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• W2006455330 abstract "The composition of chromatin-remodeling complexes dictates how these enzymes control transcriptional programs and cellular identity. In the present study we investigated the composition of SWI/SNF complexes in embryonic stem cells (ESCs). In contrast to differentiated cells, ESCs have a biased incorporation of certain paralogous SWI/SNF subunits with low levels of BRM, BAF170, and ARID1B. Upon differentiation, the expression of these subunits increases, resulting in a higher diversity of compositionally distinct SWI/SNF enzymes. We also identified BRD7 as a novel component of the Polybromo-associated BRG1-associated factor (PBAF) complex in both ESCs and differentiated cells. Using short hairpin RNA-mediated depletion of BRG1, we showed that SWI/SNF can function as both a repressor and an activator in pluripotent cells, regulating expression of developmental modifiers and signaling components such as Nodal, ADAMTS1, BMI-1, CRABP1, and thyroid releasing hormone. Knockdown studies of PBAF-specific BRD7 and of a signature subunit within the BAF complex, ARID1A, showed that these two subcomplexes affect SWI/SNF target genes differentially, in some cases even antagonistically. This may be due to their different biochemical properties. Finally we examined the role of SWI/SNF in regulating its target genes during differentiation. We found that SWI/SNF affects recruitment of components of the preinitiation complex in a promoter-specific manner to modulate transcription positively or negatively. Taken together, our results provide insight into the function of compositionally diverse SWI/SNF enzymes that underlie their inherent gene-specific mode of action. The composition of chromatin-remodeling complexes dictates how these enzymes control transcriptional programs and cellular identity. In the present study we investigated the composition of SWI/SNF complexes in embryonic stem cells (ESCs). In contrast to differentiated cells, ESCs have a biased incorporation of certain paralogous SWI/SNF subunits with low levels of BRM, BAF170, and ARID1B. Upon differentiation, the expression of these subunits increases, resulting in a higher diversity of compositionally distinct SWI/SNF enzymes. We also identified BRD7 as a novel component of the Polybromo-associated BRG1-associated factor (PBAF) complex in both ESCs and differentiated cells. Using short hairpin RNA-mediated depletion of BRG1, we showed that SWI/SNF can function as both a repressor and an activator in pluripotent cells, regulating expression of developmental modifiers and signaling components such as Nodal, ADAMTS1, BMI-1, CRABP1, and thyroid releasing hormone. Knockdown studies of PBAF-specific BRD7 and of a signature subunit within the BAF complex, ARID1A, showed that these two subcomplexes affect SWI/SNF target genes differentially, in some cases even antagonistically. This may be due to their different biochemical properties. Finally we examined the role of SWI/SNF in regulating its target genes during differentiation. We found that SWI/SNF affects recruitment of components of the preinitiation complex in a promoter-specific manner to modulate transcription positively or negatively. Taken together, our results provide insight into the function of compositionally diverse SWI/SNF enzymes that underlie their inherent gene-specific mode of action. Chromatin plays a key role in the regulation of tissue-specific gene expression during development and differentiation (1Muller C. Leutz A. Curr. Opin. Genet. Dev. 2001; 11: 167-174Crossref PubMed Scopus (108) Google Scholar). Embryonic stem cells (ESCs) 2The abbreviations used are: ESC, embryonic stem cell; RA, retinoic