Sp3 Mediates Transcriptional Activation of the Leukocyte Integrin Genes CD11C and CD11B and Cooperates with c-Jun to Activate CD11C
1997; Elsevier BV; Volume: 272; Issue: 38 Linguagem: Inglês
10.1074/jbc.272.38.24038
ISSN1083-351X
Autores Tópico(s)T-cell and B-cell Immunology
ResumoThe leukocyte integrin genes CD11cand CD11b are expressed predominately in myelomonocytic cells. In previous experiments, the −70 to −65 and −121 to −103 regions of the CD11c promoter and the −66 to −59 region of the CD11b promoter were shown to be essential for Sp1-mediated activation of these genes. In vivo genomic footprinting had also revealed cell-specific binding of protein, presumably Sp1, to these regions. In this study, electrophoretic mobility shift analysis showed that the Sp1-related factor, Sp3, also binds at or near these same regions. Cotransfection of Sp3 along withCD11c promoter-luciferase constructs into Sp-deficientDrosophila Schneider 2 cells showed that Sp3 could activate the CD11c promoter. Deletion of both the −70 to −65 and −121 to −103 regions of the CD11c promoter resulted in the loss of activation by Sp3. Both sites showed activation by Sp3; however, the −70 to −65 region was more responsive to Sp3 than to Sp1. Similar transfection analysis of the −66 to −59 region of theCD11b promoter showed Sp3-dependent expression. Further, cotransfection analysis in Drosophila cells showed that Sp3, as was previously shown for Sp1, also synergizes with c-Jun to activate CD11c. Antisense experiments that knocked out endogenous Sp3 expression in the myelomocytic cell line, HL60, revealed that Sp3 participates in activation of the CD11c andCD11b promoters in vivo. The leukocyte integrin genes CD11cand CD11b are expressed predominately in myelomonocytic cells. In previous experiments, the −70 to −65 and −121 to −103 regions of the CD11c promoter and the −66 to −59 region of the CD11b promoter were shown to be essential for Sp1-mediated activation of these genes. In vivo genomic footprinting had also revealed cell-specific binding of protein, presumably Sp1, to these regions. In this study, electrophoretic mobility shift analysis showed that the Sp1-related factor, Sp3, also binds at or near these same regions. Cotransfection of Sp3 along withCD11c promoter-luciferase constructs into Sp-deficientDrosophila Schneider 2 cells showed that Sp3 could activate the CD11c promoter. Deletion of both the −70 to −65 and −121 to −103 regions of the CD11c promoter resulted in the loss of activation by Sp3. Both sites showed activation by Sp3; however, the −70 to −65 region was more responsive to Sp3 than to Sp1. Similar transfection analysis of the −66 to −59 region of theCD11b promoter showed Sp3-dependent expression. Further, cotransfection analysis in Drosophila cells showed that Sp3, as was previously shown for Sp1, also synergizes with c-Jun to activate CD11c. Antisense experiments that knocked out endogenous Sp3 expression in the myelomocytic cell line, HL60, revealed that Sp3 participates in activation of the CD11c andCD11b promoters in vivo. Myeloid cells play a key role in a host of leukocyte-dependent functions within the immune system. A number of transcription factors including PU.1 (1Perez C. Coeffier E. Moreau-Gachelin F. Wietzerbin J. Benech P.D. Mol. Cell. Biol. 1994; 14: 5023-5031Crossref PubMed Google Scholar, 2Zhang D. Hetherington C.J. Chen H. Tenen D.G. Mol. Cell. Biol. 1994; 14: 373-381Crossref PubMed Scopus (53) Google Scholar, 3Sturrock A. Franklin K.F. Hoidal J.R. J. Biol. Chem. 1996; 271: 32392-32402Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 4Pahl H.L. Scheibe R.J. Zhang D.-E. Chen H.-M. Galson D.L. Maki R.A. Tenen D.G. J. Biol. Chem. 1993; 268: 5014-5020Abstract Full Text PDF PubMed Google Scholar), Spi-B (5Ray D. Bosselut R. Ghysdael J. Mattei M.-G. Tavitian A. Moreau-Gachelin F. Mol. Cell. Biol. 1989; 12: 4297-4304Crossref Scopus (145) Google Scholar), MZF-1 (6Hromas R. Collins S.J. Hickstein D. Raskind W. Deaven L.L. O'Hara P. Hagen F.S. Kaushansky K. J. Biol. 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Immunol. 