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Regulatory Factor for X-box Family Proteins Differentially Interact with Histone Deacetylases to Repress Collagen α2(I) Gene (COL1A2) Expression

2006; Elsevier BV; Volume: 281; Issue: 14 Linguagem: Inglês

10.1074/jbc.m511724200

ISSN

1083-351X

Autores

Yong Xu, Pritam K. Sengupta, Edward Seto, Barbara D. Smith,

Tópico(s)

Genomics and Chromatin Dynamics

Resumo

Our studies indicate that the regulatory factor for X-box (RFX) family proteins repress collagen α2(I) gene (COL1A2) expression (Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2003) J. Biol. Chem. 278, 49134–49144; Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2004) J. Biol. Chem. 279, 41319–41332). In this study, we examined the mechanism(s) underlying the repression of collagen gene by RFX proteins. Two members of the RFX family, RFX1 and RFX5, associate with distinct sets of co-repressors on the collagen transcription start site in vitro. RFX5 specifically interacts with histone deacetylase 2 (HDAC2) and the mammalian transcriptional repressor (mSin3B), whereas RFX1 preferably interacts with HDAC1 and mSin3A. HDAC2 cooperates with RFX5 to down-regulate collagen promoter activity, whereas HDAC1 enhances inhibition of collagen promoter activity by RFX1. Interferon-γ promotes the recruitment of RFX5/HDAC2/mSin3B to the collagen transcription start site but decreases the occupancy by RFX1/mSin3A as manifested by chromatin immunoprecipitation assay. RFX1 binds to the methylated collagen sequence with much higher affinity than unmethylated sequence, recruiting more HDAC1 and mSin3A. The DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine, which inhibits DNA methylation, reduces RFX1/HDAC1 binding to the collagen transcription start site in chromatin immunoprecipitation assays. Finally, both RFX1 and RFX5 are acetylated in vivo. Trichostatin A stimulates the acetylation of RFX proteins and activates the collagen promoter activity. Collectively, our data strongly indicate two separate pathways for RFX proteins to repress collagen gene expression as follows: one for RFX5/HDAC2 in interferon-γ-mediated repression, and the other for RFX1/HDAC1 in methylation-mediated collagen silencing. Our studies indicate that the regulatory factor for X-box (RFX) family proteins repress collagen α2(I) gene (COL1A2) expression (Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2003) J. Biol. Chem. 278, 49134–49144; Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2004) J. Biol. Chem. 279, 41319–41332). In this study, we examined the mechanism(s) underlying the repression of collagen gene by RFX proteins. Two members of the RFX family, RFX1 and RFX5, associate with distinct sets of co-repressors on the collagen transcription start site in vitro. RFX5 specifically interacts with histone deacetylase 2 (HDAC2) and the mammalian transcriptional repressor (mSin3B), whereas RFX1 preferably interacts with HDAC1 and mSin3A. HDAC2 cooperates with RFX5 to down-regulate collagen promoter activity, whereas HDAC1 enhances inhibition of collagen promoter activity by RFX1. Interferon-γ promotes the recruitment of RFX5/HDAC2/mSin3B to the collagen transcription start site but decreases the occupancy by RFX1/mSin3A as manifested by chromatin immunoprecipitation assay. RFX1 binds to the methylated collagen sequence with much higher affinity than unmethylated sequence, recruiting more HDAC1 and mSin3A. The DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine, which inhibits DNA methylation, reduces RFX1/HDAC1 binding to the collagen transcription start site in chromatin immunoprecipitation assays. Finally, both RFX1 and RFX5 are acetylated in vivo. Trichostatin A stimulates the acetylation of RFX proteins and activates the collagen promoter activity. Collectively, our data strongly indicate two separate pathways for RFX proteins to repress collagen gene expression as follows: one for RFX5/HDAC2 in interferon-γ-mediated repression, and the other for RFX1/HDAC1 in methylation-mediated collagen silencing. Collagen, currently consisting of more than 27 members, is a large family of extracellular matrix proteins that play vital structural and physiological roles maintaining the integrity and contributing to homeostasis of the human body (3Myllyharju J. Kivirikko K.I. Trends Genet. 