The Bromodomain of Gcn5 Regulates Site Specificity of Lysine Acetylation on Histone H3
2014; Elsevier BV; Volume: 13; Issue: 11 Linguagem: Inglês
10.1074/mcp.m114.038174
ISSN1535-9484
AutoresAnne M. Cieniewicz, Linley Moreland, Alison E. Ringel, Samuel G. Mackintosh, Ana Raman, Tonya M. Gilbert, Cynthia Wolberger, Alan J. Tackett, Sean D. Taverna,
Tópico(s)Ubiquitin and proteasome pathways
ResumoIn 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. 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. Eukaryotic DNA is packaged around the histone proteins H3, H4, H2A, and H2B to form the nucleosomal core particle that is the fundamental building block of chromatin (1Kornberg R.D. Lorch Y. 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In contrast, mass spectrometric approaches yield detailed information describing the specificity of a HAT reaction regarding acetylation rates for specific lysines (31Arnold K.M. Lee S. Denu J.M. Processing mechanism and substrate selectivity of the core NuA4 histone acetyltransferase complex.Biochemistry. 2011; 50: 727-737Crossref PubMed Scopus (9) Google Scholar). However, previous mass spectrometric studies of Gcn5 HAT kinetics predominantly analyzed combined populations of the histone acetylation states generated during a particular reaction, so it is difficult to interpret information about HAT site specificity (the acetylation of a specific lysine relative to that of a proximal lysine) (16Kuo Y.M. Andrews A.J. Quantitating the specificity and selectivity of Gcn5-mediated acetylation of histone H3.PLoS One. 2013; 8: e54896Crossref PubMed Scopus (75) Google Scholar, 32Henry R.A. Kuo Y.M. Andrews A.J. 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Tan S. Expression and purification of recombinant yeast Ada2/Ada3/Gcn5 and piccolo NuA4 histone acetyltransferase complexes.Methods. 2007; 41: 271-277Crossref PubMed Scopus (22) Google Scholar). Full-length Ada3 was generated using nested primers to extend the truncated ADA3 fragment, which was cloned into the pST44 vector (supplemental Fig. S1). The Gcn5 Y413A bromodomain mutant and the Gcn5 P371T/M372A double point mutant were generated using QuikChange site-directed mutagenesis (LaJolla, CA) on the plasmid containing full-length ADA3. The expression of full-length Gcn5/Ada2/Ada3 subcomplex and subsequent purification with Talon metal affinity resin (Clontech) and an FPLC SourceQ column (GE Healthcare) were performed as previously described (33Barrios A. Selleck W. Hnatkovich B. Kramer R. Sermwittayawong D. Tan S. Expression and purification of recombinant yeast Ada2/Ada3/Gcn5 and piccolo NuA4 histone acetyltransferase complexes.Methods. 2007; 41: 271-277Crossref PubMed Scopus (22) Google Scholar). Concentrations of the recombinant wild-type and mutant Gcn5/Ada2/Ada3 subcomplexes were normalized using SDS-PAGE gels and Coomassie Blue staining. Wild-type (WT), Y413A, and P371T/M372A yeast Gcn5 bromodomains (residues 329–438 of Gcn5p) were cloned from the respective pST44-yAda3t2HISx3-yAda2 × 3-yGcn5 × 5 polycistronic vectors containing yeast GCN5, ADA2, and ADA3 from WT, Y413A, and P371T/M372A. Bromodomains were cloned into an N-terminal thioredoxin-HIS6-S•tag tag (pET32a vector, Darmstadt, Germany). Proteins were recombinantly expressed in chemically competent BL21 Escherichia coli (Invitrogen) after overnight induction with 1 mm isopropyl 1-thio-â-D-galactopyranoside at 20 °C in LB medium. Bacteria were pelleted, freeze-thawed, and resuspended in purification buffer (50 mm Tris, pH 7.5, 500 mm NaCl, 40 mm imidazole, 10% glycerol, 2 mm B-ME, 1 mm PMSF, 2 mm benzamidine) and lysed by sonication (Thomas Scientific, Swedesboro, NJ). Lysate was incubated with nickel-nitrilotriacetic acid agarose resin (Invitrogen) for least 1 h at 4 °C. Resin was washed with purification buffer, and protein was eluted with purification buffer containing 150 mm imidazole. Protein was aliquoted, flash-frozen in liquid nitrogen, and stored at −80 °C. Recombinant histones from Xenopus laevis were expressed in E. coli, purified from inclusion bodies, and assembled into histone octamers as described previously (34Luger K. Rechsteiner T.J. Richmond T.J. Expression and purification of recombinant histones and nucleosome reconstitution.Methods Mol. Biol. 1999; 119: 1-16PubMed Google Scholar). DNA for recombinant mononucleosomes was obtained via EcoRV digestion of pST55–16xNCP601, courtesy of Dr. Song Tan (Penn State, PA), which contained 16 tandem copies of a 147-bp fragment with the 601 positioning sequence (35Lowary P.T. Widom J. