The Role of Activating Protein 1 in the Transcriptional Regulation of the Human FCGR2B Promoter Mediated by the -343 G → C Polymorphism Associated with Systemic Lupus Erythematosus
2006; Elsevier BV; Volume: 282; Issue: 3 Linguagem: Inglês
10.1074/jbc.m605808200
ISSN1083-351X
AutoresMikhail Olferiev, Emi Masuda, Shizuko Tanaka, Marissa C. Blank, Luminita Pricop,
Tópico(s)Diabetes and associated disorders
ResumoThe inhibitory receptor FcγRIIb is a negative regulator of antibody production and inflammatory responses. The -343 G → C polymorphism in the human FCGR2B promoter is associated with systemic lupus erythematosus. The -343 C mutant promoter has decreased transcriptional activity. In the present study, we show that the transcriptional change correlates with quantitative differences in the interaction of the activating protein 1 complex with the mutant FCGR2B promoter. Promoter pulldown and chromatin immunoprecipitation assays demonstrated binding of c-Jun to the FCGR2B promoter. Phosphorylation of c-Jun was accompanied by transactivation of both FCGR2B promoter variants, whereas dephosphorylation of c-Jun by an inhibitor of c-Jun N-terminal kinase, markedly decreased the promoter activities. The -343 G → C substitution enabled the specific interaction of the transcription factor Yin-Yang 1 with the mutant FCGR2B promoter. Yin-Yang 1 competed with activating protein 1 for binding at the -343 site, and contributed to the repression of the mutant FCGR2B promoter activity. This mechanism could be responsible for the decreased expression of FcγRIIb associated with the -343 C/C homozygous FCGR2B genotype in lupus patients. These findings provide a rationale for the transcriptional defect mediated by the -343 C/C FCGR2B promoter polymorphism associated with systemic lupus erythematosus, and add to our understanding of the complex transcriptional regulation of the human FCGR2B promoter. The inhibitory receptor FcγRIIb is a negative regulator of antibody production and inflammatory responses. The -343 G → C polymorphism in the human FCGR2B promoter is associated with systemic lupus erythematosus. The -343 C mutant promoter has decreased transcriptional activity. In the present study, we show that the transcriptional change correlates with quantitative differences in the interaction of the activating protein 1 complex with the mutant FCGR2B promoter. Promoter pulldown and chromatin immunoprecipitation assays demonstrated binding of c-Jun to the FCGR2B promoter. Phosphorylation of c-Jun was accompanied by transactivation of both FCGR2B promoter variants, whereas dephosphorylation of c-Jun by an inhibitor of c-Jun N-terminal kinase, markedly decreased the promoter activities. The -343 G → C substitution enabled the specific interaction of the transcription factor Yin-Yang 1 with the mutant FCGR2B promoter. Yin-Yang 1 competed with activating protein 1 for binding at the -343 site, and contributed to the repression of the mutant FCGR2B promoter activity. This mechanism could be responsible for the decreased expression of FcγRIIb associated with the -343 C/C homozygous FCGR2B genotype in lupus patients. These findings provide a rationale for the transcriptional defect mediated by the -343 C/C FCGR2B promoter polymorphism associated with systemic lupus erythematosus, and add to our understanding of the complex transcriptional regulation of the human FCGR2B promoter. Fc γ-receptors (FcγR) 2The abbreviations used are: FcγR, Fc γ-receptors; SNP, single nucleotide polymorphism; SLE, systemic lupus erythematosus; AP-1, activating protein 1; YY1, Yin-Yang 1; JNK, Jun N-terminal kinase; MEKK, mitogen-activated/extracellular signal-regulated kinase kinase kinase; EMSA, electrophortic mobility shift