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Chromatin Affinity Purification and Quantitative Mass Spectrometry Defining the Interactome of Histone Modification Patterns

2011; Elsevier BV; Volume: 10; Issue: 11 Linguagem: Inglês

10.1074/mcp.m110.005371

ISSN

1535-9484

Autores

Miroslav Nikolov, Alexandra Stützer, Kerstin Mosch, Andrius Krasauskas, Szabolcs Soeroes, Holger Stark, Henning Urlaub, Wolfgang Fischle,

Tópico(s)

Protein Degradation and Inhibitors

Resumo

DNA and histone modifications direct the functional state of chromatin and thereby the readout of the genome. Candidate approaches and histone peptide affinity purification experiments have identified several proteins that bind to chromatin marks. However, the complement of factors that is recruited by individual and combinations of DNA and histone modifications has not yet been defined. Here, we present a strategy based on recombinant, uniformly modified chromatin templates used in affinity purification experiments in conjunction with SILAC-based quantitative mass spectrometry for this purpose. On the prototypic H3K4me3 and H3K9me3 histone modification marks we compare our method with a histone N-terminal peptide affinity purification approach. Our analysis shows that only some factors associate with both, chromatin and peptide matrices but that a surprisingly large number of proteins differ in their association with these templates. Global analysis of the proteins identified implies specific domains mediating recruitment to the chromatin marks. Our proof-of-principle studies show that chromatin templates with defined modification patterns can be used to decipher how the histone code is read and translated. DNA and histone modifications direct the functional state of chromatin and thereby the readout of the genome. Candidate approaches and histone peptide affinity purification experiments have identified several proteins that bind to chromatin marks. However, the complement of factors that is recruited by individual and combinations of DNA and histone modifications has not yet been defined. Here, we present a strategy based on recombinant, uniformly modified chromatin templates used in affinity purification experiments in conjunction with SILAC-based quantitative mass spectrometry for this purpose. On the prototypic H3K4me3 and H3K9me3 histone modification marks we compare our method with a histone N-terminal peptide affinity purification approach. Our analysis shows that only some factors associate with both, chromatin and peptide matrices but that a surprisingly large number of proteins differ in their association with these templates. Global analysis of the proteins identified implies specific domains mediating recruitment to the chromatin marks. Our proof-of-principle studies show that chromatin templates with defined modification patterns can be used to decipher how the histone code is read and translated. DNA methylation and histone post-translational modifications (PTM) 1The abbreviations used are: PTMpost translational modificationSILACstable isotope labeling by amino acids in cell cultureH3K4me3histone H3 lysine 4 trimethylationH3K9me3histone H3 lysine 9 trimethylationPHDplant homeodomainMudPITmultidimensional protein identification technologyTAFTATA box binding protein (TBP)-associated factorPHF8PHD finger protein 8CHD1chromodomain-helicase-DNA-binding protein 1ING2inhibitor of growth family member 2WDR5WD repeat domain 5HP1heterochromatin protein 1CBXchromobox homologUHRF1ubiquitin-like with PHD and ring finger domains 1CDYLchromodomain protein Y-likeMPHOSP8M-phase phosphoprotein 8SPIN1spindling 1FANCFFanconi anemia, complementation group FADNPactivity-dependent neuroprotector homeoboxZMYM3zinc finger MYM-type 3ACTL8actin-like 8SMCHD1structural maintenance of chromosomes flexible hinge domain containing 1TFIIDTATA binding proteinPOGZpogo transposable element with ZnF domainZnFzinc fingerDAXXdeath-domain associated proteinDNMT1DNA (cytosine-5-)-methyltransferase 1H3K27me3histone H3 lysine 27 trimethylationSRASET and RING associatedHAThistone acetyl transferaseSNF2sucrose non fermentable 2ARIDAT-rich