acid; BAF, BRG1-associated factor; PBAF, Polybromo-associated BAF; MudPIT, multidimensional protein identification technique; shRNA, short hairpin RNA; RNAi, RNA interference; ES, embryonic stem; HA, hemagglutinin; ChIP, chromatin immunoprecipitation; TF, transcription factor; POLII, RNA polymerase II; IP, immunoprecipitation; MS, mass spectrometry; BCL7, B-cell leukemia protein 7; BRD7, bromodomain-containing protein 7; TRH, thyroid releasing hormone; PIC, preinitiation complex. possess a distinctive global chromatin structure that is characterized by hyperdynamic architectural proteins (2Meshorer E. Yellajoshula D. George E. Scambler P.J. Brown D.T. Misteli T. Dev. Cell. 2006; 10: 105-116Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar) and bivalent domains (3Bernstein B.E. Mikkelsen T.S. Xie X. Kamal M. Huebert D.J. Cuff J. Fry B. Meissner A. Wernig M. Plath K. Jaenisch R. Wagschal A. Feil R. Schreiber S.L. Lander E.S. Cell. 2006; 125: 315-326Abstract Full Text Full Text PDF PubMed Scopus (4106) Google Scholar), ultimately resulting in elevated global transcription compared with differentiated cells (4Efroni S. Duttagupta R. Cheng J. Dehghani H. Hoeppner D.J. Dash C. Bazett-Jones D.P. Le Grice S. McKay R.D. Buetow K.H. Gingeras T.R. Misteli T. Meshorer E. Cell Stem Cell. 2008; 2: 437-447Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). This chromatin structure is dictated by stem cell-specific transcription factors, chromatin architecture, and epigenetic regulation (5Niwa H. Development. 2007; 134: 635-646Crossref PubMed Scopus (663) Google Scholar) and is a prerequisite for self-renewal and the capacity to differentiate into the three germ layers (6Meshorer E. Misteli T. Nat. Rev. Mol. Cell Biol. 2006; 7: 540-546Crossref PubMed Scopus (558) Google Scholar). Important determinants of this unique genomic plasticity are ATP-dependent chromatin-remodeling complexes. These multisubunit enzymes catalyze non-covalent eviction, restructuring or repositioning of nucleosomes to modulate the accessibility of transcription factors and other regulatory proteins to chromosomal DNA (7Becker P.B. Horz W. Annu. Rev. Biochem. 2002; 71: 247-273Crossref PubMed Scopus (625) Google Scholar). Multiple distinct families of chromatin-remodeling complexes exist, some of which have been implicated in developmental processes (8de la Serna I.L. Ohkawa Y. Imbalzano A.N. Nat. Rev. Genet. 2006; 7: 461-473Crossref PubMed Scopus (313) Google Scholar, 9Kaeser M.D. Emerson B.M. Curr. Opin. Genet. Dev. 2006; 16: 508-512Crossref PubMed Scopus (13) Google Scholar). For example, genomic disruption of specific chromatin-remodeling components results in early embryonic lethality (10Stopka T. Skoultchi A.I. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14097-14102Crossref PubMed Scopus (164) Google Scholar, 11Bultman S. Gebuhr T. Yee D. La Mantia C. Nicholson J. Gilliam A. Randazzo F. Metzger D. Chambon P. Crabtree G. Magnuson T. Mol. Cell. 2000; 6: 1287-1295Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar, 12Klochendler-Yeivin A. Fiette L. Barra J. Muchardt C. Babinet C. Yaniv M. EMBO Rep. 2000; 1: 500-506Crossref PubMed Scopus (339) Google Scholar, 13Kim J.K. Huh S.O. Choi H. Lee K.S. Shin D. Lee C. Nam J.S. Kim H. Chung H. Lee H.W. Park S.D. Seong R.H. Mol. Cell. Biol. 2001; 21: 7787-7795Crossref PubMed Scopus (170) Google Scholar, 14Gao X. Tate P. Hu P. Tjian R. Skarnes W.C. Wang Z. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 6656-6661Crossref PubMed Scopus (270) Google Scholar). Other remodeling modules are required to maintain the balance between ESC self-renewal and differentiation (15Fazzio T.G. Huff J.T. Panning B. Cell. 2008; 134: 162-174Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 16Kaji K. Caballero I.M. MacLeod R. Nichols J. Wilson V.A. Hendrich B. Nat. Cell Biol. 2006; 8: 285-292Crossref PubMed Scopus (299) Google Scholar, 17Yan Z. Wang Z. Sharova L. Sharov A.A. Ling C. Piao Y. Aiba K. Matoba R. Wang W. Ko M.S. Stem Cells (Dayton). 