assay; ChIP, chromatin immunoprecipitation; shRNA, short hairpin RNA; PMA, phorbol 12-myristate 13-acetate; ION, ionomycin; WT, wild type; Bt2cAMP, dibutyryl-cAMP. 2The abbreviations used are: FcγR, Fc γ-receptors; SNP, single nucleotide polymorphism; SLE, systemic lupus erythematosus; AP-1, activating protein 1; YY1, Yin-Yang 1; JNK, Jun N-terminal kinase; MEKK, mitogen-activated/extracellular signal-regulated kinase kinase kinase; EMSA, electrophortic mobility shift assay; ChIP, chromatin immunoprecipitation; shRNA, short hairpin RNA; PMA, phorbol 12-myristate 13-acetate; ION, ionomycin; WT, wild type; Bt2cAMP, dibutyryl-cAMP. bind IgG-containing immune complexes and mediate important immune functions such as phagocytosis, degranulation, antibody-dependent cellular cytotoxicity, and production of inflammatory mediators. Human hematopoetic cells express several FcγR isoforms encoded by seven separate genes. Unlike activating FcγR, FcγRIIb is unique in that it contains an immunoreceptor tyrosine-based inhibitory motif in the intracellular domain. A specific amino acid sequence in the immunoreceptor tyrosine-based inhibitory motif domain of FcγRIIb allows the recruitment of phosphatases and the initiation of inhibitory signaling. Cross-linking of FcγRIIb with activating receptors on B cells and mononuclear phagocytes leads to down-regulation of antibody production, phagocytosis, and cytokine secretion. Several lines of evidence demonstrate that FcγRIIb is important in the maintenance of self-tolerance. FcγRIIb deficiency is associated with spontaneous development of autoimmune manifestations in several mouse genetic backgrounds (1Bolland S. Ravetch J.V. Immunity. 2000; 13: 277-285Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). Autoimmune prone mouse strains share an Fcgr2 promoter haplotype containing deletions and polymorphisms associated with reduced expression of FcγRIIb on the surface of activated B cells and macrophages (2Luan J.J. Monteiro R.C. Sautes C. Fluteau G. Eloy L. Fridman W.H. Bach J.F. Garchon H.J. J. Immunol. 1996; 157: 4707-4716PubMed Google Scholar, 3Jiang Y. Hirose S. Abe M. Sanokawa-Akakura R. Ohtsuji M. Mi X. Li N. Xiu Y. Zhang D. Shirai J. Hamano Y. Fujii H. Shirai T. Immunogenetics. 2000; 51: 429-435Crossref PubMed Scopus (146) Google Scholar, 4Pritchard N.R. Cutler A.J. Uribe S. Chadban S.J. Morley B.J. Smith K.G. Curr. Biol. 2000; 10: 227-230Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Whereas the engineered deletion and the natural deficiency in FcγRIIb confer susceptibility to development of lupus-like disease, transplantation of bone marrow cells transduced with FcγRIIb-expressing retrovirus restored the healthy phenotype (5McGaha T.L. Sorrentino B. Ravetch J.V. Science. 2005; 307: 590-593Crossref PubMed Scopus (230) Google Scholar). This body of data created the impetus for the study of FcγRIIb regulation and function in humans.Genetic studies revealed a significant association of a single nucleotide polymorphism (SNP) encoding the amino acid substitution I232T in the transmembrane of FcγRIIb with systemic lupus erythematosus (SLE) in several racial groups (6Kyogoku C. Dijstelbloem H.M. Tsuchiya N. Hatta Y. Kato H. Yamaguchi A. Fukazawa T. Jansen M.D. Hashimoto H. van de Winkel J.G. Kallenberg C.G. Tokunaga K. Arthritis Rheum. 2002; 46: 1242-1254Crossref PubMed Scopus (280) Google Scholar, 7Siriboonrit U. Tsuchiya N. Sirikong M. Kyogoku C. Bejrachandra S. Suthipinittharm P. Luangtrakool K. Srinak D. Thongpradit R. Fujiwara K. Chandanayingyong D. Tokunaga K. Tissue Antigens. 2003; 61: 374-383Crossref PubMed Scopus (140) Google Scholar, 8Chu Z.T. Tsuchiya N. Kyogoku C. Ohashi J. Qian Y.P. Xu S.B. Mao C.Z. Chu J.Y. Tokunaga K. Tissue Antigens. 