interaction domainMybmyeloblastosis oncogene likeSANTswitching-defective protein 3 (Swi3) adaptor 2 (Ada2) nuclear receptor co-repressor (N-CoR) transcription factor (TF)IIIBH4K20me3histone H4 lysine 20 trimethylationH3K27me1histone H3 lysine 27 monomethylationH3R2me2histone H3 arginine 2 dimethylationH4R3me2histone H4 arginine 3 dimethylation. play important roles in regulating chromatin states and thereby the use and readout of the genome. Trimethylation of lyinse 4 (H3K4me3) and lysine 9 (H3K9me3) of histone H3 have, for example, been connected to transcriptional activation and repression, respectively. They therefore present a prototypic pair of antagonistic histone PTMs. post translational modification stable isotope labeling by amino acids in cell culture histone H3 lysine 4 trimethylation histone H3 lysine 9 trimethylation plant homeodomain multidimensional protein identification technology TATA box binding protein (TBP)-associated factor PHD finger protein 8 chromodomain-helicase-DNA-binding protein 1 inhibitor of growth family member 2 WD repeat domain 5 heterochromatin protein 1 chromobox homolog ubiquitin-like with PHD and ring finger domains 1 chromodomain protein Y-like M-phase phosphoprotein 8 spindling 1 Fanconi anemia, complementation group F activity-dependent neuroprotector homeobox zinc finger MYM-type 3 actin-like 8 structural maintenance of chromosomes flexible hinge domain containing 1 TATA binding protein pogo transposable element with ZnF domain zinc finger death-domain associated protein DNA (cytosine-5-)-methyltransferase 1 histone H3 lysine 27 trimethylation SET and RING associated histone acetyl transferase sucrose non fermentable 2 AT-rich interaction domain myeloblastosis oncogene like switching-defective protein 3 (Swi3) adaptor 2 (Ada2) nuclear receptor co-repressor (N-CoR) transcription factor (TF)IIIB histone H4 lysine 20 trimethylation histone H3 lysine 27 monomethylation histone H3 arginine 2 dimethylation histone H4 arginine 3 dimethylation. Generally, chromatin marks either influence chromatin packaging directly or via recruitment of specific proteins and multiprotein complexes that mediate downstream effects (1Fischle W. Wang Y. Allis C.D. Histone and chromatin cross-talk.Curr. Opin. Cell Biol. 2003; 15: 172-183Crossref PubMed Scopus (986) Google Scholar, 2Campos E.I. Reinberg D. Histones: annotating chromatin.Annu. Rev. Genet. 2009; 43: 559-599Crossref PubMed Scopus (652) Google Scholar). Candidate approaches of individual factors or using targeted libraries of protein families together with histone tail peptide affinity purification experiments carried out in isolation or on peptide arrays have identified a number of proteins that specifically interact with individual chromatin marks (see for example ref. 3Bua D.J. Kuo A.J. Cheung P. Liu C.L. Migliori V. Espejo A. Casadio F. Bassi C. Amati B. Bedford M.T. Guccione E. Gozani O. Epigenome microarray platform for proteome-wide dissection of chromatin-signaling networks.PLoS One. 2009; 4e6789Crossref PubMed Scopus (84) Google Scholar, 4Chan D.W. Wang Y. Wu M. Wong J. Qin J. Zhao Y. Unbiased proteomic screen for binding proteins to modified lysines on histone H3.Proteomics. 2009; 9: 2343-2354Crossref PubMed Scopus (38) Google Scholar, 5Liu H. Galka M. Iberg A. Wang Z. Li L. Voss C. Jiang X. Lajoie G. Huang Z. Bedford M.T. Li S.S. Systematic identification of methyllysine-driven interactions for histone and nonhistone targets.J. Proteome Res. 2010; 9: 5827-5836Crossref PubMed Scopus (33) Google Scholar, 6Vermeulen M. Eberl H.C. Matarese F. Marks H. Denissov S. Butter F. Lee K.K. Olsen J.V. Hyman A.A. Stunnenberg H.G. Mann M. Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers.Cell. 2010; 142: 967-980Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar). These include factors containing methyl-DNA binding domains as well as chromodomains, plant homeodomain (PHD) fingers, tudor domains, and ankyrin repeats interacting with histone methyl-lysine residues. Further, 14-3-3 proteins interacting with histone phospho-serine residues and bromodomain containing factors binding to histone acetyl-lysine residues have been described (7Taverna 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 (1175) Google Scholar). In vitro studies have characterized the exact binding specificities of several proteins containing these domains. Also, structural insights are now available for a number of chromatin mark binding complexes (7Taverna 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 (1175) Google Scholar, 8Adams-Cioaba M.A. Min J. Structure and function of histone methylation binding proteins.Biochem. Cell Biol. 2009; 87: 93-105Crossref PubMed Scopus (128) Google Scholar). Interestingly, the interactions of individual domains of chromatin modification binding proteins with their cognate marks are rather weak (interaction strength in the micromolar range) (9Fischle W. Wang Y. Allis C.D. Binary switches and modification cassettes in histone biology and beyond.Nature. 2003; 425: 475-479Crossref PubMed Scopus (551) Google Scholar). Although the study of interactions of individual proteins with DNA methylation or distinct histone PTM marks has been central to our current understanding of chromatin mediated processes, it is emerging that patterns of marks rather than individual modifications direct functional states of chromatin (10Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7666) Google Scholar, 11Sims 3rd, R.J. Reinberg D. Is there a code embedded in proteins that is based on post-translational modifications?.Nat. Rev. Mol. Cell Biol. 2008; 9: 815-820Crossref PubMed Scopus (254) Google Scholar). Here, factors containing multiple domains interacting with different chromatin marks have gained high interest (12Ruthenburg A.J. Li H. Patel D.J. Allis C.D. Multivalent engagement of chromatin modifications by linked binding modules.Nat. Rev. Mol. Cell Biol. 2007; 8: 983-994Crossref PubMed Scopus (820) Google Scholar). Multivalent binding might not only allow for stronger and thereby more discriminatory interaction than single domain binding, but could also direct readout of complex patterns of modifications. Also, multiprotein complexes appear to contain several factors with the same or distinct chromatin mark recognition functionality thereby possibly establishing more stable interaction. Gaining global insight into the relationship of chromatin modifications and functional states of chromatin ultimately requires isolation and characterization of intact chromatin domains from cells. In absence of such experimental systems in vitro approaches that mimic and incorporate different DNA methylation and histone PTM configurations will likely be extremely useful in defining the complement of factors that targets a given pattern of chromatin marks. Here, DNA and/or histone tail peptide affinity purification experiments can only be of limited value as only individual or shortly spaced combinatorial patterns of modifications can be analyzed (see for example ref. 13Fischle W. Tseng B.S. Dormann H.L. Ueberheide B.M. Garcia B.A. Shabanowitz J. Hunt D.F. Funabiki H. Allis C.D. Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation.Nature. 2005; 438: 1116-1122Crossref PubMed Scopus (739) Google Scholar). Nonquantitative mass spectrometry (e.g. MudPIT, ref. 14Liu H. Lin D. Yates 3rd, J.R. Multidimensional separations for protein/peptide analysis in the post-genomic era.BioTechniques. 2002; (898, 900, 902 passim): 32PubMed Google Scholar) analysis of differential affinity purification reactions has been useful in identifying proteins binding a given target (4Chan D.W. Wang Y. Wu M. Wong J. Qin J. Zhao Y. Unbiased proteomic screen for binding proteins to modified lysines on histone H3.Proteomics. 2009; 9: 2343-2354Crossref PubMed Scopus (38) Google Scholar). However, because these methods do not provide sufficient quantitative information on the proteins recovered in separate experiments in the first place, factors that bind two separate matrices (e.g. sample and control) with different strength will not be necessarily recognized as specific interaction partners of either one. Therefore, different mass spectrometry methods have been introduced that allow identification and sensitive quantification of proteins in matched experiments (15Wilm M. Quantitative proteomics in biological research.Proteomics. 