1996; 156: 3780-3787PubMed Google Scholar), some of which are lineage-restricted, have been shown to control expression of myeloid genes. The leukocyte integrin genes CD11c (13Springer T.A. Miller L.J. Anderson D.C. J. Immunol. 1986; 136: 230-245PubMed Google Scholar) and CD11b(14Springer T. Galfre G. Secher D.S. Milstein C. Eur. J. Immunol. 1979; 9: 301-306Crossref PubMed Scopus (870) Google Scholar), which encode the alpha subunits for the dimeric receptors p150,95 and Mac-1, respectively, are among the myeloid-specific genes that are transcriptionally activated during myeloid differentiation (15Noti J.D. Reinemann B.C. Mol. Immunol. 1995; 32: 361-369Crossref PubMed Scopus (29) Google Scholar, 16Rosmarin A.G. Weil S.C. Rosner G.L. Griffin J.D. Arnaout M.A. Tenen D.G. Blood. 1989; 73: 131-136Crossref PubMed Google Scholar). Recent reports by my laboratory and others have shown that both theCD11c (17Noti J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar) and CD11b genes (18Chen H.-M. Pahl H.L. Scheibe R.J. Zhang D.-E. Tenen D.G. J. Biol. Chem. 1993; 268: 8230-8239Abstract Full Text PDF PubMed Google Scholar) are controlled by the ubiquitous transcription factor Sp1, which binds these respective promoters in a cell-specific manner. The mechanism for cell-specific binding of Sp1 to either of these promoters is unknown. Since Sp1 binding in other gene systems is affected either directly or indirectly by other transcription factors such as Egr-1 (19Ebert S.N. Wong D.L. J. Biol. Chem. 1995; 270: 17299-17305Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), GATA (20Fischer K.-D. Haese A. Nowock J. J. Biol. Chem. 1993; 268: 23915-23923Abstract Full Text PDF PubMed Google Scholar, 21Merika M. Orkin S.A. Mol. Cell. Biol. 1995; 15: 2437-2447Crossref PubMed Scopus (437) Google Scholar), NF-E1 (22Yu C.-Y. Chen J. Lin L.-I. Tam M. Shen C.-K.J. Mol. Cell. Biol. 1990; 10: 282-294Crossref PubMed Google Scholar), and Pit-1 (23Schaufele F. West B.L. Reudelhuber T.L. J. Biol. Chem. 1990; 265: 17189-17196Abstract Full Text PDF PubMed Google Scholar), it is conceivable that binding of Sp1 toCD11c and CD11b may also be influenced by other factors. Sp1-related genes have recently been cloned based on their sequence homology to Sp1 (24Kingsley C. Winoto A. Mol. Cell. Biol. 1992; 12: 4251-4261Crossref PubMed Scopus (488) Google Scholar, 25Hagen G. Muller S. Beato G. Suske G. Nucleic Acids Res. 1992; 20: 5519-5525Crossref PubMed Scopus (523) Google Scholar). Both Sp2 and Sp3 are widely expressed; however, Sp4 expression is restricted to certain cells of the brain. Sp4 (26Hagen G. Dennig J. Preiß A. Beato M. Suske G. J. Biol. Chem. 1995; 270: 24989-24994Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), like Sp1, activated Sp1-responsive promoters, whereas transfection of an Sp3 expression plasmid into various cell lines repressed the activity of the uteroglobin promoter (27Dennig J. Hagen G. Beato M. Suske G. J. Biol. Chem. 1995; 270: 12737-12744Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), the SV40 enhancer/promoter (28Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (655) Google Scholar), and the HIV-I promoter (29De Luca P. Majello B. Lania L. J. Biol. Chem. 1996; 271: 8533-8536Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Further, Sp1-mediated activation of promoter constructs containing the E1B TATA box fused to either two Sp1 binding sites from element II in the uteroglobin promoter (27Dennig J. Hagen G. Beato M. Suske G. J. Biol. Chem. 1995; 270: 12737-12744Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) or the two Sp1 binding sites from the HTLV-III promoter (28Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (655) Google Scholar) were reversed when Sp3 was cotransfected, and it was proposed that Sp3 acts by competitively binding to the Sp1 binding site. The finding that Sp3 can act as a transcriptional repressor prompted us to examine whether cell-specific expression of CD11c andCD11b was a result of the repressor activity of Sp3 on these promoters in nonmyeloid