2004; 63: 21-27Crossref PubMed Scopus (124) Google Scholar). The SLE-associated transmembrane polymorphism (FcγRIIb-Thr232) induced loss of inhibitory function of FcγRIIb through exclusion from sphingolipid rafts (9Floto R.A. Clatworthy M.R. Heilbronn K.R. Rosner D.R. MacAry P.A. Rankin A. Lehner P.J. Ouwehand W.H. Allen J.M. Watkins N.A. Smith K.G. Nat. Med. 2005; 11: 1056-1058Crossref PubMed Scopus (257) Google Scholar). In addition, several SNPs present in the FCGR2B promoter correlated with differences in regulatory function and associated with autoimmunity in certain racial groups (10Su K. Wu J. Edberg J.C. Li X. Ferguson P. Cooper G.S. Langefeld C.D. Kimberly R.P. J. Immunol. 2004; 172: 7186-7191Crossref PubMed Scopus (149) Google Scholar, 11Blank M.C. Stefanescu R.N. Masuda E. Marti F. King P.D. Redecha P.B. Wurzburger R.J. Peterson M.G. Tanaka S. Pricop L. Hum. Genet. 2005; 117: 220-227Crossref PubMed Scopus (129) Google Scholar). The -343 G → C substitution identified by us in the proximal FCGR2B promoter correlates with decreased transcriptional activity and associates with SLE in European-Americans (11Blank M.C. Stefanescu R.N. Masuda E. Marti F. King P.D. Redecha P.B. Wurzburger R.J. Peterson M.G. Tanaka S. Pricop L. Hum. Genet. 2005; 117: 220-227Crossref PubMed Scopus (129) Google Scholar).In the present study, we investigated the regulation of the human proximal FCGR2B promoter activity, and the mechanism underlying the defect mediated by the -343 C SNP associated with SLE. The -343 G → C substitution correlated with decreased promoter activity in transiently transfected cells. Electrophoretic mobility shift assays (EMSA) and supershift experiments indicated differences in the binding of activating protein 1 (AP-1) family members to the two FCGR2B promoter sequence variants, with decreased binding of c-Jun to the mutant sequence. The -343 G → C substitution created a new binding site for the nuclear factor Yin-Yang 1 (YY1) that interacted specifically with the mutant sequence competing with c-Jun for DNA binding. The inhibition of YY1 synthesis with specific short hairpin RNA (shRNA) resulted in the up-regulation of the mutant promoter activity. We identify a shift in the binding of YY1 versus c-Jun to the mutant promoter, and this mechanism could be responsible for the repression of FCGR2B promoter activity in SLE patients bearing the -343 C/C genotype. Our study has implications for the modulation of FCGR2B gene expression in SLE and other diseases having altered expression and function of inhibitory FcγRIIb as pathogenic factor.EXPERIMENTAL PROCEDURESCell Cultures—Human U937, Raji, and CL-01 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (Invitrogen) (12Cerutti A. Zan H. Schaffer A. Bergsagel L. Harindranath N. Max E.E. Casali P. J. Immunol. 1998; 160: 2145-2157PubMed Google Scholar). Cells were stimulated with 1 ng/ml phorbol 12-myristate 13-acetate (PMA) plus 1 mm ionomycin (ION) or with 1 mm dibutyryl-cAMP (Bt2cAMP) (Sigma). The peptide inhibitor of c-Jun NH2-terminal kinase (JNKIII) Ac-YGRKKRRQRRR-gaba-ILKQSMTLNLADPVGSLKPHLRAKN-NH2 (Calbiochem) was added to the cell cultures at the concentration of 10 μm.Plasmids—The -537 to +43 fragment of the human FCGR2B promoter was amplified with primers containing recognition sites for KpnI in the forward primer (5′-GCGCGGTACCGCCATCCTGACATACCTCCTT-3′) and XhoI in the reverse primer (5-GCGCCTCGAGCACTCCCTGGAGCGACGTGGC-3′). The products were directionally ligated using a rapid DNA ligation kit (Roche) into the KpnI/XhoI site of the pGL3-Enhancer vectors (Promega, Madison, WI). Constructs containing G at position -343 were used as template to create -343 C constructs by site-directed mutagenesis using the QuikChange