2004; 20: 33-43Abstract Full Text Full Text PDF PubMed Scopus (884) Google Scholar). Because of their diverse structures and distributions as well as complex interactions with other components of the extracellular matrix, expression and regulation of collagen proteins are extremely complicated, yet critical processes, which occur at multiple levels as follows: transcriptional, post-transcriptional, translational, and post-translational. Type I collagen, composed of two α1(I) (COL1A1) chains and one α2(I) (COL1A2) chain, is the most abundantly expressed member of the collagen genes, thereby playing a significant role in maintaining homeostasis. 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Acetylation of histones is a dynamic and reversible process that is catalyzed by two groups of antagonistic enzymes called histone acetyltransferase and histone deacetylase (HDAC). 2The abbreviations used are: HDAC, histone deacetylase; RFX, regulatory factor for X-box; ChIP, chromatin immunoprecipitation; aza-dC,5-aza-2′-deoxycytidine; TSA, trichostatin A; IFN-γ, interferon-γ; PMSF, phenylmethylsulfonyl fluoride; ANOVA, analysis of variance; MHC, major histocompatibility; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; siHDAC, silencing HDACs; CIITA, class II transactivator. 2The abbreviations used are: HDAC, histone deacetylase; RFX, regulatory factor for X-box; ChIP, chromatin immunoprecipitation; aza-dC,5-aza-2′-deoxycytidine; TSA, trichostatin A; IFN-γ, interferon-γ; PMSF, phenylmethylsulfonyl fluoride; ANOVA, analysis of variance; MHC, major histocompatibility; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; siHDAC, silencing HDACs; CIITA, class II transactivator. So far, 18 different HDACs have been identified and categorized into three groups based on their homology to three yeast HDACs. Class I HDACs, containing HDAC1, -2, -3, and -8, are homologous to yeast yRPD3 and are expressed in most tissues (20Laherty C.D. Yang W.M. Sun J.M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar, 21Gao L. Cueto M.A. Asselbergs F. Atadja P. J. Biol. Chem. 2002; 277: 25748-25755Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar, 22Dangond F. Henriksson M. Zardo G. Caiafa P. Ekstrom T.J. Gray S.G. Int. J. Oncol. 2001; 19: 773-777PubMed Google Scholar, 23Kasten M.M. Dorland S. Stillman D.J. Mol. Cell. Biol. 1997; 17: 4852-4858Crossref PubMed Scopus (114) Google Scholar, 24Buggy J.J. Sideris M.L. Mak P. Lorimer D.D. McIntosh B. Clark J.M. Biochem. J. 2000; 350: 199-205Crossref PubMed Scopus (151) Google Scholar, 25Dangond F. Hafler D.A. Tong J.K. Randall J. Kojima R. 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For example, the NuRD complex is composed of HDAC1, HDAC2, and methyl group binding protein (33Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2240) Google Scholar, 34Zhang Y. Ng H.H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Genes Dev. 1999; 13: 1924-1935Crossref PubMed Scopus (933) Google Scholar). Another commonly found HDAC-containing complex is the Sin3 complex that consists of HDAC1 and HDAC2 in addition to Sin3-associated polypeptides SAP18 and SAP30 (35Hassig C.A. Tong J.K. Fleischer T.C. Owa T. Grable P.G. Ayer D.E. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3519-3524Crossref PubMed Scopus (332) Google Scholar, 36Zhang Y. Iratni R. Erdjument-Bromage H. Tempst P. Reinberg D. Cell. 1997; 89: 357-364Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, 37Zhang Y. Sun Z.W. Iratni R. Erdjument-Bromage H. Tempst P. Hampsey M. Reinberg D. Mol. Cell. 1998; 1: 1021-1031Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). The major focus of research at this laboratory has been toward understanding the transcriptional events occurring at the transcription start site of type I collagen genes. Earlier investigations have led to the discovery of a binding site for the regulatory factor for X-box (RFX) at the transcription start site of both α1(I) and α2(I) genes, COL1A1 and COL1A2 (38Sengupta P. Xu Y. Wang L. Widom R. Smith B.D. J. Biol. Chem. 2005; 280: 21004-21014Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 39Sengupta P.K. Fargo J. Smith B.D. J. Biol. Chem. 2002; 277: 24926-24937Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Binding of two members of RFX proteins, RFX1 and RFX5, to the start sites of the type I collagen genes represses their expression. RFX1 is able to form dimers with itself as well as with two other RFX members, RFX2 and RFX3, with which RFX1 shares significant homology, including the dimerization domain that mediates the complex formation. On the other hand, RFX5, lacking the dimerization domain, is less homologous to other family members and forms a trimeric complex with two other proteins, RFXB and RFXAP (1Xu Y. Wang L. Buttice G. Sengupta P.K. Smith B.D. J. Biol. Chem. 2003; 278: 49134-49144Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). RFX5 is also responsible for recruiting class II transactivator (CIITA), the master regulator for major histocompatibility II (MHC II) expression, to the collagen transcription start site during IFN-γ response (2Xu Y. Wang L. Buttice G. Sengupta P.K. Smith B.D. J. Biol. Chem. 2004; 279: 41319-41332Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Although both RFX1 and RFX5 down-regulate collagen expression, their distinct association with other transcription factors suggests that they are involved in different physiological and pathophysiological events leading to the repression of collagen synthesis. Our results presented here demonstrate that RFX1 and RFX5 differentially interact with class I HDACs, underlying the different pathways when repressing collagen synthesis. Cell Culture Maintenance and Treatment Protocols—Human lung fibroblasts, IMR-90, (IMR, NJ), human kidney cells 293FT (Invitrogen), and human fibrosarcoma cells HT1080 (ATCC, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin G/streptomycin (Sigma). In several studies, IMR-90 cells were treated with IFN-γ and/or TSA (Sigma). IMR-90 fibroblasts were plated in p150 tissue culture dishes at 4 × 106 cells/dish and maintained in DMEM with 10% FBS for 16–24 h. Cells were pretreated in DMEM with 0.4% FBS for 16 h prior to IFN-γ treatment (100 units/ml in 0.4% DMEM for 0, 8, 16, or 24 h) and/or TSA treatment (0.5–2 μm for 24 h where applicable). IFN-γ and TSA was added together. In other studies, HT1080 cells were plated at a density of 5 × 105 cells per p35 tissue culture dish and treated with 5-aza-2′-deoxycytidine (aza-dC) (500 nm and 1 μm) for 3 days in DMEM adding fresh aza-dC each day. In some studies HT1080 cells were treated with TSA (300 nm) for 24 h. DNA Affinity Pull-down Assay—The collagen sequence (COL1A2 –25/+30, GenBank™ accession number AF004877) with a HindIII overhang was synthesized as complementary strands and annealed as described previously (40Sengupta P.K. Erhlich M. Smith B.D. J. Biol. Chem. 1999; 274: 36649-36655Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Double-stranded collagen DNA was biotin-labeled by incubating with Klenow fragment (New England Biolabs, Beverly, MA) and biotin-14-dATP (Invitrogen) supplemented with regular dCTP, dTTP, and dGTP at room temperature for 30 min. The reaction mixture was phenol/chloroform-extracted and alcohol-precipitated to remove unincorporated biotin. Nuclear protein extracts from IMR-90 cells were obtained as described previously using 450 mm sodium chloride (1Xu Y. Wang L. Buttice G. Sengupta P.K. Smith B.D. J. Biol. Chem. 2003; 278: 49134-49144Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 41Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3916) Google Scholar). The streptavidin beads (Promega, Madison, WI) were washed three times with ice-cold phosphate-buffered saline supplemented with 1 mm PMSF. Nuclear proteins (100–200 