2008; 26: 1155-1165Crossref PubMed Scopus (139) Google Scholar). Specific components of chromatin remodelers confer specialized activities and selective gene targeting to distinct complexes. For example, the evolutionarily conserved SWI/SNF family exists in several subgroups and is implicated in regulating cellular processes such as transcription, differentiation, development, and tumorigenesis (8de la Serna I.L. Ohkawa Y. Imbalzano A.N. Nat. Rev. Genet. 2006; 7: 461-473Crossref PubMed Scopus (313) Google Scholar, 18Simone C. J. Cell. Physiol. 2006; 207: 309-314Crossref PubMed Scopus (83) Google Scholar, 19Mohrmann L. Verrijzer C.P. Biochim. Biophys. Acta. 2005; 1681: 59-73Crossref PubMed Scopus (276) Google Scholar). Each subgroup contains one of two highly homologous ATPases, BRG1 or BRM, and a variable composition of BRG1-associated factors (BAFs) (20Wang W. Xue Y. Zhou S. Kuo A. Cairns B.R. Crabtree G.R. Genes Dev. 1996; 10: 2117-2130Crossref PubMed Scopus (573) Google Scholar, 21Wang W. Cote J. Xue Y. Zhou S. Khavari P.A. Biggar S.R. Muchardt C. Kalpana G.V. Goff S.P. Yaniv M. Workman J.L. Crabtree G.R. EMBO J. 1996; 15: 5370-5382Crossref PubMed Scopus (686) Google Scholar). BRG1- and BRM-SWI/SNF complexes have very different functions as shown by gene knock-out experiments in which loss of BRG1, but not BRM, is embryonic lethal (11Bultman S. Gebuhr T. Yee D. La Mantia C. Nicholson J. Gilliam A. Randazzo F. Metzger D. Chambon P. Crabtree G. Magnuson T. Mol. Cell. 2000; 6: 1287-1295Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar, 22Reyes J.C. Barra J. Muchardt C. Camus A. Babinet C. Yaniv M. EMBO J. 1998; 17: 6979-6991Crossref PubMed Scopus (375) Google Scholar). BAF subunits that constitute “core” SWI/SNF complexes include BAFs 170, 155, 60, 57, 53, and 47 (SNF5/INI1) and actin (20Wang W. Xue Y. Zhou S. Kuo A. Cairns B.R. Crabtree G.R. Genes Dev. 1996; 10: 2117-2130Crossref PubMed Scopus (573) Google Scholar, 21Wang W. Cote J. Xue Y. Zhou S. Khavari P.A. Biggar S.R. Muchardt C. Kalpana G.V. Goff S.P. Yaniv M. Workman J.L. Crabtree G.R. EMBO J. 1996; 15: 5370-5382Crossref PubMed Scopus (686) Google Scholar). Additionally “specificity” subunits like ARID2 (AT-rich interactive domain 2, BAF200) and Polybromo (BAF180) distinguish Polybromo-associated BAF (PBAF) from BAF complexes, which are specified by ARID1 (BAF250) (23Nie Z. Xue Y. Yang D. Zhou S. Deroo B.J. Archer T.K. Wang W. Mol. Cell. Biol. 2000; 20: 8879-8888Crossref PubMed Scopus (245) Google Scholar). Orthologous “signature” subunits also have been shown to confer specialized activities to SWI/SNF complexes in Drosophila (24Moshkin Y.M. Mohrmann L. van Ijcken W.F. Verrijzer C.P. Mol. Cell. Biol. 2007; 27: 651-661Crossref PubMed Scopus (105) Google Scholar). Interestingly some subunits exist as paralogues that are differentially expressed in a tissue-restricted manner, such as BAF60A, -B, and -C, and impart specific functions to SWI/SNF (18Simone C. J. Cell. Physiol. 