2009; 9: 4590-4605Crossref PubMed Scopus (69) Google Scholar). Especially, isotope labeling by amino acids in cell culture (SILAC) has proven useful in various proteomics based approaches (16Ong S.E. Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC).Nat. Protoc. 2006; 1: 2650-2660Crossref PubMed Scopus (686) Google Scholar). Here, we set out to establish an in vitro system usable for the analysis of complex chromatin modification patterns based on recombinant, uniformly modified chromatin templates in combination with quantitative SILAC-based mass spectrometry analysis. In this manner, we defined the interactome of the H3K4me3 and H3K9me3 chromatin marks. Surprisingly, only some factors were also recruited to corresponding histone N-terminal peptides in parallel experiments. Our results set the stage for using chromatin-based affinity approaches to investigate how the histone code is read and translated on a global scale. HeLa S3 cells were grown in lysine- and arginine-deficient Dulbecco's modified Eagle's medium supplemented with 10% dialyzed fetal bovine serum (PAA, Pasching, Austria). One cell population was supplemented with normal isotope containing l-lysine and l-arginine (Sigma, Munich, Germany) and another with heavy isotope labeled 13C6-lysine and 13C615N4-arginine (Euriso-Top, Saint-Aubin Cedex, France) generating mass shifts of +6 and +10 Da, respectively. Cells were grown for at least six passages at smaller volumes and then expanded to 2 l in spinner flasks (0.5–1.0 × 106 cells/ml) (16Ong S.E. Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC).Nat. Protoc. 2006; 1: 2650-2660Crossref PubMed Scopus (686) Google Scholar). The cells were then transferred to a 5 l fermenter (Applikon, Schiedam, Netherlands) and grown under standard conditions (2.5–5.0 × 106 cells/ml). Harvested cells were used to prepare nuclear extract according to standard procedures (17Dignam J.D. Martin P.L. Shastry B.S. Roeder R.G. Eukaryotic gene transcription with purified components.Methods Enzymol. 1983; 101: 582-598Crossref PubMed Scopus (745) Google Scholar). Peptides containing the 20 N-terminal amino acids of histone H3 were synthesized in unmodified and modified form using Fmoc (N-(9-fluorenyl)methoxycarbonyl)-based solid-phase synthesis H3unmodified: ARTKQTARKSTGGKAPRKQL; H3K4me3: ARTK(me3)QTARKSTGGKAPRKQL; H3K9me3: ARTKQTARK(me3)STGGKAPRKQL. Peptides contained a C-terminal non-native lysine biotinylated at the ε-amino group for affinity purification reactions or were transformed to thioacetamidthiophenylesters for native protein ligation (18Biancalana S. Hudson D. Songster M.F. Thompson S.A. Fmoc chemistry compatible thio-ligation assembly of proteins.Lett. Peptide Sci. 2001; 7: 291-297Crossref Scopus (24) Google Scholar, 19Futaki S. Sogawa K. Maruyama J. Asahara T. Niwa M. Hojo H. Preparation of peptide thioesters using Fmoc-solid-phase peptide synthesis and its application to the construction of a template-assembled synthetic protein (TASP).Tetrahedron Letters. 1997; 38: 6237-6240Crossref Scopus (112) Google Scholar, 20von Eggelkraut-Gottanka R. Klose A. Beck-Sickinger A.G. Beyermann M. Peptide (alpha)thioester formation using standard Fmoc-chemistry.Biopolymers. 2003; 71: 352-353Google Scholar). Histone modifications were achieved by native protein ligation using histone H3 (1Fischle W. Wang Y. Allis C.D. Histone and chromatin cross-talk.Curr. Opin. Cell Biol. 2003; 15: 172-183Crossref PubMed Scopus (986) Google Scholar, 2Campos E.I. Reinberg D. Histones: annotating chromatin.Annu. Rev. Genet. 2009; 43: 559-599Crossref PubMed Scopus (652) Google Scholar, 3Bua D.J. Kuo A.J. Cheung P. Liu C.L. Migliori V. Espejo A. Casadio F. Bassi C. Amati B. Bedford M.T. Guccione E. Gozani O. Epigenome microarray platform for proteome-wide dissection of chromatin-signaling networks.PLoS One. 2009; 4e6789Crossref PubMed Scopus (84) Google Scholar, 4Chan D.W. Wang Y. Wu M. Wong J. Qin J. Zhao Y. Unbiased proteomic screen for binding proteins to modified lysines on histone H3.Proteomics. 