μg) were pre-cleared by incubating with the washed beads for 30 min at 4 °C on a shaking platform as described previously (2Xu Y. Wang L. Buttice G. Sengupta P.K. Smith B.D. J. Biol. Chem. 2004; 279: 41319-41332Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Pre-cleared nuclear proteins were prepared by capturing the beads on a magnetic stand and removing the supernatant. The supernatant was then incubated with biotin-labeled collagen DNA probe (–25/+30) for 1 h at room temperature in binding buffer (60 mm NaCl, 20 mm HEPES, pH 7.9, 0.1 mm EDTA, 4% glycerol, 2 mm dithiothreitol) supplemented with bovine serum albumin, poly(dI-dC), and sonicated salmon sperm DNA to remove nonspecific binding. DNA-protein complex formed was captured by the magnetic beads and washed extensively with binding buffer supplemented with 0.01% Triton X and 100 mm KCl. The bound proteins were eluted with 1× electrophoresis sample buffer by incubating at 90 °C for 10 min and analyzed by SDS-polyacrylamide gels. Plasmids, Transfections, and Luciferase Assays—The COL1A2-luciferase construct (pH20) (42Goldberg H. Helaakoski T. Garrett L.A. Karsenty G. Pellegrino A. Lozano G. Maity S. de Crombrugghe B. J. Biol. Chem. 1992; 267: 19622-19630Abstract Full Text PDF PubMed Google Scholar) contains sequences from –221 to +54 bp of mouse COL1A2 promoter fused to the luciferase reporter gene. Full-length class I HDAC expression constructs (HDAC1, HDAC2, and HDAC3) in pcDNA3 and silencing HDACs (43Zhang X. Wharton W. Yuan Z. Tsai S.C. Olashaw N. Seto E. Mol. Cell. Biol. 2004; 24: 5106-5118Crossref PubMed Scopus (77) Google Scholar) were kindly provided by Dr. Edward Seto. Full-length FLAG-RFX5 were kindly provided by Dr. Jenny Ting. RFX1 cDNA was excised from pHISB-RFX-1 plasmid, respectively, and inserted into the pIRES-hrGFP-2A construct (Stratagene) that has green fluorescent protein-coding sequence. RFX1 plasmid was digested with NotI and XhoI to produce the 5-kb vector along with 3-kb fragment. The RFX1 fragment was inserted into the EcoRI/XhoI site of the pIRES-hrGFP-2A plasmid. This bicistronic plasmid can encode the protein along with the green fluorescent protein when expressed in mammalian cell lines. Cells were plated at the density of 3 × 105 cells/well in 6-well tissue culture dishes (for IMR-90 cells) or 5 × 106 cells per p100 tissue culture dish (for 293FT cells). Transfections were performed with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. Cells were harvested 48 h post-transfection, and luciferase assays were performed with a luciferase reporter assay system (Promega). Immunoprecipitations—To investigate whether factors interact in vivo, co-immunoprecipitations were performed. Whole cell lysates (IMR-90 or 293FT with transfected constructs as indicated where applicable) were obtained by resuspending cell pellets in RIPA buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche Applied Science) and PMSF (100 μg/ml RIPA). Anti-RFX5 (194, Rockland) or anti-RFX1 (I-19; Santa Cruz Biotechnology) antibody was added to and incubated with IMR-90 cell lysate overnight before being absorbed by protein A/G PLUS-agarose beads (Santa Cruz Biotechnology). Precipitated immune complex was released by boiling with 1× SDS electrophoresis sample buffer. Alternatively, FLAG-conjugated beads (M2; Sigma) were added to and incubated with 293FT cell lysate overnight. Precipitated immune complex was eluted with 3× FLAG peptide (Sigma). Westerns—Proteins were separated by 8 or 10% polyacrylamide gel electrophoresis with pre-stained markers (Bio-Rad) for estimating molecular weight and efficiency of transfer to blots. Proteins were transferred to nitrocellulose membranes (Bio-Rad) in a Mini-Trans-Blot Cell (Bio-Rad). The membranes were blocked with 5% milk powder in Tris-buffered saline buffer (TBST: 0.05% Tween 20, 150 mm NaCl, 100 mm Tris-HCl, pH 7.4) at 4 °C overnight and incubated for 3 h to monoclonal anti-FLAG (1:1000) (Sigma), polyclonal anti-RFX5 (194, 1:1000) (Rockland), polyclonal anti-mSin3A (K-20, 1:200) (Santa