kit (Stratagene). The c-Jun trans-Reporting System PathDetect Kit containing the pFC-MEKK plasmid expressing mitogen-activated/extracellular signal-regulated kinase kinase kinase (MEKK) and the pFA2-cJUN plasmid expressing c-Jun was obtained from Stratagene. The pCMV-YY1 vector expressing full-size YY1 was obtained from OriGene.Cell Transfection and Reporter Assays—Transient transfection of CL-01 cells was performed with Lipofectamine (Invitrogen) according to the manufacturer's instructions. CL-01 cells (1.6 × 106 cell/ml) in Opti-MEM I medium (Invitrogen) were incubated with plasmid-Lipofectamine complexes for 5 h, and then supplemented with complete medium and cultured for 48 h. Firefly and Renilla luciferase activities were measured in cell lysates by the dual luciferase assay kit (Promega) using a Wallac MicroBetta Trilux 1450 microplate luminometer. Where indicated, CL-01 cells were co-transfected with shRNA against YY1 1 day prior to the luciferase reporter assay. The TranSilent YY1 shRNA and TranSilent control vector (Panomics) were transfected into CL-01 using AMAXA nucleofector kit. Where indicated, co-transfection with pFC-MEKK, pFA2-cJUN, and pCMV-YY1 was performed.EMSA—Nuclear extracts from CL-01 and U937 cells were prepared as previously described (11Blank M.C. Stefanescu R.N. Masuda E. Marti F. King P.D. Redecha P.B. Wurzburger R.J. Peterson M.G. Tanaka S. Pricop L. Hum. Genet. 2005; 117: 220-227Crossref PubMed Scopus (129) Google Scholar). Oligonucleotides (Operon, Inc.) were annealed and radiolabeled with [α-32P]dCTP. Specific oligonucleotide competitors against AP-1, YY1, and SP1 were purchased from Santa Cruz. Nuclear extracts from resting and activated CL-01 and U937 cells were incubated on ice for 10 min in EMSA binding buffer (10 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.5 mm dithiothreitol, 1 mm EDTA, 0.05% Nonidet P-40, 4% glycerol) with 0.5 μg of poly(dI-dC), followed by addition of labeled probe. In EMSA competition studies, nuclear extracts were incubated with 200-fold excess of unlabeled competitor. Anti-c-Jun (number 06–225, Upstate Biolabs), anti-c-Fos, anti-JunB, anti-YY1, and anti-SP1 antibodies (Santa Cruz Biotechnology) were used in supershift assays.Promoter Pulldown Assay—Biotinylated WT, MUT, and AP-1 oligonucleotides (Operon, Inc.) were annealed and coupled to streptavidin-agarose CL-4B (Sigma). Nuclear extracts from activated CL-01 cells were incubated overnight with agarose-coupled oligonucletides. Agarose suspensions containing bound nuclear factors were applied to minicolumns, washed, and the bound nuclear proteins were eluted with 400 mm NaCl and separated in 10% SDS-PAGE. Immunoblotting was carried out with anti-YY1, -c-Fos, and –phospho-c-JunP73 antibodies.Chromatin Immunoprecipitation (ChIP) Assay—ChIP assay was performed according to a published procedure, with several modifications (13Hayakawa J. Mittal S. Wang Y. Korkmaz K.S. Adamson E. English C. Ohmichi M. McClelland M. Mercola D. Mol. Cell. 2004; 16: 521-535Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Briefly, CL-01 cells, harvested at baseline or after activation, were treated with 1% formaldehyde for 15 min. The cells were lysed and sonicated with SONICATOR 3000 (MISONIX). Supernatants were incubated overnight at 4 °C with anti c-Jun antibodies (sc-45X, Santa Cruz Biotechnology) and anti-XBP (sc-7160X, Santa Cruz Biotechnology), followed by incubation with protein A-agarose in the presence of salmon sperm DNA. The chromatin fraction was eluted, treated with proteinase K, and precipitated. The DNA fragments were amplified by PCR using primers spanning regions -495 to -472 and -278 to -258 of the FCGR2B promoter.RESULTSWe investigated the binding of nuclear factors to the nucleotide sequence (-369 to -330) containing the -343 G → C substitution (dbSNP number rs3219018) identified by us in the human FCGR2B promoter (11Blank M.C. Stefanescu R.N. Masuda E. Marti F. King P.D. Redecha P.B. Wurzburger R.J. Peterson M.G. Tanaka S. Pricop L. Hum. Genet. 