2006; 207: 309-314Crossref PubMed Scopus (83) Google Scholar). Interestingly incorporation of distinct, mutually exclusive paralogues of the ARID1 protein family into SWI/SNF complexes determines whether SWI/SNF functions as a corepressor (ARID1A) or coactivator (ARID1B) of cell cycle control genes (25Nagl Jr., N.G. Wang X. Patsialou A. Van Scoy M. Moran E. EMBO J. 2007; 26: 752-763Crossref PubMed Scopus (196) Google Scholar). Given the diverse nature of SWI/SNF enzymes and the requirement for some, but not all, of its subunits in embryonic stem cells, we investigated which complexes exist in pluripotent cells. Our results indicate that several BAF subunits form a core that is contained in the majority of SWI/SNF enzymes. Surprisingly in ESCs specific paralogues predominate in SWI/SNF complexes, and incorporation of related proteins is restricted by transcriptional repression of their genes. This indicates a reduced diversity of SWI/SNF complexes in pluripotent cells that is reversed upon differentiation, probably reflecting the need to regulate more intricate transcriptional programs. Our functional analysis of BRG1 in pluripotent cells revealed that SWI/SNF can both repress and activate target genes. We also identified novel stoichiometric components of SWI/SNF complexes, among them the PBAF-specific bromodomain-containing protein 7 (BRD7) protein. Using an RNAi-based approach for BRD7 and ARID1A, we showed that both BAF and PBAF complexes can play important roles in gene-specific repression and activation. Overall our results add new insights into how the composition of SWI/SNF complexes imposes transcriptional regulation on individual target genes. Cell Culture and Differentiation—293T and R1 mouse ESCs were obtained from ATCC. ES cells were cultivated on feeder cells according to ATCC guidelines. R201 cells, R218 cells, and their derivatives were maintained on gelatinized tissue culture dishes in Dulbecco's modified Eagle's medium supplied with 15% fetal bovine serum and leukemia-inhibitory factor. For retinoic acid (RA) differentiation, leukemia-inhibitory factor was omitted, and medium was supplied with 1 μm all-trans-retinoic acid and changed daily. Lentiviral Production and Infection—Lentiviral expression vectors are based on pWPT-GFP, which contain an SV40-puro cassette. All transgenes are expressed by an EF1α promoter. Detailed maps are available upon request. Lentiviral shRNAs were constructed as described previously (26Tiscornia G. Singer O. Ikawa M. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1844-1848Crossref PubMed Scopus (500) Google Scholar) for BRG1 and the control hairpin targeting GLUT4 (27Liao W. Nguyen M.T. Imamura T. Singer O. Verma I.M. Olefsky J.M. Endocrinology. 2006; 147: 2245-2252Crossref PubMed Scopus (54) Google Scholar) or purchased from Sigma (ARID1A, BRD7, and scrambled hairpin control). The targeted sequence in BRG1 is AAGCACCAGGAGTACCTCAAC. Lentiviral particles were produced and concentrated as described previously (28Tiscornia G. Singer O. Verma I.M. Nat. Protoc. 2006; 1: 241-245Crossref PubMed Scopus (729) Google Scholar). To establish stably expressing cell lines, 105 ESCs were infected at low multiplicity and selected with puromycin (0.25 mg/liter) for 7 days. Cell lines R201 and R218 were derived by infection of R1 with viruses expressing Nanog-HA and Nanog-MYC, respectively, and single cell-cloned. Antibodies, Protein Purification, and ATPase Assay—For immunodetection or ChIP, the following antibodies were used: BRG1 (G7), BAF155 (H76), BAF170 (H116), HA probe (F9), TFIIB (C-18), TFIID (N-12), and RNA polymerase II (POLII) (H-224) from Santa Cruz Biotechnology; BAF47 (612110) and BAF60 (611728) from BD Transduction Laboratories; FLAG probe (M2) from Sigma; BRM (ab15597) from Abcam; and BRD7 (51009-2-AP) from Proteintech. Antibodies recognizing ARID2, BAF57, BAF53, and J1 were generous gifts from Dr. Weidong Wang; ARID1A was from Dr. Elisabeth Moran. For protein purification and co-IPs, cells were lysed in DBP-IP (20 mm HEPES, pH 7.9, 1.5 mm MgCl2, 140 mm NaCl, 0.1% Nonidet P-40, 10% glycerol, 0.2 mm ZnCl2, 1 mm dithiothreitol, protease inhibitors). After disruption by brief sonication, chromatin proteins were extracted by increasing NaCl to 420 mm. After pelleting insoluble material, protein complexes were purified using M2 FLAG affinity matrix (Sigma). Immunocomplexes were washed six times with DBP-IP containing 420 mm NaCl and 20 μg/ml ethidium bromide and eluted in BC0.1 buffer (20 mm HEPES, pH 7.9, 2 mm EDTA, 20% glycerol, 100 mm KCl) and a 0.5 mg/ml concentration of the corresponding peptide. ATPase assays were essentially performed as described previously (29Logie C. Peterson C.L. Methods Enzymol. 