cells. Surprisingly, Sp3 was found to activate rather than repress the promoters of both CD11c andCD11b. This study shows that intact Sp1 sites are necessary for activation of both promoters by Sp3 and that Sp3 apparently competes for binding with Sp1 for the same or overlapping sites. Further, antisense studies indicate that both endogenous Sp1 and Sp3 proteins are functionally active on these promoters in myeloid cells. Lastly, Sp3 can cooperate with c-Jun for activation of theCD11c promoter. HL60 (promyelocytic leukemia, ATCC CCL 240), U937 (histiocytic lymphoma, ATCC CRL 1593), and Molt4 cells (T cell lymphoblastic leukemia, ATCC CRL 1582) were grown in RPMI 1640 medium containing 10% fetal calf serum (Biofluids, Rockville, MD). HeLa cells (cervical epitheloid carcinoma, ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Biofluids). Schneider's Drosophila 2 cells (Drosophila melanogaster embryo, ATCC CRL 1963) were grown in Schneider's medium containing 10% insect-tested fetal calf serum (Sigma). All media contained 100 units/ml of penicillin and 100 units/ml streptomycin. The −133 to +66 region of the CD11cpromoter was prepared by the polymerase chain reaction (PCR) 1The abbreviations used are: PCR, polymerase chain reaction; wt, wild type; PMA, phorbol 12-myristate 13-acetate; EMSA, electrophoretic mobility shift analysis; pPacO,Drosophila actin promoter expression plasmid; Rb, retinoblastoma protein. with oligonucleotide primers specific to this region and fused to the luciferase gene. The primers used in the PCR were initially synthesized with aHindIII restriction site for cloning of the final PCR product into the HindIII site immediately upstream of the luciferase gene in plasmid pGL2-Basic (Promega, Madison, WI) to create a CD11c-luciferase reporter plasmid (referred to as wild type, wt). The −500 to +50 region of the CD11b promoter was prepared in a similar manner and ligated into the HindIII site immediately upstream of the luciferase gene in plasmid pGL3-Basic (Promega) to create a wt. Primers containing deletions of Sp1/Sp3 binding sites were used in the PCR to construct additionalCD11c-luciferase reporter plasmids and aCD11b-luciferase reporter plasmid containing specific deletions of these sites (refer to figure legends for details). The plasmid pPacSp1, which expresses Sp1 from the Drosophilaactin promoter, and the control plasmid pPacO containing only theDrosophila actin promoter was generously provided by Dr. R. Tjian. To construct plasmids that express Sp2 and Sp3 from the actin promoter (plasmids pPacSp2 and pPacSp3, respectively), cDNA clones for Sp2 and Sp3 (Ref. 24Kingsley C. Winoto A. Mol. Cell. Biol. 1992; 12: 4251-4261Crossref PubMed Scopus (488) Google Scholar; ATCC 95507 and ATCC 95505, respectively) were obtained from ATCC and used as templates in the PCR to generate DNAs containing the complete coding sequences for these factors. The primers contained XhoI restriction sites that were used to clone the Sp2 and Sp3 DNAs into the XhoI site of pPacO. The plasmid pCMV-Hrb (30Udvadia A.J. Rogers K.T. Higgins P.D.R. Murata Y. Martin K.H. Humphrey P.A. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3265-3269Crossref PubMed Scopus (187) Google Scholar), which expresses the human retinoblastoma gene from the cytomegalovirus early promoter, was generously provided by Dr. J. M. Horowitz. To construct plasmid pPacJun, which expresses c-Jun from the Drosophila actin promoter, the plasmid pCMV c-Jun, kindly provided by Dr. R. Tjian, was used as a template in the PCR to generate DNA corresponding to the complete coding sequence for c-jun. XhoI sites were incorporated into the PCR primers to facilitate cloning of c-jun into the XhoI site of pPacO. To construct plasmid pPac-luciferase, which expresses luciferase from the Drosophila actin promoter, pGL3-Basic was used as a template in the PCR to generate DNA corresponding to the complete coding sequence for luciferase that was subsequently ligated into the pPacO plasmid. The integrity of all constructs was verified by DNA sequence analysis. Transfections of human