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.J. Mol. Biol. 1998; 276: 19-42Crossref PubMed Scopus (1203) Google Scholar). Prior to nucleosome reconstitution, the 147-bp DNA fragments were purified as described previously (36Dyer P.N. Edayathumangalam R.S. White C.L. Bao Y. Chakravarthy S. Muthurajan U.M. Luger K. Reconstitution of nucleosome core particles from recombinant histones and DNA.Methods Enzymol. 2004; 375: 23-44Crossref PubMed Scopus (535) Google Scholar). Nucleosome core particles were assembled using the salt gradient dialysis method followed by HPLC purification on a TSKgel DEAE-5PW column with 13-μm particle size (34Luger K. Rechsteiner T.J. Richmond T.J. Expression and purification of recombinant histones and nucleosome reconstitution.Methods Mol. Biol. 1999; 119: 1-16PubMed Google Scholar). Nucleosomes were dialyzed into low-salt buffer (10 mm Tris-HCl, pH 7.5, 5 mm KCl, 1 mm DTT) and concentrated to 25 to 50 μm for storage at 4 °C, and they were used within a month of preparation. HAT assays were performed with full-length recombinant Gcn5/Ada2/Ada3 subcomplex using 1 μg of recombinantly expressed Saccharomyces cerevisiae free histone H3 and 30 μm acetyl CoA in HAT reaction buffer (20 mm Tris, pH 7.5, 50 mm NaCl, 5% glycerol) in a total volume of 60 μl. For HAT assays with variable enzyme concentrations, samples were incubated for 30 min at 30 °C and then flash-frozen in liquid nitrogen to stop the reaction. Samples were then lyophilized. Acetyl CoA was omitted from the control reactions. HAT assays run over a time course were first incubated at 30 °C for 2 min prior to the addition of enzymatic subcomplex at a concentration of 50 nm, except for the Time 0 assay, which was flash frozen immediately upon the addition of enzyme. Reactions were carried out for the respective time course at 30 °C and flash-frozen and lyophilized upon completion. A non-enzymatic reaction was carried out for 8 h at 30 °C to control for spontaneous acetyl CoA acetylation or histone degradation (supplemental Fig. S4). HAT reactions analyzed via quantitative mass spectrometry were performed in triplicate using 150 nm of WT or Y413A ADA subcomplex and carried out at 30 °C for 30 min. Assays containing nucleosomal substrates consisted of 0.55 μm nucleosome, 150 nM of either WT or Y413A subcomplex, and 30 μm acetyl CoA carried out in a total 30 μl reaction volume. Reactions were run for 30 min at 30 °C and flash-frozen upon completion. ADA subcomplex was omitted from the non-enzymatic negative controls. The pET3a vector containing yeast histone H3 sequence was obtained courtesy of Dr. Gregory Bowman (The Johns Hopkins University, Baltimore, MD). Both H3K14R and H3K23R were generated using QuikChange site-directed mutagenesis (Stratagene). Histones were expressed, purified from inclusion bodies, and further purified over HPLC. To obtain a maximally acetylated histone H3 control, we subjected 200 μg of recombinant S. cerevisiae histone H3 to in vitro acetylation as previously outlined (37Papazyan R. Taverna S.D. Separation and purification of multiply acetylated proteins using cation-exchange chromatography.Methods Mol. Biol. 2013; 981: 103-113Crossref PubMed Scopus (4) Google Scholar). Acetylated histone H3 was purified and desalted using HPLC purification, and fractions were combined, flash-frozen, and lyophilized. Concentrations were normalized using a Ponceau stained dot blot. All lyophilized samples were resuspended in Laemmli loading buffer. Running chambers were washed with methanol to prevent keratin contamination. NuPAGE SDS-PAGE 12% gels (Invitrogen) were used to resolve histone H3. Acid-urea gels were assembled and run as previously described (38Shechter D. Dormann H.L. Allis C.D. Hake S.B. Extraction, purification and analysis of histones.Nat. Protoc. 2007; 2: 1445-1457Crossref PubMed Scopus (716) Google Scholar). Gels were washed with nano-pure water and stained with SimplyBlue Safe Stain (Invitrogen). Bands were individually excised using a clean scalpel and frozen for subsequent mass spectrometric analysis. Histone H3 samples were resolved by means of SDS-PAGE and transferred to a PVDF membrane using a semi-dry transfer system. Samples resolved on acid urea gels were transferred to a PVDF membrane as previously described (38Shechter D. Dormann H.L. Allis C.D. Hake S.B. Extraction, purification and analysis of histones.Nat. Protoc. 