1999; 304: 726-741Crossref PubMed Scopus (53) Google Scholar) with the following modifications: 200 ng of purified SWI/SNF were incubated with trace amounts of 32P-labeled γ-ATP and incubated at 37 °C. DNA stimulation was performed with 20 ng of genomic DNA. Different phosphate species were separated by thin layer chromatography and quantified using a PhosphorImager. Multidimensional Protein Identification Technique (MudPIT) Analysis—Trichloroacetic acid-precipitated IP samples were resuspended in 8 m urea, 100 mm Tris, pH 8.5; reduced; alkylated; diluted to 2 m urea, 1 mm CaCl2, 100 mm Tris, pH 8.5; and digested with trypsin. The tryptic digests were supplemented with formic acid to 5% and analyzed using the anion and cation exchange MudPIT method as described previously (30Motoyama A. Xu T. Ruse C.I. Wohlschlegel J.A. Yates III, J.R. Anal. Chem. 2007; 79: 3623-3634Crossref PubMed Scopus (145) Google Scholar). Briefly the samples were loaded onto a 250-μm-inner diameter column with a Kasil frit containing a 2.5-cm reverse phase section packed with 5-μm, 125-Å Aqua C18 resin (Phenomenex, Torrance, CA) and a 2.5-cm anion and cation exchange section proximal to the frit. The anion and cation exchange section was packed with a 1:2 mixture of strong cation exchange resin (Partisphere 5-μm SCX resin from Whatman) and anion exchange resin (PolyWAX LP from PolyLC Inc., Columbia, MD). After desalting, this biphasic column was connected to a 10-cm-long, 100-μm-inner diameter analytical reverse phase column made of 3-μm, 125-Å Aqua C18 resin (Phenomenex). MS analysis was performed on a linear trap quadrupole mass spectrometer (ThermoFisher Scientific) using a three-step MudPIT method with salt pulses at 0, 40, and 100% buffer C. Each full MS scan was followed by five MS/MS scans. The MS/MS spectra were searched with SEQUEST against a mouse International Protein Index protein data base using a 3-atomic mass unit mass tolerance. The search results were filtered with a modified version of DTASelect (31Tabb D.L. McDonald W.H. Yates III, J.R. J. Proteome Res. 2002; 1: 21-26Crossref PubMed Scopus (1144) Google Scholar) with a 5% false positive cutoff at the spectrum level, requiring peptides to be half- or fully tryptic and a minimum of two peptides per protein identification. The false positive rate for protein identification is 2% or lower. SWI/SNF subunits were absent in the control sample (Fig. 1, no tagged subunit) except BRG1 (three peptides) and BAF53A (two peptides). Mowse protein scores are derived from peptide scores as a non-probabilistic basis for ranking protein hits. Peptide score is −10 × log(p) where p is the probability that the observed match is a random event. Individual peptide scores >27 indicate identity or extensive homology (p < 0.05). Chromatin IP—ChIP was performed as described previously (32Kaeser M.D. Iggo R.