2009; 9: 2343-2354Crossref PubMed Scopus (38) Google Scholar, 5Liu H. Galka M. Iberg A. Wang Z. Li L. Voss C. Jiang X. Lajoie G. Huang Z. Bedford M.T. Li S.S. Systematic identification of methyllysine-driven interactions for histone and nonhistone targets.J. Proteome Res. 2010; 9: 5827-5836Crossref PubMed Scopus (33) Google Scholar, 6Vermeulen M. Eberl H.C. Matarese F. Marks H. Denissov S. Butter F. Lee K.K. Olsen J.V. Hyman A.A. Stunnenberg H.G. Mann M. Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers.Cell. 2010; 142: 967-980Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 7Taverna 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 (1175) Google Scholar, 8Adams-Cioaba M.A. Min J. Structure and function of histone methylation binding proteins.Biochem. Cell Biol. 2009; 87: 93-105Crossref PubMed Scopus (128) Google Scholar, 9Fischle W. Wang Y. Allis C.D. Binary switches and modification cassettes in histone biology and beyond.Nature. 2003; 425: 475-479Crossref PubMed Scopus (551) Google Scholar, 10Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7666) Google Scholar, 11Sims 3rd, R.J. Reinberg D. Is there a code embedded in proteins that is based on post-translational modifications?.Nat. Rev. Mol. Cell Biol. 2008; 9: 815-820Crossref PubMed Scopus (254) Google Scholar, 12Ruthenburg A.J. Li H. Patel D.J. Allis C.D. Multivalent engagement of chromatin modifications by linked binding modules.Nat. Rev. Mol. Cell Biol. 2007; 8: 983-994Crossref PubMed Scopus (820) Google Scholar, 13Fischle W. Tseng B.S. Dormann H.L. Ueberheide B.M. Garcia B.A. Shabanowitz J. Hunt D.F. Funabiki H. Allis C.D. Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation.Nature. 2005; 438: 1116-1122Crossref PubMed Scopus (739) Google Scholar, 14Liu H. Lin D. Yates 3rd, J.R. Multidimensional separations for protein/peptide analysis in the post-genomic era.BioTechniques. 2002; (898, 900, 902 passim): 32PubMed Google Scholar, 15Wilm M. Quantitative proteomics in biological research.Proteomics. 2009; 9: 4590-4605Crossref PubMed Scopus (69) Google Scholar, 16Ong S.E. Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC).Nat. Protoc. 2006; 1: 2650-2660Crossref PubMed Scopus (686) Google Scholar, 17Dignam J.D. Martin P.L. Shastry B.S. Roeder R.G. Eukaryotic gene transcription with purified components.Methods Enzymol. 1983; 101: 582-598Crossref PubMed Scopus (745) Google Scholar, 18Biancalana S. Hudson D. Songster M.F. Thompson S.A. Fmoc chemistry compatible thio-ligation assembly of proteins.Lett. Peptide Sci. 2001; 7: 291-297Crossref Scopus (24) Google Scholar, 19Futaki S. Sogawa K. Maruyama J. Asahara T. Niwa M. Hojo H. Preparation of peptide thioesters using Fmoc-solid-phase peptide synthesis and its application to the construction of a template-assembled synthetic protein (TASP).Tetrahedron Letters. 1997; 38: 6237-6240Crossref Scopus (112) Google Scholar, 20von Eggelkraut-Gottanka R. Klose A. Beck-Sickinger A.G. Beyermann M. Peptide (alpha)thioester formation using standard Fmoc-chemistry.Biopolymers. 2003; 71: 352-353Google Scholar) thioester peptides and recombinant X. laevis histone H3Δ1–20,A21C as described (21Shogren-Knaak M.A. Peterson C.L. Creating designer histones by native chemical ligation.Methods Enzymol. 2004; 375: 62-76Crossref PubMed Scopus (33) Google Scholar). Reactions were carried out in 100 mm potassium phosphate (pH 7.9), 3 m guanidine-HCl, 0.5% v/v benzyl mercaptan, 0.5% v/v thiophenol at 25 °C with vigorous mixing. Crude reaction mixture was diluted 50-fold into SAU-200 buffer (7 m deionized urea, 20 mm sodium acetate (pH 5.2), 1 mm EDTA, 1 mm dithiotreitol, 200 mm NaCl), applied to a 5 ml Hi-Trap SP-Sepharose high performance cation exchange column (GE Healthcare, Munich, Germany), and eluted with a linear NaCl gradient from 200 to 600 mm in 10 column volumes. Protein was dialyzed extensively against 2 mm dithiotreitol at 4 °C, lyophilized and stored at −80 °C. Routinely we set up ligation reactions containing 27 mg histone (2 μmol), 23 mg thioacetamidthiophenylester histone H3 peptide (10 μmol) in 10 ml reaction volume. After purification on average 10 mg ligated histone H3 was obtained (0.6 μmol). Purity and