2005; 117: 220-227Crossref PubMed Scopus (129) Google Scholar). Two FcγRIIb-expressing cell types, the B lymphoma cell line CL-01 and the myelomonocytic leukemia cell line U937, were used as sources for nuclear extracts. EMSA experiments were carried out with double-stranded oligonucleotide probes labeled with [α-32P]dCTP. The WT probe corresponded to the sequence of the common FCGR2B promoter that contained -343 G, whereas the MUT probe contained -343 C (sequences are shown in Table 1). Analysis of the EMSA pattern revealed that the WT and MUT probes formed distinct DNA-protein complexes (Fig. 1, A and B). The WT probe formed complex b, and the MUT probe formed complexes b and c with nuclear extract from resting CL-01 cells (Fig. 1A, lanes 2 and 3). Nuclear extracts from U937 incubated with the MUT probe allowed the formation of complex c (Fig. 1B, lanes 2 and 3). Complex b was not evident with nuclear extracts from U937 cells, suggesting cell-specific differences in the binding of nuclear factors to the FCGR2B promoter probes.TABLE 1Probes and competitors used for EMSANameSequenceFCGR2B-343 SNP|-369 GGA GAA GAT TGC TGG TGC ACG CTG TCC TGC ATC ACC CTT TCT -330ProbesWT5'-GGA GAA GAT TGC TGG TGC ACG CTG TCC TGC ATC ACC CTT TCT GG-3'MUT5'-GGA GAA GAT TGC TGG TGC ACG CTG TCC TCC ATC ACC CTT TCT GG-3'AP-15'-GGA GAA GAT TGC TGG TGC ACG CTG TCC TGA CTC ACC CTT TCT GG-3'AP-1neg5'-GGA GAA GAT TGC TGG TGC ACG CTG TCC TAA GAC ACC CTT TCT GG-3'CompetitorsCL 5'-A GAA GAT TGC TGG TGC ACG CTG T-3'CWT5'-GG TCC TGC ATC ACC CTT TCT CC-3'CMUT5'-GG TCC TCC ATC ACC CTT TCT CC-3'Specific competitors of transcription factor bindingCAP-15'-CGC TTG ATG ACT CAG CCG GAA-3'CYY15'-CGC TCC CCG GCC ATC TTG GCG GCT GGT-3'CSP15'-ATT CGA TCG GGG CGG GGC GAG-3' Open table in a new tab To test the effect of cell activation on the binding of nuclear factors to the WT and MUT probes, we treated CL-01 cells and U937 cells with PMA:ION. Two slow migrating DNA-protein complexes, a1 and a2, were formed when the WT probe was incubated with nuclear extracts from PMA:ION-treated CL-01 and U937 cells (Fig. 1, A and B, lane 5). Complex a1 was predominant in activated CL-01 cells, whereas complex a2 was predominant in activated U937 cells, indicating differences in the formation of the activation-induced complexes in the two cell types (Fig. 1, A and B, lane 5). The formation of complex a1 and a2 with the MUT probe was weak in activated CL-01 and U937 cells (Fig. 1, A and B, lane 6).Computer-based analysis of the FCGR2B sequence containing the -343 site indicated similarity with the core consensus binding sequence for AP-1 (TGACTCA) (Fig. 1C). We compared the migration pattern of complex a1 and a2 with that of AP-1 by generating a probe (AP-1 probe) containing the canonical AP-1 binding motif (TGACTCA) within the sequence of the FCGR2B promoter (Fig. 1C). The AP-1 probe formed bands with similar mobility with complexes a1 and a2 in activated CL-01 and U937 cells (Fig. 1, A and B, lane 7). These results suggested that AP-1 participated in the formation of activation-induced complex a1 and a2. The expression level of the AP-1 family members, c-Jun, JunB, and c-Fos in nuclear extracts was analyzed in Western blots. The activation of CL-01 and U937 cells with PMA:ION induced the phosphorylation and nuclear translocation of c-Jun, JunB, and c-Fos (Fig. 1D).To verify the interaction of complexes a1 and a2 with the AP-1 binding sequence, we