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 95-100Crossref PubMed Scopus (272) Google Scholar) with buffers described by Upstate with the following modifications: ∼40 × 106 cross-linked cells were resuspended in 2 ml of SDS lysis buffer and sonicated 4 × 8 s, power 4. Soluble complexes were diluted in 3 volumes of ChIP dilution buffer, and lysate corresponding to ∼107 cells was incubated with 2 μg of antibody (HA, TFIIB, TFIID, or POLII) or 2 μl of J1 prebound on 20 μl of Dynal protein G beads. After two washes in high salt wash buffer, two washes in LiCl wash buffer, and two washes in high salt wash buffer, complexes were decross-linked at 65 °C for 6 h. DNA was precipitated using 10 μg of yeast tRNA and 10 μg of glycogen. RNA Extraction and Quantitative PCR—Total RNA was extracted using TRIzol (Invitrogen). Reverse transcription was performed with 0.5 μg of total RNA, random hexamers, and SuperScript III polymerase (Invitrogen). Quantitative PCR was performed on a Stratagene Mx3005P system using SYBR Green (Applied Biosystems). The error bars shown represent duplicate measurements from independent biological duplicates. Primer sequences are listed in supplemental materials. Composition of SWI/SNF in Pluripotent ES Cells—Initially we investigated which forms of the SWI/SNF multisubunit complex exist in ESCs. We used a strategy in which epitope-tagged subunits are virally integrated into the ESC genome. Affinity purification is then accomplished in a simple one-step procedure, resulting in native multisubunit protein preparations of high yield and purity (33Sif S. Stukenberg P.T. Kirschner M.W. Kingston R.E. Genes Dev. 1998; 12: 2842-2851Crossref PubMed Scopus (227) Google Scholar). We expressed several well characterized subunits to increase our ability to purify most of the compositionally distinct forms of SWI/SNF that may be present in ESCs. BAF47, BAF57, BAF155, BAF170, and BRG1 are all core subunits that associate with both BAF and PBAF complexes (Fig. 1A). To purify SWI/SNF from substantial amounts of homogeneously undifferentiated ESCs, we created the cell lines R201 and R218. Both were derived from the R1 ESC line by infection with lentiviruses expressing Nanog-HA and Nanog-MYC, respectively. Both cell lines exhibit morphological features similar to pluripotent ES cells and differentiate upon leukemia-inhibitory factor withdrawal or RA treatment (supplemental Fig. 1). We also compared gene expression in parental R1 and R218 cells in response to RA and found them to be very similar albeit with a slower Oct4 mRNA decrease in R218 cells. Additionally the R218 line was capable of differentiating into spontaneously beating cardiomyocytes with efficiency similar to that reported previously (34Wobus A.M. Kaomei G. Shan J. Wellner M.C. Rohwedel J. Ji G. Fleischmann B. Katus H.A. Hescheler J. Franz W.M. J. Mol. Cell. Cardiol. 1997; 29: 1525-1539Abstract Full Text PDF PubMed Scopus (359) Google Scholar) (data not shown). SWI/SNF in Embryonic Stem Cells Is Composed of a Limited Subset of Components—To prepare purified SWI/SNF complexes, R201 cells were transduced with lentiviruses expressing a C-terminal FLAG-tagged cDNA of Baf47, Baf57, Baf155, Baf170, or Brg1. Except for BAF170, ectopic expression of SWI/SNF subunits did not lead to an overall increase in protein levels (Fig. 1B). To examine SWI/SNF composition in an unbiased manner, we purified sufficient material to analyze by silver staining and MudPIT. As shown in Fig. 1C, anti-FLAG eluates from ESCs expressing individual FLAG-tagged BAFs 47, 57, 155, or 170 or BRG1 contained a similar set of proteins, which resembles the subunit pattern observed in the initial SWI/SNF purifications (35Kwon H. Imbalzano A.N. Khavari P.A. Kingston R.E. Green M.R. Nature. 