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Functional Proteomic Analysis of Repressive Histone Methyltransferase Complexes Reveals ZNF518B as a G9A Regulator*

2015; Elsevier BV; Volume: 14; Issue: 6 Linguagem: Inglês

10.1074/mcp.m114.044586

ISSN

1535-9484

Autores

Verena Maier, Caitlin M. Feeney, Jordan E. Taylor, Amanda L. Creech, Jana Qiao, Attila Szántó, Partha Pratim Das, Nicholas Chevrier, Catherine Cifuentes‐Rojas, Stuart H. Orkin, Steven A. Carr, Jacob D. Jaffe, Philipp Mertins, Jeannie T. Lee,

Tópico(s)

RNA modifications and cancer

Resumo

Cell-type specific gene silencing by histone H3 lysine 27 and lysine 9 methyltransferase complexes PRC2 and G9A-GLP is crucial both during development and to maintain cell identity. Although studying their interaction partners has yielded valuable insight into their functions, how these factors are regulated on a network level remains incompletely understood. Here, we present a new approach that combines quantitative interaction proteomics with global chromatin profiling to functionally characterize repressive chromatin modifying protein complexes in embryonic stem cells. We define binding stoichiometries of 9 new and 12 known interaction partners of PRC2 and 10 known and 29 new interaction partners of G9A-GLP, respectively. We demonstrate that PRC2 and G9A-GLP interact physically and share several interaction partners, including the zinc finger proteins ZNF518A and ZNF518B. Using global chromatin profiling by targeted mass spectrometry, we discover that even sub-stoichiometric binding partners such as ZNF518B can positively regulate global H3K9me2 levels. Biochemical analysis reveals that ZNF518B directly interacts with EZH2 and G9A. Our systematic analysis suggests that ZNF518B may mediate the structural association between PRC2 and G9A-GLP histone methyltransferases and additionally regulates the activity of G9A-GLP. Cell-type specific gene silencing by histone H3 lysine 27 and lysine 9 methyltransferase complexes PRC2 and G9A-GLP is crucial both during development and to maintain cell identity. Although studying their interaction partners has yielded valuable insight into their functions, how these factors are regulated on a network level remains incompletely understood. Here, we present a new approach that combines quantitative interaction proteomics with global chromatin profiling to functionally characterize repressive chromatin modifying protein complexes in embryonic stem cells. We define binding stoichiometries of 9 new and 12 known interaction partners of PRC2 and 10 known and 29 new interaction partners of G9A-GLP, respectively. We demonstrate that PRC2 and G9A-GLP interact physically and share several interaction partners, including the zinc finger proteins ZNF518A and ZNF518B. Using global chromatin profiling by targeted mass spectrometry, we discover that even sub-stoichiometric binding partners such as ZNF518B can positively regulate global H3K9me2 levels. Biochemical analysis reveals that ZNF518B directly interacts with EZH2 and G9A. Our systematic analysis suggests that ZNF518B may mediate the structural association between PRC2 and G9A-GLP histone methyltransferases and additionally regulates the activity of G9A-GLP. Multicellular organisms consist of a plethora of divergent cell types of vastly different appearance and function, even though the cells share a common genome. To achieve phenotypic variety, genes whose expression would interfere with the physiology of particular cell types must be epigenetically silenced. Chromatin modifying complexes play a major role in this epigenetic regulation. Two histone methyltransferase complexes of particular importance in early mammalian development are Polycomb repressive complex 2 (PRC2) 1The abbreviations used are:PRC2polycomb repressive complex 2G9Ahistone-lysine N-methyltransferase EHMT2GLPhistone-lysine N-methyltransferase EHMT1ZNF518Bzinc finger protein 518BSILACstable isotope labeling of amino acids in cell cultureAPMSaffinity proteomics mass sepctrometrymESCsmouse embryonic stem cells. 1The abbreviations used are:PRC2polycomb repressive complex 2G9Ahistone-lysine N-methyltransferase EHMT2GLPhistone-lysine N-methyltransferase EHMT1ZNF518Bzinc finger protein 518BSILACstable isotope labeling of amino acids in cell cultureAPMSaffinity proteomics mass sepctrometrymESCsmouse embryonic stem cells. and histone-lysine N-methyltransferase EHMT2 (G9A)-histone-lysine N-methyltransferase EHMT1 (GLP), which together promote cell type specific gene silencing by adding repressive posttranslational modifications to histone tails at promoters of target genes (1Simon J.A. Kingston R.E. Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put.Mol. Cell. 2013; 49: 808-824Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar). PRC2 contains one of two alternative methyltransferase subunits, EZH2 or EZH1. In addition, PRC2 comprises EED and SUZ12, which regulate the RNA-binding and methyltransferase activities of EZH2, as well as one of two histone binding proteins, RBBP4 or RBBP7 (2Di Croce L. Helin K. Transcriptional regulation by Polycomb group proteins.Nat. Struct. Mol. Biol. 2013; 20: 1147-1155Crossref PubMed Scopus (611) Google Scholar). PRC2 catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3), a repressive histone mark found on facultative heterochromatin. On the other hand, the G9A-GLP complex dimethylates H3 at lysine 9 (H3K9me2) and works together with the widely interspersed zinc finger protein WIZ. 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Cells were grown on a layer of irradiated DR4 mouse embryonic fibroblasts under selective pressure of 400 μg/ml neomycin, 1 μg/ml puromycin and/or 200 μg/ml hygromycin where applicable and split every second day. For the last three passages before harvesting, mESCs were grown in Dulbecco's Modified Eagle's Medium devoid of arginine and lysine (Invitrogen, Grand Island, NY), supplemented with 15% fetal bovine serum (Gibco, Grand Island, NY) dialyzed with a cut-off of 10 kDa, 15 mm Hepes pH 7.6, 45 mm NaHCO3, glucose to a final concentration of 4.5 mg/ml, 0.1 mm non-essential amino acids (Gibco), 2 mm Glutamine (Gibco), 100 units/ml penicillin/streptomycin (Gibco), 50 μm β-mercaptoethanol (Gibco), 1000 units/ml leukemia inhibitory factor (Millipore, Billerica, MA) and 30 g/l methionine, 42g/l arginine and 73g/l lysine (Cambridge Isotope Laboratories, Tewksbury, MA). The latter two were added either in light (Arg0/Lys0), medium (13C6 arginine (Arg6)/D4 lysine (Lys4)) or heavy (13C6-15N4 arginine (Arg10)/13C6-15N2 lysine (Lys8)) form. G9A-3xFLAG expression was induced with 1 μg/ml doxycycline for 24 h before harvesting. Per condition, 2 × 15 cm plates were grown to 80% confluency, washed once with and scraped in 20 ml ice cold PBS (8.1 mm Na2HPO4, 1.45 mm KH2PO4, 137 mm NaCl, 2.7 mm KCL, pH 7.4). Pelleted cells were incubated in 10 ml buffer A (10 mm Hepes pH 7.5, 10 mm KCl, 1.5 mm MgCl2, 0.5 mm PMSF) for 15 min with frequent vortexing. Nuclei were pelleted (10 min, 2500 g), lysed in 500 μl RIPA buffer (50 mm TrisCl pH 7.5, 150 mm NaCl, 0.5% Na-deoxycholate, 0.5% IGPAL-CA-630 (Sigma-Aldrich, St. Louis, MO), 0.1% SDS, 5% glycerol, complete proteinase inhibitors (Roche, Indianapolis, IN)) and debris was removed by centrifugation (10 min, 16,000 g). Protein content of supernatants was determined with the Pierce© BCA protein assay. 60 μl Dynabeads MyOne streptavidin C1 (Invitrogen) or M2 FLAG agarose beads (Sigma-Aldrich), equilibrated in RIPA buffer, were added to 2 mg of supernatant and samples were rotated for 1 h at 4 °C. Beads were captured on a magnetic rack or collected by centrifugation (3 min, 2.5 rpm), washed once with 1 ml wash buffer (50 mm TrisCl pH 7.5, 150 mm NaCl, 5% glycerol, complete proteinase inhibitors (Roche)) + 0.05% IGPAL-CA-630, resuspended in 1 ml wash buffer + 1 mm MgCl2, + 0.1 mm CaCl2, + 20 units Turbo DNase (Ambion, Grand Island, NY), incubated at 25 °C for 20 min and washed once more with 1 ml wash buffer. Beads incubated with light, medium and heavy labeled lysates were combined in 80 μl of freshly prepared trypsin-urea buffer (2 m urea, 50 mm TrisCl pH 7.5, 1 mm DTT, 5 μg/ml sequencing grade trypsin (Promega, Madison, WI)) and incubated for 1 h at 25 °C with 1000 rpm. The supernatant was collected and the beads were washed twice with 2 m urea, 50 mm TrisCl pH 7.5. Elution and washes were combined and reduced by adding 4 mm DTT (30 min, 25 °C, 1000 rpm). Proteins were alkylated with 10 mm iodoacetamide (Sigma-Aldrich) (45 min, 25 °C, 1000 rpm) protected from light, digested overnight at 25 °C with 0.5 μg trypsin and acidified with formic acid (1% final concentration). Samples were applied on C18 StageTips (45Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.Nat. Protoc. 