created an additional probe AP-1neg bearing a mutated sequence (TAAGAC) of the AP-1 core consensus binding motif (Table 1). The AP-1neg probe did not form activation-induced complexes a1 and a2 (Fig. 2A, lanes 1 and 4). The nuclear factors that participate in the formation of complex b interacted with the AP-1neg probe, indicating that complex b bound outside the -343 to -340 nucleotide sequence (Fig. 2A, lane 4).FIGURE 2Analysis of nuclear factor binding to the sequence containing the -343 G → C SNP in the FCGR2B promoter by EMSA and ChIP assay. A, representative EMSA (n = 5–8) with nuclear extract from PMA:ION-activated CL-01 cells incubated with 32P-labeled WT (lane 1), MUT (lane 2), AP-1 (line 3), and the AP-1neg probe (lane 4). Unlabeled competitor probe (CL) was used in 200-fold molar excess with 32P-labeled WT (lane 5) and MUT probe (lane 6). Unlabeled competitor probes CWT and CMUT were used in 200-fold molar excess with 32P-labeled WT and MUT probes (lanes 7–10). In competition EMSA (n = 5–8), the WT probe was incubated with 200-fold excess of unlabeled competitors CAP-1 (lane 13) and CSP1 (lane 14), and the MUT probe was incubated with 200-fold excess of unlabeled competitors CAP-1 (lane 15), CYY1 (lane 16), or CSP1 (lane 17). B, EMSA (n = 3–5) with nuclear extract from PMA:ION-activated CL-01 cells with the WT probe was performed in the presence of anti-SP1 (lane 1), anti-c-Fos (lane 2), anti-c-Jun (lane 3), anti-JunB (lane 4), and with the MUT probe in the presence of anti-SP1 (lane 5) and anti-YY1 antibodies (lane 6). Supershifted (ss) complexes are indicated by arrows (left panel). Western blots with nuclear extract from resting and activated CL-01 cells overlaid with anti-YY1 antibodies (right panel). C, ChIP assay with CL-01 cells in the presence of anti-c-Jun antibodies. Chromatin immunoprecipitation from resting and PMA:ION-activated CL-01 cells was performed in the presence of anti-c-Jun and control antibodies (n = 3). The immunoprecipitated chromatin fraction was amplified by PCR with primers specific for the FCGR2B promoter.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To localize the interaction of nuclear proteins with the WT and MUT promoter sequences, we synthesized a competitor probe corresponding to the FCGR2B promoter left flanking sequence excluding the -343 site (CL). In addition, we synthesized competitor probes corresponding to the FCGR2B promoter right flanking sequence containing -343 G (CWT) and -343 C (CMUT) (Table 1). We performed EMSA with 200-fold excess of each unlabeled competitor probe. The addition of competitor CL abolished the formation of complex b with the WT and MUT probes (Fig. 2A, lanes 5 and 6), suggesting that the interaction of complex b with the FCGR2B promoter sequence was upstream and outside of the -343 G/C polymorphic region. Competitor CL added in excess did not abolish the formation of complexes a1 and c (Fig. 2A, lanes 5 and 6). Competitors CWT and CMUT did not influence the formation of complex b (Fig. 2A, lanes 7–10). CWT eliminated complex a1, but did not affect the formation of the MUT-specific complex c (Fig. 2A, lanes 7 and 8). CMUT eliminated the formation of complex a1 with the WT, and the formation of complex c with the MUT probe (Fig. 2A, lanes 9 and 10). Complex a1 was eliminated by 200-fold excess of AP-1 oligonucleotide competitor, confirming the interaction of AP-1 factors with the WT promoter sequence at that site (Fig. 2A, lanes 13 and 15). The excess SP1 competitor did not affect the formation of complex a1 (Fig. 2A, lanes 14 and 17).We sought to identify the nuclear factors participating in the formation of the AP-1 transcriptional complex. Antibodies against SP1, used as control, did not form