1994; 370: 477-481Crossref PubMed Scopus (648) Google Scholar). Using Western blotting, mass spectroscopy on the individual bands, and differences in migration upon expression of the FLAG-tagged subunits (supplemental Fig. 2), we co-localized the known SWI/SNF subunits ARID1A, ARID2, BRG1, BAF170, BAF155, BAF60, BAF57, and BAF53 with the indicated bands. The Majority of SWI/SNF Complexes Contain the Core Subunits BAFs 47, 57, and 155 and BRG1—We then examined by Western blotting whether any subunits were preferentially assembled into particular complexes by unique associations with other BAF proteins. We observed that the bait for the purification was in general slightly overrepresented (Fig. 1D). However, with the exception of the BAF170 sample, the preparations showed a remarkably similar ratio between the other examined core components. This is consistent with the notion that the majority of purified complexes contain all four subunits: BAFs 47, 57, and 155 and BRG1. BAF170 was below detection in all samples except where it was ectopically expressed, suggesting that it is rare in ESCs and that the lentiviral expression increased its abundance above normal, endogenous levels. As expected, its ectopic expression and incorporation in SWI/SNF complexes resulted in the replacement of one or both molecules of its paralogue BAF155, explaining why BAF155 levels are lower in complexes purified through BAF170. Because of the overexpression of BAF170, we did not analyze this sample any further. Several Paralogous Subunits Are Overrepresented by Specific Forms—A comparison of the samples by MudPIT revealed unique peptides of 14 previously documented SWI/SNF components in the four subunit-specific ESC and HeLa purifications (Table 1). The control sample only contained a total of five peptides from two different subunits (see “Experimental Procedures”). Because certain groups of paralogous subunits (BRG1/BRM, BAF155/BAF170, and ARID1A/ARID1B) share substantial sequence homology (>50% in each group), we only included peptides in our analysis that could be unambiguously attributed to one specific polypeptide. Because BRM and BRG1 are mutually exclusive subunits (21Wang W. Cote J. Xue Y. Zhou S. Khavari P.A. Biggar S.R. Muchardt C. Kalpana G.V. Goff S.P. Yaniv M. Workman J.L. Crabtree G.R. EMBO J. 1996; 15: 5370-5382Crossref PubMed Scopus (686) Google Scholar), the single unique BRM peptide in our BRG1 purification reflects the expected false positive identification rate of 2% or lower. We expected roughly similar amounts of unambiguous peptides when comparing the paralogous subunits within their groups as observed in our analysis of SWI/SNF from HeLa cells. However, we found that in ESCs specific paralogous subunits were preferentially incorporated. In the case of BRG1/BRM, our results suggest that the underrepresented protein BRM might exist in negligible quantities in ESCs in agreement with its low expression during early development (36Dauvillier S. Ott M.O. Renard J.P. Legouy E. Mech. Dev. 2001; 101: 221-225Crossref PubMed Scopus (11) Google Scholar). This is also the case for the postmitotic neuron-specific BAF53B of which we could not find any unambiguous peptides (37Olave I. Wang W. Xue Y. Kuo A. Crabtree G.R. Genes Dev. 2002; 16: 2509-2517Crossref PubMed Scopus (122) Google Scholar). Also in contrast to HeLa cells, BAF170 and ARID1B had considerably fewer unambiguous peptides in ESCs than their counterparts BAF155 and ARID1A, implying that these subunits are less abundant. ARID1B-containing BAF enzymes were shown to interact with transcriptional activators as opposed to complexes with ARID1A that associate with transcriptional repressors (25Nagl Jr., N.G. Wang X. Patsialou A. Van Scoy M. Moran E. EMBO J. 2007; 26: 752-763Crossref PubMed Scopus (196) Google Scholar), suggesting that in ESCs SWI/SNF might be compositionally better suited for a repressive role.TABLE 1Biased paralogue usage in ES cellsHeLa