cells were performed by electroporation as described previously (31Noti J.D. Gordon M. Hall R.E. DNA Cell Biol. 1992; 11: 123-138Crossref PubMed Scopus (10) Google Scholar). Approximately 2 × 107 cells were transfected with 15 μg of reporter plasmid and either 0.5 or 5 μg of each expression plasmid when used (see figure legends for specific details). The total volume of the plasmid transfection mix was adjusted to 30 μg with the control plasmid, pPacO. Electroporated cells were transferred to tissue culture dishes containing 15 ml of medium, and phorbol 12-myristate 13-acetate (PMA, 10 ng/ml final concentration) was added 24 h later. The cells were harvested 72 h post-transfection, and luciferase activity was assayed using a kit supplied by Promega. HL60 cells treated with Sp3 antisense oligonucleotides received PMA immediately after electroporation for a total of 24 h. Luciferase light output was measured in a Beckman scintillation counter and normalized against the total protein concentration in the cellular extract. DNA was introduced into Drosophila cells by calcium phosphate-mediated transfection as described previously (17Noti J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar). Approximately 3 × 106 Drosophila cells were transfected with 10 μg of a specific luciferase reporter plasmid and either 0.5 or 5 μg of each expression plasmid (see figure legends for specific details). The total volume of the plasmid transfection mix was adjusted to 30 μg with pPacO. The calcium phosphate-DNA precipitates were left on the cells for 48 h before harvesting and assaying for luciferase activity. Most transfections were performed in triplicate and repeated four to five times to ensure reproducibility and to monitor for transfection efficiency. Statistical analysis was performed using Microsoft Excel (Microsoft Corp., Roselle, IL). Data from individual experiments were pooled and expressed as the mean ± S.D. EMSA was performed as described previously (7Noti J.D. Reinemann B.C. Petrus M.N. Mol. Immunol. 1996; 33: 115-117Crossref PubMed Scopus (21) Google Scholar). Nuclear extracts for use in the EMSA were prepared from all untransfected human cell lines as described previously (7Noti J.D. Reinemann B.C. Petrus M.N. Mol. Immunol. 1996; 33: 115-117Crossref PubMed Scopus (21) Google Scholar). Nuclear extracts were prepared fromDrosophila cells transfected with pPacSp1, pPacSp2, or pPacSp3 as described by Andrews and Faller (32Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2211) Google Scholar) with the following modifications. Forty-eight hours after transfection of 3 × 106 Drosophila cells, the cells were harvested and pelleted for 10 min at 248 × g in a bench-top centrifuge. The cells were resuspended in cold phosphate-buffered saline, transferred to a 2-ml Eppendorf tube, and pelleted for 3 min at 735 × g. The cells were resuspended in 400 μl of cold Buffer A (10 mm HEPES-KOH, pH 7.9, at 4 °C, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride) and incubated on ice for 20 min. The cells were vortexed at top speed for 10 s and pelleted at top speed in an Eppendorf centrifuge for 10 s. The pelleted cells were resuspended in 100 μl of cold low salt buffer (20 mm HEPES-KOH, pH 7.9, at 4 °C, 25% glycerol, 1.5 mm MgCl2, 20 mm KCl, 0.5 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride), and 150 μl of high salt buffer (low salt buffer but with 1.6 m KCl) were slowly added. The nuclei were gently mixed on a rocker shaker for 30 min at 4 °C to extract nuclear proteins then pelleted at top speed in an Eppendorf centrifuge for 2 min at 4 °C, and the supernatant fraction was stored at −80 °C. The following double-stranded oligonucleotide probes were used: 5′ GTACTCTGCCCGCCCCCT 3′, which corresponds to the −79 to −62 region of the CD11c promoter (this probe contains site D (shown in bold; see Fig. 1 and Ref. 17Noti J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar), which shows homology to the Sp1 consensus binding sequence); 5′ CTCCGGTTGGGGGGTGGGGGCGTGTGGGAGGCCGAGC 3′, which corresponds to the −135 to −99 region of theCD11c promoter (this probe contains sites A, B, and C (shown in bold; see Fig. 1 and Ref. 17Noti J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar), which show homology to the Sp1 consensus binding sequence); and 5′ TGTGCTCACTGAGCCTCCGCCCTCTTCCTTTGA 3′, which corresponds to the −80 to −48 region of the CD11b promoter (this probe contains an Sp1 binding site (shown in bold; Ref. 18Chen H.