identity of thioester peptides and ligated proteins was confirmed by analytical high-performance liquid chromatography, mass spectrometry, and SDS-PAGE (see supplemental Fig. S1). Recombinant chromatin was prepared essentially as described (22Franz H. Mosch K. Soeroes S. Urlaub H. Fischle W. Multimerization and H3K9me3 binding are required for CDYL1b heterochromatin association.J. Biol. Chem. 2009; 284: 35049-35059Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Briefly, recombinant wild type Xenopus laevis histones were expressed in Escherichia coli and purified as described (23Luger K. Rechsteiner T.J. Richmond T.J. Preparation of nucleosome core particle from recombinant histones.Methods Enzymol. 1999; 304: 3-19Crossref PubMed Scopus (567) Google Scholar). Assembly of histone octamers containing modified and unmodified histone H3 as well as nucleosome array reconstitution was performed by salt dialysis on biotinylated 12 × 200 × 601 DNA template as described (23Luger K. Rechsteiner T.J. Richmond T.J. Preparation of nucleosome core particle from recombinant histones.Methods Enzymol. 1999; 304: 3-19Crossref PubMed Scopus (567) Google Scholar, 24Huynh V.A. Robinson P.J. Rhodes D. A method for the in vitro reconstitution of a defined "30 nm" chromatin fibre containing stoichiometric amounts of the linker histone.J. Mol. Biol. 2005; 345: 957-968Crossref PubMed Scopus (146) Google Scholar). Quality of chromatin reconstitution was monitored by native agarose gel electrophoresis, MNase digest, and analytical ultracentrifugation (see supplemental Fig. S2). Affinity purifications were performed essentially as described using two separate preparations of nuclear extract (22Franz H. Mosch K. Soeroes S. Urlaub H. Fischle W. Multimerization and H3K9me3 binding are required for CDYL1b heterochromatin association.J. Biol. Chem. 2009; 284: 35049-35059Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Each experiment was performed in "forward" (light extract, unmodified chromatin and peptide; heavy extract, modified chromatin and peptide) and "reverse" (light extract, modified chromatin and peptide; heavy extract, unmodified chromatin and peptide) label swap affinity purification. For peptide affinity purifications, 40 μl prewashed streptavidin coated paramagnetic beads (Pierce, Rockford, IL) were saturated with 10 μg biotinylated histone peptide overnight at 4 °C. A 0.5 ml aliquot of precleared HeLa S3 nuclear extract (light or heavy isotope labeled) was incubated with the peptide-bound paramagnetic beads for 4 h while rotating. Beads were washed three times with 1 ml of PD150 buffer (20 mm HEPES pH 7.9, 150 mm NaCl, 0.1% Triton X-100, 5% glycerol). Beads from parallel affinity purification reactions using unmodified and modified peptides were mixed (25Schulze W.X. Mann M. A novel proteomic screen for peptide-protein interactions.J. Biol. Chem. 2004; 279: 10756-10764Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar) and bound proteins were eluted with LDS sample buffer (Invitrogen, Carlsbad, CA). Chromatin affinity purifications were performed accordingly using 50 μg chromatin and 200 μl paramagnetic beads. To improve SDS-PAGE resolution, eluates of chromatin affinity purification reactions were incubated with 1 kU benzonase (Calbiochem, San Diego, CA) nuclease for 1 h at 37 °C. Primary antibodies used were: αH3K4me3 (1:2,000, Abcam, Cambridge, UK), αH3K9me3 (1:1,000, Millipore, Billerica, MA), αFLAG (1:1,000, Sigma, Munich, Germany), and αSMCHD1 (1:1,000, Abcam, Cambridge, UK). Eluted proteins were separated on 4–12% gradient SDS-PAGE gels (Invitrogen, Carlsbad, CA) and stained with Colloidal Coomassie Blue. Each gel lane was cut into 23 equal gel slices and proteins therein were in-gel digested with trypsin (Promega, Madison, WI) as described (26Shevchenko A. Tomas H. Havlis J. Olsen J.V. Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes.Nat. Protoc. 