supershifts and did not eliminate the formation of the activation-induced complexes a1 and a2 (Fig. 2B, lane 1). Antibodies specific for c-Fos, c-Jun, and Jun-B enabled the formation of supershift bands with the WT probe (Fig. 2B, lanes 2–4). The incubation of nuclear extract from CL-01 cells with anti-c-Jun antibodies eliminated complex a1 (Fig. 2B, lane 3), whereas anti-c-Fos antibodies eliminated complex a2 (Fig. 2B, lane 2). These results suggested that in CL-01 cells, complex a1 is formed preferentially by c-Jun, and that c-Fos participates mainly in the formation of complex a2.We investigated the direct interaction of c-Jun with the FCGR2B promoter by ChIP assay. Nuclear extracts were prepared from resting CL-01 cells (0 h) and CL-01 cells activated with PMA:ION for 3, 7, and 30 h (Fig. 2C). Immunoprecipitation with anti-c-Jun antibodies (sc-45X, Santa Cruz Biotechnology) generated a PCR product with specific primers for the FCGR2B promoter (Fig. 2C). We did not detect PCR products by amplification with FCGR2B-specific primers following chromatin immunoprecipitation with control antibody (anti-XBP sc-7160X, Santa Cruz Biotechnology). These results demonstrate the direct interaction of c-Jun with the FCGR2B promoter and suggest that c-Jun could act as transcriptional regulator of the FCGR2B promoter activity at this site.The formation of complex c was specific for the MUT probe in both CL-01 and U937 cells (Fig. 1, A and B, lanes 3 and 6). The nuclear factor YY1, also known as NF-E1 and UCRBP, has the core consensus binding sequence CCATNTT (14Shrivastava A. Calame K. Nucleic Acids Res. 1994; 22: 5151-5155Crossref PubMed Scopus (279) Google Scholar, 15Yao Y.L. Dupont B.R. Ghosh S. Fang Y. Leach R.J. Seto E. Nucleic Acids Res. 1998; 26: 3776-3783Crossref PubMed Scopus (39) Google Scholar). The -343 G → C mutation created a possible YY1 binding site (CCATCAC) to the FCGR2B promoter sequence (Fig. 1C). The MUT-specific complex c was eliminated by an excess of CYY1 competitor in both U937 cells (data not shown) and CL-01 cells (Fig. 2A, lane 16) without affecting other bands. Complex c was not eliminated by addition of the 200-fold excess of CSP1 competitor, used as control (Fig. 2A, lane 17). Supershift formation with YY1 antibodies, but not with SP1 antibodies, established the identity of complex c formed with the MUT probe as being YY1 (Fig. 2B, lanes 5 and 6). Western blot experiments did not show changes in the level of YY1 expression in resting or activated cells (Fig. 2B, right panel). These results suggested that the -343 G to C nucleotide substitution in the FCGR2B promoter created a new binding site for YY1 that may alter the transcriptional activity of the MUT promoter.Complex a1 and a2 had the highest intensity with the AP-1 probe (Figs. 1, A and B, lane 7, and 2A, lane 3). The WT probe formed more intense a1 and a2 complexes compared with the MUT probe (Figs. 1, A and B, lanes 5 and 6, and 2A, lanes 1 and 2). These results pointed out a lower ability of the MUT sequence to bind AP-1 compared with the WT sequence.To confirm that the MUT probe interacted less efficiently with the AP-1 complex, we performed EMSA experiments with nuclear extract from activated CL-01 cells in which 32P-labeled AP-1-specific probes were preincubated with the competitor oligonucleotides CWT and CMUT (Fig. 3A). The concentration of unlabeled CWT or CMUT ranged from 62.5- to 1000-fold excess. Analysis of the intensity of the AP-1 band indicated that 4 times more CMUT was necessary to attain 50% inhibition of AP-1 complex formation compared with using CWT (Fig. 3A,CWT lanes 2–6, and CMUT, lanes 7–11). The graphic representation of the inhibition of optical density of the AP-1 band by increasing