BAF47ESCBRG1BAF47BAF57BAF155BAF471823212115BRG13476484550BRM341122BAF1554467526237BAF170601214105BAF60A1934303227BAF60B171091511BAF60C25332BAF53A2538152518BAF572217212817PB5139373125ARID1A4861496450ARID1B3611141512ARID23018241416 Open table in a new tab Differentiation Increases Incorporation of Previously Under-represented SWI/SNF Components—Because we observed a surprisingly biased usage of paralogous SWI/SNF subunits in pluripotent cells, we examined the changes in SWI/SNF composition during ESC differentiation. We used RA for 2 and 6 days to differentiate pluripotent stem cells into restricted descendants. SWI/SNF complexes were purified from differentiating cells using a tagged version of BAF47 as described previously (33Sif S. Stukenberg P.T. Kirschner M.W. Kingston R.E. Genes Dev. 1998; 12: 2842-2851Crossref PubMed Scopus (227) Google Scholar) and compared with a preparation from undifferentiated cells (Fig. 2A). To identify subunit-specific changes, we performed Western blot analyses (Fig. 2B). Each SWI/SNF preparation was standardized to give approximately the same levels of the core components BAF47 and BAF57. Throughout the course of differentiation, we observed considerable induction of some subunits, such as BAF53, BAF170, and BRM. Conversely ARID2 and ARID1A decreased by day 6 of differentiation. BAF155 exhibited a decrease in the immunoreactive 155-kDa band but an increase in the signal at 120 kDa. Mass spectroscopy of this silver-stainable band at 120 kDa identified 18 individual peptides of BAF155, supporting the notion of a splice variant, specific cleavage, or degradation product of BAF155, which may represent the previously observed BAF110 (21Wang W. Cote J. Xue Y. Zhou S. Khavari P.A. Biggar S.R. Muchardt C. Kalpana G.V. Goff S.P. Yaniv M. Workman J.L. Crabtree G.R. EMBO J. 1996; 15: 5370-5382Crossref PubMed Scopus (686) Google Scholar). RA-mediated Differentiation Increases ARID1B at the Expense of ARID1A—Several distinct mechanisms could be responsible for the changes in SWI/SNF composition, such as differences in subunit transcription, protein stability, or variations in subunit incorporation rates. It was shown previously that differentiation leads to changes in cellular protein levels that are very similar to the variations we observed in SWI/SNF composition (17Yan Z. Wang Z. Sharova L. Sharov A.A. Ling C. Piao Y. Aiba K. Matoba R. Wang W. Ko M.S. Stem Cells (Dayton). 2008; 26: 1155-1165Crossref PubMed Scopus (139) Google Scholar). We therefore tested whether differences in subunit transcription might explain the changes in SWI/SNF composition by measuring their individual RNA expression levels (Fig. 2C). We observed little change, if any, in expression of Baf155 and Brg1, whereas considerable increases in Brm and Baf170 transcripts were apparent. This corroborated our measurements of protein levels and the indications obtained from MudPIT analysis of these samples (Table 2). We also detected a reduction in Arid1a message, confirming the decrease observed in Western blotting by 6 days of differentiation. Interestingly Arid1b exhibits an opposing phenotype revealed by the increase in both transcript and unique peptides in mass spectroscopy. Unfortunately we could not confirm this by Western blotting because we lacked antibodies that recognized ARID1B under these conditions. The compositional changes we observed by different approaches (and suggested by MudPIT for ARID1B) are summarized in Fig. 2D.TABLE 2Differentiation increases usage of previously underrepresented paraloguesESCDay 2Day 6BAF47443746BRG1606887BRM2120BAF15598123108BAF170215576BAF60A413839BAF60B122130BAF60C3731BAF53A212933BAF57262832PB927575ARID1A584966ARID1B71941ARID2453722 Open table in a new tab B-cell Leukemia Protein 7 (BCL7) Fa" @default.
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