Cruz Biotechnology), polyclonal anti-mSin3B (AK-12, 1:200) (Santa Cruz Biotechnology), anti-HDAC1 (H-51, 1:200) (Santa Cruz Biotechnology), polyclonal anti-HDAC2 (C-19, 1:200) (Santa Cruz Biotechnology), polyclonal anti-HDAC3 (H-99, 1:200) (Santa Cruz Biotechnology), polyclonal N-acetyl-lysine (1:2000) (Cell Signaling), and polyclonal anti-RFX1 (I-19, 1:100) (Santa Cruz Biotechnology) antibodies at room temperature. After three washes with TBST, the membranes were incubated with appropriate secondary antibodies, either anti-goat IgG (Sigma), anti-mouse IgG, or anti-rabbit IgG (Amersham Biosciences) conjugated to horseradish peroxidase, for another 1 h at room temperature. Then protein blots were visualized using ECL reagent (PerkinElmer Life Sciences) on a Kodak image station (PerkinElmer Life Sciences). Chromatin Immunoprecipitation (ChIP)—Chromatin in control and IFN-γ treated cells were cross-linked with 1% formaldehyde for 8 min at room temperature, sequentially washed with phosphate-buffered saline, Solution I (10 mm HEPES, pH 7.5, 10 mm EDTA, 0.5 mm EGTA, 0.75% Triton X-100), and Solution II (10 mm HEPES, pH 7.5, 200 mm NaCl, 1 mm EDTA, 0.5 mm EGTA). Cells were incubated in lysis buffer (150 mm NaCl, 25 mm Tris, pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet (Roche Applied Science) and PMSF. DNA was fragmented into ∼500-bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-RFX5 (194; Rockland), anti-RFX1 (I-19; Santa Cruz Biotechnology), anti-mSin3A (K-20; Santa Cruz Biotechnology), anti-mSin3B (AK-12; Santa Cruz Biotechnology), anti-HDAC1 (H-51; Santa Cruz Biotechnology), anti-HDAC2 (C-19; Santa Cruz Biotechnology), and anti-HDAC3 (H-99; Santa Cruz Biotechnology) antibodies followed by adsorption to protein A/G PLUS-agarose beads (Santa Cruz Biotechnology). Precipitated DNA-protein complexes were washed sequentially with RIPA buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 0.1% SDS, 0.5% deoxycholate, 1% Nonidet P-40, 1 mm EDTA), high salt buffer (50 mm Tris, pH 8.0, 500 mm NaCl, 0.1% SDS, 0.5% deoxycholate, 1% Nonidet P-40, 1 mm EDTA), LiCl buffer (50 mm Tris, pH 8.0, 250 mm LiCl, 0.1% SDS, 0.5% deoxycholate, 1% Nonidet P-40, 1 mm EDTA), and TE buffer (10 mm Tris, 1 mm EDTA pH 8.0), respectively. DNA-protein cross-link was reversed by heating the samples to 65 °C overnight. Proteins were digested with proteinase K (Sigma), and DNA was phenol/chloroform-extracted and precipitated by 100% ethanol. Dried DNA was dissolved in 50 μl of deionized distilled water, and 10 μl was used for each real time PCR. The primers surrounding the collagen start site for real time PCR have been described previously (1Xu Y. Wang L. Buttice G. Sengupta P.K. Smith B.D. J. Biol. Chem. 2003; 278: 49134-49144Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). RNA Isolation and Real Time PCR—Cells were harvested, and RNA was extracted using an RNeasy RNA isolation kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Reverse transcriptase reactions were performed using a SuperScript first-strand synthesis system (Invitrogen) according to the manufacturer's protocol. Real time PCRs were performed on a ABI Prism 7700 sequence detection PCR machine (Applied Biosystems, Foster City, CA) according to manufacturer's protocol. The oligonucleotide forward and reverse PCR primers and fluorescent probes are described in Table 1.TABLE 1Primers for mRNA and hnRNA real time PCRGeneAmplicon locationExonSequencesCOL1A2 mRNAaGenBank™ accession number AF004877.7568-7650/8929-8965Forward primerExon 55′-GCCCCCCAGGCAGAGA-3′TaqMan probeExon 5/66FAM-CCTGGTCTCGGTGGGAACTTTGCTG-TAMRAbThe TaqMan probes contained a fluorescent dye (FAM) and a quencher (TAMRA).Reverse primerExon 65′-CCAACTCCTTTTCCATCATACTGA-3′COL1A2 hnRNAaGenBank™ accession number AF004877.2468-2552Forward primerExon15′-CTTGCAGTAACCTTATGCCTAGCA-3′TaqMan probeExon 1/Intron 16FAM-CATGCCAATGTAAGTGCCTTCAGCTTGTT-TAMRAbThe TaqMan probes contained a fluorescent dye (FAM) and a quencher (TAMRA).Reverse primerIntron 15′-CCCATCTAACCTCTCTACCCAGTCT-3′a GenBank™ accession number AF004877.b The TaqMan probes contained a fluorescent dye (FAM) and a quencher (TAMRA). Open table in a new tab RFX1 and RFX5 Differentially Interact with Class I HDACs on COL1A2 Transcription Start Site—Previously, we reported that two members of the RFX family, RFX1 and RFX5, bind to the transcription start site of the COL1A2 gene and repress its expression (39Sengupta P.K. Fargo J. Smith B.D. J. Biol. Chem. 2002; 277: 24926-24937Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). During IFN-γ treatment, when RFX5 occupies the COL1A2 transcription start site, there is a coordinate decrease in acetylation of histones (1Xu Y. Wang L. Buttice G. Sengupta P.K. Smith B.D. J. Biol. Chem. 2003; 278: 49134-49144Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Because transcriptional repression is usually associated with the recruitment of HDACs, we examined whether RFX1 and/or RFX5 is responsible for recruiting the HDACs to the collagen site. To this end, DNA affinity pull-down experiments were performed, as described previously (2Xu Y. Wang L. Buttice G. Sengupta P.K. Smith B.D. J. Biol. Chem. 2004; 279: 41319-41332Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), with nuclear proteins extracted from human IMR-90 cells and a biotin-labeled double-stranded DNA probe that spans from –25 to +30 of the collagen promoter containing the RFX-binding site. Different DNA oligonucleotides were also used as competitors for RFX binding to test the specificity of the interactions. Streptavidin-conjugated magnetic beads were used to sequester the biotinylated probe with bound nuclear proteins. After extensive washing, bound proteins were eluted with SDS electrophoresis buffer, separated on SDS-10% PAGE, and transferred to membranes for Western analysis using specific antibodies for RFX and HDAC family members. No proteins were present in the eluates without a DNA probe (Fig. 1, A, lane 2, and B, lane 3). In the presence of the DNA probe, RFX1 is detected in the eluates along with HDAC1 (Fig. 1, A, lane 3, and B, lane 4). When a methylated sequence, which acts as a competitor specifically eliminating the binding of RFX1 (mpBR), is added as demonstrated previously (40Sengupta P.K. Erhlich M. Smith B.D. J. Biol. Chem. 1999; 274: 36649-36655Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), HDAC1 binding is also lost (Fig. 1, A, lane 4, and B, lane 5). This indicates that RFX1 selectively interacts with HDAC1 on the collagen start site. In contrast, RFX5 is able to bind to DNA in the absence of RFX1 along with HDAC2 (Fig. 1, A, lane 4, and B, lane 5), suggesting a selective interaction between RFX5 and HDAC2. The MHC II X-box sequence abolishes the binding of RFX1 (Fig. 1A, lane 5) and RFX5 (Fig. 1B, lane 6), as well as the two HDACs, whereas neither Sp1 nor Ets consensus sequence alters the binding of RFX1, HDAC1, RFX5, or HDAC2, implying that the interactions are specific. A third class I HDAC, HDAC3, is not detectable under any of these circumstances, probably because it is not recruited to the collagen transcription start site through RFX proteins. IFN-γ Enhances the Interaction between RFX5 and Co-repressors—Earlier studies indicated that IFN-γ increased RFX5 protein levels, nuclear localization, and occupation on the collagen start site in IMR-90 cells (1Xu Y. Wang L. Buttice G. Sengupta P.K. Smith B.D. J. Biol. Chem. 2003; 278: 49134-49144Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Because there was an interaction between RFX5 and HDACs, RFX5 may recruit more HDACs and IFN-γ could enhance the association between RFX5 and HDACs at the collagen transcription start site. Increased HDACs could inhibit histone acetylation and ultimately repress collagen transcription. To test this hypothesis, DNA affinity pull-down experiments were performed first with nuclear proteins extracted from IMR-90 cells treated with or without IFN-γ (100 units) for 24 h. Both RFX1 and HDAC1 bind to the collagen sequence with similar af

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