concentrations of CWT and CMUT is depicted in Fig. 3B.FIGURE 3Interaction of transcription factors AP-1 and YY1 with the FCGR2B promoter WT and MUT probes. A, EMSA with nuclear extract from PMA:ION-activated CL-01 cells and 32P-labeled AP-1 probe in the absence of competitor (lane 1) and in the presence of increasing amounts (62.5–1000-fold) of unlabeled WT (lanes 2–6) or MUT competitor (lane 7–11). B, the optical density of bands on radiographs was measured using Scion Image Software 4.0. The graph represents the inhibition of the density of the AP-1 complex by increasing concentrations of unlabeled WT and MUT competitor. C, promoter pulldown assay of transcription factors bound to the AP-1, WT, and MUT probes. Biotinylated WT, MUT, and AP-1 probes coupled to streptavidin-agarose beads were incubated with nuclear extract from PMA:ION-activated CL-01 cells. The eluted proteins were analyzed by Western blotting using anti-c-Jun P73, anti-c-Fos, and anti-YY1 antibodies (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We tested the ability of AP-1 and YY1 nuclear factors to bind to the WT, MUT, and AP-1 probes in promoter pulldown assays. Biotinylated probes coupled to streptavidin-agarose beads were incubated with nuclear extract from activated CL-01 cells. Bound nuclear factors were eluted and analyzed by Western blotting with antibodies against YY1, c-Fos, and phosphorylated c-Jun-P73 (Fig. 3C). YY1 was eluted only from the MUT probe, and was undetectable in pull-down assays using AP-1 and WT probes (Fig. 3C, upper panel). c-Fos was eluted from the AP-1 and WT probes, and was not detected in the eluted fraction obtained from the MUT probe (Fig. 3C, middle panel). A higher amount of phosphorylated c-Jun-P73 protein was eluted from the WT probe compared with the MUT probe (Fig. 3C, lower panel). The highest amount of phosphorylated c-Jun-P73 was eluted from the AP-1 probe.We tested whether the -343 G/C polymorphic region participates in transcriptional regulation of the FCGR2B promoter in luciferase reporter assays. WT and MUT promoter constructs (-537 to +43) were inserted into the pGL3-Enhancer vector upstream of the luciferase gene, and transfected into CL-01 cells. The -343 C promoter construct (MUT) had reduced promoter activity (0.6 ± 0.1, p < 0.01, n = 8) compared with the -343 G promoter construct (WT) in CL-01 cells (Fig. 4A).FIGURE 4Transcriptional regulation of WT and MUT FCGR2B promoter constructs measured by luciferase reporter assay. CL-01 cells were transiently transfected with FCGR2B promoter constructs. The firefly luciferase activity measured at 48 h was normalized to Renilla activity. A, luciferase activity measured in the presence and absence of JNKIII. The luciferase activity mediated by the WT promoter construct was significantly down-regulated (*, p < 0.01, n = 9) by treatment with JNKIII. B, the phosphorylation of c-Jun in the presence and absence of JNKIII was analyzed in Western blotting using anti-c-Jun P73 antibodies. C, relative luciferase activity measured in non-activated and PMA:ION-activated CL-01 cells in the presence and absence of JNKIII. Down-regulation of the luciferase activity mediated by the WT (*, p < 0.01, n = 8) and MUT (*, p < 0.01, n = 8) promoter constructs in the presence of JNKIII. D, relative luciferase activity measured in resting and activated CL-01 cells with Bt2cAMP (1 mm) in the presence and absence of JNKIII. Significant reduction of the WT (*, p < 0.01, n = 8) and MUT (*, p < 0.01, n = 8) promoter activities in the presence of JNKIII. E, the expression of c-Jun in CL-01 cells transiently transfected with pFA2-cJUN was analyzed in Western blotting using anti-c-Jun ant
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