23R, substrates in wild-type HAT reactions, further suggesting that Gcn5-catalyzed acetylation of H3K14 and bromodomain binding to H3K14ac are important steps preceding H3K18ac. In sum, this work details a previously uncharacterized cross-talk between the Gcn5 bromodomain "reader" function and enzymatic HAT activity that might ultimately affect gene expression. Future studies of how mutations in bromodomains or other histone post-translational modification readers can affect chromatin-templated enzymatic activities will yield unprecedented insight into a potential "histone/epigenetic code." MS data are available via ProteomeXchange with identifier PXD001167.
authors.affiliation: From the ‡Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; §Center for Epigenetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205;
authors.forename: Anne M
authors.initials: AM
authors.lastname: Cieniewicz
authors.affiliation: ¶Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205;
authors.forename: Linley
authors.initials: L
authors.lastname: Moreland
authors.affiliation: ‖Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; **Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ringel
authors.affiliation: ¶Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205;
authors.forename: Samuel G
authors.initials: SG
authors.lastname: Mackintosh
authors.affiliation: From the ‡Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; §Center for Epigenetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205;
authors.forename: Ana
authors.initials: A
authors.lastname: Raman
authors.affiliation: From the ‡Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; §Center for Epigenetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205;
authors.forename: Tonya M
authors.initials: TM
authors.lastname: Gilbert
authors.affiliation: §Center for Epigenetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; ‖Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; **Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Wolberger
authors.affiliation: ¶Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; staverna@jhmi.edu ajtackett@uams.edu.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Tackett
authors.affiliation: From the ‡Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; §Center for Epigenetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; staverna@jhmi.edu ajtackett@uams.edu.
authors.forename: Sean D
authors.initials: SD
authors.lastname: Taverna
mesh_terms: D000107:Acetylation; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D051548:Histone Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D000099224:p300-CBP-Associated Factor
chemical_list: D051548:Histone Acetyltransferases; D006657:Histones; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C090575:ADA2 protein, S cerevisiae; C083323:NGG1 protein, S cerevisiae; C501392:GCN5 protein, S cerevisiae; D000099224:p300-CBP-Associated Factor
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