2007; 2: 1896-1906Crossref PubMed Scopus (2570) Google Scholar) that had been conditioned with 100 μl 90% acetonitrile/0.1% formic acid and equilibrated twice with 100 μl 0.1% formic acid (3 min, 4000 g). StageTips were washed twice with 100 μl 0.1% formic acid and peptides were eluted with 60 μl 90% acetonitrile/0.1% formic acid and dried in a speed vac. APMS peptide samples were reconstituted in 9 μl of solvent A (3% acetonitrile/0.1% formic acid) and 4 μl were analyzed on an EASY-nLC 1000 UHPLC system (Thermo Fisher Scientific, Waltham, MA) coupled via a 20 cm C18 column (Picofrit, New Objective, Woburn, MA, PF360–75-10-N-5; packed in-house with 1.9 μm ReproSil-Pur C18-AQ medium, Dr. Maisch GmbH, r119.aq) to a benchtop Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific) as described (46Mertins P. Qiao J.W. Patel J. Udeshi N.D. Clauser K.R. Mani D.R. Burgess M.W. Gillette M.A. Jaffe J.D. Carr S.A. Integrated proteomic analysis of post-translational modifications by serial enrichment.Nat. Methods. 2013; 10: 634-637Crossref PubMed Scopus (432) Google Scholar). Peptides were separated at a flow rate of 200 nL/min with a linear 84 min gradient from 6 to 30% solvent B (90% acetonitrile, 0.1% formic acid), followed by a linear 9 min gradient from 30 to 60% solvent B. Each sample was run for 150 min, including sample loading and column equilibration times. Data was acquired in data dependent mode using Xcalibur 2.2 software. MS1 Spectra were measured with a resolution of 70,000, an AGC target of 3e6 and a mass range from 300 to 1800 m/z. Up to 12 MS2 spectra per duty cycle were triggered at a resolution of 17,500, an AGC target of 5e4, an isolation window of 2.5 m/z and a normalized collision energy of 25. All raw data were analyzed with MaxQuant software version 1.3.0.5 (47Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9154) Google Scholar) using a mouse UniProt Mouse database (release 2013_12; containing 51,195 entries), and MS/MS searches were performed with the following parameters: Oxidation of methionine, deamidation of asparagine and protein N-terminal acetylation as variable modifications; carbamidomethylation as fixed modification; Trypsin/P as the digestion enzyme; precursor ion mass tolerances of 20 p.p.m. for the first search (used for nonlinear mass re-calibration) and 6 p.p.m. for the main search, and a fragment ion mass tolerance of 20 p.p.m. For identification, we applied a maximum FDR of 1% separately on protein and peptide level. We required 2 or more unique/razor peptides for protein identification and a ratio count of 2 or more for protein quantification per biological replicate measurement. To determine relative enrichment of proteins in bait versus control samples, SILAC protein ratios were calculated as the median of all unique/razor peptides for each protein group. To identify significant interactors we filtered for proteins that were quantified in at least 2 or more biological replicates and calculated moderated t test p values corrected by the Benjamini Hochberg method, as described previously (48Udeshi N.D. Mani D.R. Eisenhaure T. Mertins P. Jaffe J.D. Clauser K.R. Hacohen N. Carr S.A. Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition.Mol. Cell. Proteomics. 2012; 11: 148-159Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). To determine stoichiometries of interactors relative to the bait protein we used an approach adapted from (49Smits A.H. Jansen P.W. Poser I. Hyman A.A. Vermeulen M. Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics.Nucleic Acids Res. 2013; 41: e28Crossref PubMed Scopus (177) Google Scholar). iBAQ intensities were calculated using MaxQuant by summing the intensities of all tryptic peptides for each protein and dividing this number by the number of theoretically observable peptides. Within each SILAC triple labeling experiment, iBAQ intensities of enriched proteins in the medium and heavy channels were corrected for background binding intensities in the light SILAC control channel by subtracting the light channel iBAQ intensities from the medium and heavy channel iBAQ intensities, respectively. Intensities of peptides assigned to different isoforms were combined. To obtain molar ratios of interactors to bait proteins, corrected interactor iBAQ intensities were divided by bait iBAQ intensities in each SILAC experiment. For biological interpretation we required that protein stoichiometries were calculated independently in n-1 replicates. The raw mass spectrometry data have been deposited in the public proteomics repository MassIVE and are accessible at ftp://[email protected] when providing the username "MSV000078980" an

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