2007; 2: 1445-1457Crossref PubMed Scopus (716) Google Scholar). Membranes were blocked overnight in 5% milk at 4 °C and washed in Tris-buffered saline (TBS). Primary antibodies were diluted in 1% milk in TBS and 0.1% Tween as follows for nucleosomes (Fig. 6 and supplemental Fig. S2): anti-H3 (Cambridge, MA ab1791, 1/50,000), anti-H4 (Abcam ab10158–25, 1/5000), anti-H2B (Abcam ab1790–25, 1/20,000), anti-H3K9ac (Abcam, 1/5000), anti-H3ac (Millipore, 1/5000), anti-H3K14ac (ab52946, Abcam, 1/5000), anti-H3K18ac (Carlsbad, CA 39130, 1/10,000), anti-H3K23ac (Active Motif 39132, 1/20,000), anti-H3K27ac (Active Motif 39134, 1/10,000), anti-H3K56ac (Millipore, Darmstadt Germany, 1/10,000). The following antibodies were used for histone K to R mutants: anti-H3K14ac (ab46984, Abcam, 1/500), anti-H3K18ac (Millipore 07–354, 1/7500), anti-H3K23ac (Millipore 07–355, 1/5000). Each primary antibody was applied for 1 h at room temperature and then washed in TBS and 0.1% Tween. Goat anti-rabbit IgG-horseradish peroxidase secondary antibody (Amersham Biosciences) was diluted to 1/4000 in 1% milk and TBS–0.1% Tween, applied for 1 h at room temperature, and washed in TBS and 0.1% Tween. Blots were developed using Pierce ECL Western Blotting Substrate (Thermo Scientific) and exposed using film. Peptide pulldowns were essentially performed as by Taverna et al. (10Taverna S.D. Li H. Ruthenburg A.J. Allis C.D. Patel D.J. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers.Nat. Struct. Mol. Biol. 2007; 14: 1025-1040Crossref PubMed Scopus (1169) Google Scholar), with the following modifications. Briefly, streptavidin-coupled dynabeads (20 μl per sample) (Invitrogen M-280) were incubated with biotinylated histone peptides (1 μg per sample) in PBS and washed in PBS. Peptide-coated beads were then incubated with purified bromodomain (1 μg) in the presence of BSA competitor (1 μg) in binding buffer (20 mm HEPES, pH 7.9, 100 mm NaCl, 0.2% Triton X-100, 0.5 mm DTT, 10% glycerol) for 3 h at room temperature. Beads were washed three times for 5 min each time with high-salt wash buffer (20 mm HEPES, pH 7.9, 300 mm NaCl, 0.2% Triton X-100, 0.5 mm DTT, 10% glycerol) and one time with low-salt wash buffer (4 mm HEPES, pH 7.9, 20 mm NaCl). Peptide bound protein was eluted off beads with boiling 1× SDS-PAGE sample buffer, resolved on 12% SDS-polyacrylamide gels, transferred to PVDF, and probed with antibodies recognizing S-tag (ab18588, Abcam, 1/500) and Streptavidin-HRP (Molecular Probes S-911, 1/1000). Input lanes represent 0.1% bromodomain protein used in the pulldown. H3 peptides: H3 1–20 unmod biotin: ARTKQTARKSTGGKAPRKQL-K(Biotin)-NH2. H3K14ac 1–20 biotin: ARTKQTARKSTGGK(ac)APRKQL-K(Biotin)-NH2. Gel bands excised from acid-urea gels were de-stained (serially washed with 50 mm ammonium bicarbonate in 50% methanol), treated with 30% d6-acetic anhydride in 100 mm ammonium bicarbonate to chemically acetylate lysines (39Tackett A.J. Dilworth D.J. Davey M.J. O'Donnell M. Aitchison J.D. Rout M.P. Chait B.T. Proteomic and genomic characterization of chromatin complexes at a boundary.J. Cell Biol. 2005; 169: 35-47Crossref PubMed Scopus (121) Google Scholar), and subjected to in-gel trypsin digestion (100 ng of trypsin at 37 °C for 15 h). Treatment with d6-acetic anhydride adds isotopically heavy acetyl groups (+45 Da) to unmodified and monomethylated lysines, which serves to prevent trypsin digestion at lysine residues with a distinguishable synthetic modification. This heavy acetylation enhances the identification of histone peptides (39Tackett A.J. Dilworth D.J. Davey M.J. O'Donnell M. Aitchison J.D. Rout M.P. Chait B.T. 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MS data were acquired using the FTMS analyzer in profile mode at a resolution of 60,000 over a range of 375 to 1500 m/z. MS/MS data were acquired for the top 15 peaks from each MS scan using the ion trap analyzer in centroid mode and normal mass range with a normalized collision energy of 35.0. Mass spectrometric data were database searched with Mascot using heavy and light acetylation of lysines as variable modifications. The percentage of site-specific lysine acetylation was determined with spectral count comparisons of peptides with light or heavy acetylation at the given lysine (% light acetylation for a lysine in a given peptide = (light spectral counts/light + heavy spectral counts) × 1
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