2006; 1: 2856-2860Crossref PubMed Scopus (3554) Google Scholar). Tryptic peptides from each gel slice were extracted and analyzed by nanoflow HPLC (Agilent, Boeblingen, Germany) coupled to nanoelectrospray LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA) operating in positive ion mode. Peptides were first loaded and desalted onto an in-line trap column (1.5 cm length, 150 μm inner diameter, packed in-house with Reprosil AQ-5 μm/300Å) and then separated on analytical column (15 cm length, 75 μm inner diameter, as trap column) at flow-rate 250 nL/min and linear gradient from 7.5 to 37.5% acetonitrile in 0.1% (v/v) formic acid for 50 min. Data-dependent acquisition of eluting peptides was applied and consisted of one survey scan in Orbitrap (with resolution set to 30,000 at m/z 400 and automatic gain control target at 106) followed by MS/MS of the five most intense precursors in the LTQ using collision-induced decay fragmentation with previously fragmented ions dynamically excluded for 90 s. Each sample was analyzed in three technical replicates. Raw MS files from LTQ-Orbitrap XL were analyzed by MaxQuant software (version 1.0.13.13) (27Cox 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 (9262) Google Scholar). Peak lists generated by Quant.exe (the first module of MaxQuant) were searched by Mascot search engine (Mascot Daemon 2.2.2; Matrix Science, London, UK) using International Protein Index (IPI) Human protein database (version 3.73, June 2010, containing 89739 entries) supplemented with 179 common contaminants (e.g. keratins, serum albumin) and concatenated with the reverse sequences of all entries. Mascot search parameters were used as follows: Carbamidomethylation of cystein and oxidation of methionine were set as variable modifications, tryptic specificity with no proline restriction and up to two missed cleavages was used. The initial mass tolerance used was 7 ppm and for MS/MS 0.6 Da. Only peptides with minimal length of six amino acids were considered. Peptides were filtered for maximum false discovery rate of 1% in MaxQuant. Only unique and razor peptides with posterior error probability of less than 0.05 and proteins with ratio count of at least three were accepted and used for quantification (27Cox 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 (9262) Google Scholar). Results from MaxQuant were analyzed and visualized with R (script details are available in supplemental Experimental Procedures). Proteins showing opposite ratios between forward and reverse label-swap experiments were manually validated using MaxQuant Viewer, marked as potential false positives and included into a separate list that was not used for plotting with R. All proteins are listed in supplemental Tables S1–S4 with accession numbers, number of unique peptides, % sequence coverage, quantification significance, and variability, as reported by MaxQuant. For enrichment analysis-based hierarchical clustering, the quantified proteins from each experiment were divided into five lists corresponding to enrichment ratio cutoffs of below –4 to –4, –4 to –2, –2 to 2, 2 to 4, and above 4. Proteins from each list were searched for enriched protein domains terms using DAVID (28Huang da W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat. Protoc. 2009; 4: 44-57Crossref PubMed Scopus (25565) Google Scholar) without enrichment score cutoff (databases used: UniProt, Sequence Feature, InterPro, PIR Superfamily, PFAM, SMART). The resulting lists were then collated using a Python script (script details are available in supplemental Experimental Procedures; http://www.python.org). All terms being enriched with EASE score from DAVID of better than 0.1 in at least one of the lists were included into a combined list. Hierarchical clustering was done in the R statistical environment using the Euclidean distance function and combined linkage method. cDNAs encoding mACTL8 (IMAGE ID:IRATp970D1240D), mFANCF (IMAGE ID:IRATp970B10123D), mSPIN1 (IMAGE ID:IRAVp968H0931D), and mADNP (FANTOM3 ID:6330563C07) were obtained from Imagenes. cDNA encoding mZmym3 was amplified from cDNA isolated from NIH3T3 cells (reference sequence GenBank NM_019831.3). cDNAs were cloned into a modified pcDNA3.1 vector fusing the 3′ end to a 2xHA-2XFLAG tag. The following primer pairs were used for PCR amplification

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