-M. Pahl H.L. Scheibe R.J. Zhang D.-E. Tenen D.G. J. Biol. Chem. 1993; 268: 8230-8239Abstract Full Text PDF PubMed Google Scholar)). The probes were labeled with [γ-32P]ATP to a specific activity of 2–4 × 108 cpm/μg. Approximately 2–4 × 104 cpm of each probe were incubated for 20 min at room temperature with either 10 μg of nuclear extract prepared from untransfected PMA-stimulated HL60 cells or 10 μg of nuclear extract prepared from Drosophila cells transfected with pPacSp1, pPacSp2, or pPacSp3 as described previously (7Noti J.D. Reinemann B.C. Petrus M.N. Mol. Immunol. 1996; 33: 115-117Crossref PubMed Scopus (21) Google Scholar). For supershift analysis, 1 μl of antibody specific for Sp1, Sp3, or Sp4 (Santa Cruz Biotechnology, Santa Cruz, CA) was added for 1 h after incubation of the probe with protein. The reaction products were analyzed by acrylamide gel electrophoresis as described (7Noti J.D. Reinemann B.C. Petrus M.N. Mol. Immunol. 1996; 33: 115-117Crossref PubMed Scopus (21) Google Scholar). Transfected Drosophila cells were resuspended in lysis buffer (50 mm Tris-HCl, pH 7.4, 120 mmNaCl, 2.5 mm EDTA, 1 mm dithiothreitol, 0.5% SDS) and boiled for 5 min, and the lysate from 3 × 105 cells was loaded onto each lane of an SDS-polyacrylamide gel. Western blotting was performed as described (33Williams C.L. Phelps S.H. Porter R.A. Biochem. Pharmacol. 1996; 51: 707-715Crossref PubMed Scopus (41) Google Scholar). Proteins were transferred to a polyvinylidene difluoride membrane and probed with Sp1, Sp3, and c-Jun antibodies (Santa Cruz) at a final concentration of either 1 or 2 μg/ml for 1.5 h at 4 °C. The filter was washed then incubated with horseradish peroxidase-labeled anti-rabbit immunoglobulins diluted 1:4000, and bound antibody was visualized with an ECL Western blotting kit (Amersham Corp.). The following phosphorothioate-modified nucleotides (34Eckstein F. Annu. Rev. Biochem. 1985; 54: 367-402Crossref PubMed Google Scholar) were prepared and HPLC-purified: 5′ AAGTAGCAGCACTTGGAATCTGGACT, an Sp3-specific antisense oligonucleotide that overlaps the Ile translational initiation codon of the Sp3 mRNA (24Kingsley C. Winoto A. Mol. Cell. Biol. 1992; 12: 4251-4261Crossref PubMed Scopus (488) Google Scholar) and 5′ ATTGCATCTATCGGCTTGATTTACCT 3′, a nonsense oligonucleotide. HL60 cells were incubated in complete medium with either nonsense or antisense oligonucleotide at a final concentration of 20 μm for 48 h (fresh oligonucleotides were added after 24 h), and Northern blotting was performed as described previously (15Noti J.D. Reinemann B.C. Mol. Immunol. 1995; 32: 361-369Crossref PubMed Scopus (29) Google Scholar) and analyzed on a Storm phosphoimager to determine the extent of down-regulation of mRNAs for Sp3, CD11c, andCD11b. Western blotting was performed to determine the effect of these oligonucleotides on expression of Sp3 and Sp1 protein. My laboratory has recently shown using in vitro DNase I footprinting that purified Sp1 can protect the −132 to −104 and −72 to −63 regions of the CD11c promoter (Fig.1 A) and that sites within both regions are essential for promoter activation (17Noti J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar). EMSA of these two regions of the CD11c promoter with nuclear extract protein from HL60 cells revealed multiple DNA·protein complexes (Ref. 17Noti J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar and Fig. 2). Of the three complexes formed using a probe that spans the −72 to −63 region (CD11cprobe −79 to −62), complex 1 was supershifted with anti-Sp1 antibody (Fig. 2, compare lanes 2 and 3). Similarly, of the four complexes formed using a probe that spans the −132 to −104 region (CD11c probe −135 to −99), supershift analysis with anti-Sp1 antibody showed tht Sp1 was bound to complex 1 (Fig. 2, compare lanes 7 and 8). The same complex formations and supershifts with anti-Sp1 antibody were seen when nuclear extract protein from nonmyeloid cell lines, Molt4 and HeLa, were analyzed (data not shown). Since Sp1-related proteins have recently been isolated, analysis of these complexes with antibodies to Sp3 and Sp4 was performed to determine whether these factors were present in any of the other complexes. When anti-Sp3 antibody was included in an EMSA using CD11c probe −79 to −62, formation of complex 3 was inhibited (rather than supershifted) when HL60 nuclear extract protein was assayed (Fig. 2, compare lanes 2and 4). Similarly, formation of complex 4 was inhibited when the CD11c probe −135 to −99 was used (Fig. 2, comparelanes 7 and 9). The same complex formations and inhibition of specific complex formations by anti-Sp3 antibody were seen when Molt4 and HeLa nuclear extract protein were analyzed (data not shown). These results also indicate that Sp1 and Sp3 are not co-bound on the same DNA molecule, since anti-Sp3 antibody did not affect the formation of the Sp1-specific complexes and anti-Sp1 antibody did not supershift the Sp3-specific complexes. Previous results showed that an Sp1-specific oligonucleotide could effectively compete with the formation of all complexes, including the Sp3-specific ones (17Noti J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar). Taken together, these results suggest that Sp3 competes with Sp1 for binding to the same or overlapping sites in theCD11c promoter. In contrast, anti-Sp4 antibody had no effect on any complex formation, indicating that Sp4 does not bind to these regions of the CD11c promoter (Fig. 2, lanes 5and 10). Chen et al. (18Chen H.-M. Pahl H.L. Scheibe R.J. Zhang D.-E. Tenen D.G. J. Biol. Chem. 1993; 268: 8230-8239Abstract Full Text PDF PubMed Google Scholar) show that the −66 to −59 region of theCD11b promoter was essential for Sp1-specific promoter activity (Fig. 1 B). EMSA using nuclear extract protein from HL60 cells and a DNA probe spanning this region (CD11b probe −80 to −48) revealed three DNA·protein complexes (Fig. 2). Similar to what was seen in the analysis of the CD11c promoter, anti-Sp1 antibody supershifted complex 1 (Fig. 2, compare lanes 12 and 13), and inclusion of anti-Sp3 antibody in the EMSA inhibited formation of complex 3 (Fig. 2, compare lanes 12 and 14), indicating the presence of bound Sp3 to this or an overlapping region of the CD11b promoter. Complex formations and reactions to anti-Sp1 and anti-Sp3 antibodies were similar when nuclear extract protein from Molt4 and HeLa cells were used (data not shown). Anti-Sp4 antibody had no effect on complex formation and, thus, Sp4 does not appear to interact within this region (Fig. 2, lane 15). To determine whether transfected Sp3 can bind to and activate the CD11c and CD11b promoters,Drosophila cells, which are deficient in Sp-related proteins (26Hagen G. Dennig J. Preiß A. Beato M. Suske G. J. Biol. Chem. 1995; 270: 24989-24994Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 35Coury A.J. Tjian R. Cell. 1988; 55: 887-898Abstract Full Text PDF PubMed Scopus (1079) Google Scholar), were initially cotransfected with 5 μg of pPacSp3 along with either the wt CD11c promoter-luciferase plasmid (containing the −133 to +66 region of CD11c) or the wtCD11b promoter-luciferase plasmid (containing the −500 to +50 region of CD11b) (Fig. 3). For comparison, the role of transfected Sp1 and Sp2 from expression constructs pPacSp1 and pPacSp2, respectively, was similarly analyzed. EMSA revealed that Sp3 in these transfected Drosophila cells was expressed and could bind efficiently to CD11c probes −79 to −62 and −135 to −99 and CD11b probe −80 to −48 (Fig. 3, lanes 5, 12, and 19). Transfected Sp1 in these Drosophila cells could also bind these three probes (Fig. 3, lanes 3, 10, and 17). When pPacSp1 and pPacSp3 were transfected together, both Sp1 and Sp3 were expressed and could bind CD11c probes −79 to −62 and −135 to −99 (Fig.3, lanes 7 and 14) and CD11b probe −80 to −48 (data not shown). As expected, transfected Sp1 bound to the CD11b probe was supershifted with anti-Sp1 antibody (Fig. 3, compare lanes 17 and 18), and binding of transfected Sp3 to the CD11b probe was blocked with anti-Sp3 antibody (Fig. 3, compare lanes 19 and 20). In contrast, transfected Sp2 did not bind either the CD11cprobes (Fig. 3, lanes 4 and 11) or theCD11b probe (data not shown). Sp3-dependent luciferase activity from the CD11cpromoter was shown to increase linearly with up to 1 μg of cotransfected pPacSp3 (Fig.4 A). As was previously shown (17Noti J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar), CD11c promoter activity was also stimulated by Sp1, and this activity was also increased linearly with up to 1 μg of cotransfected pPacSp1 (Fig. 4 A). Maximal CD11cpromoter activity was attainable with either 10 μg of pPacSp3 (23-fold induction) or 5 μg of pPacSp4 (27-fold induction). A similar response of the CD11b promoter to Sp3 and Sp1 was seen (Fig.4 B). CD11b promoter activity increased linearly with up to 1 μg of either pPacSp3 or pPacSp1, and maximal activity was reached with either 5 μg of pPacSp3 (28-fold induction) or 5 μg of pPacSp1 (35-fold induction). Since antibodies to Sp1 and Sp3 affected formation of different complexes in EMSA using either the CD11c or CD11bprobes (Fig. 2), this indicated that Sp1 and Sp3 are not co-bound to the same DNA molecule. Instead, this result indicated that Sp1 and Sp3 compete for the same or overlapping sites when interacting either on the CD11c or CD11b promoter. To confirm this,Drosophila cells were transfected with the appropriate reporter construct and either 1 or 5 μg each of pPacSp1 and pPacSp3. If Sp1 and Sp3 act at distinctly different sites on either promoter, then cotransfection of pPacSp1 and pPacSp3 should result in higher promoter activity than that obtained when either expression construct was transfected separately. Luciferase activity from either the wt CD11c-luciferase plasmid (Fig. 4 A) or the wt CD11b-luciferase plasmid (Fig. 4 B) was not increased above that seen in the presence of only pPacSp1. This indicates that Sp1 and Sp3 compete for the same sites, and as subsequent rounds of transcription initiate from these promoters, either Sp1 or Sp3 is bound at each site, depending on relative binding affinities. Sequence analysis of the −135 to −99 and −79 to −62 regions of theCD11c promoter previously revealed four putative Sp1 binding sites (referred to as sites A, B, C, and D; Fig. 1 A and Ref.17Noti J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar). My laboratory reported that deletion of either the −121 to −103 (containing sites B and C) or the −70 to −65 (containing site D) region led to loss of PMA-inducibility of the CD11c promoter in HL60 cells and that Sp1 interacted at these sites (17Noti J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar). To confirm that Sp3 interacts at the same sites as Sp1 in these promoters,CD11c-luciferase plasmids containing deletions (Δ) of either the −121 to −103 (ΔBC) or −70 to −65 region (ΔD) or both (ΔBCD) were cotransfected into Drosophila cells along with pPacSp1 or pPacSp3 (Fig. 5 A). Consistent with previous results, deletions of either site resulted in significantly lowered Sp1-mediated CD11c promoter activity. In contrast, although deletion of either site led to significantly decreased Sp3-mediated promoter activity, deletion of the −70 to −65 region had a much greater effect on activation of the CD11cpromoter by Sp3. Compared with the induction of the wt CD11cpromoter by Sp3 and Sp1 (18.1- and 23.2-fold, respectively), the induction by Sp3 was disproportionately lower than that by Sp1 when the −70 to −65 region was deleted (2.1-fold versus 10.2-fold, respectively). Further, interaction of Sp3 at each site is noncooperative, with each site responsible for a portion of the intact promoter activity. Analysis of the CD11b promoter indicated t
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