The Zinc Finger Transcription Factor Transforming Growth Factor β-Inducible Early Gene-1 Confers Myeloid-specific Activation of the Leukocyte Integrin CD11d Promoter

Abstract

CD11d encodes the αD subunit for a leukocyte integrin that is expressed on myeloid cells. In this study we show that the –100 to –20 region of the CD11d promoter confers myeloid-specific activation of the CD11d promoter. Transforming growth factor β-inducible early gene-1 (TIEG1) was isolated in a yeast one-hybrid screen using the –100 to –20 region of the CD11d promoter as bait. Purified GST·TIEG1 protein was able to bind within the –61 to –45 region that overlaps a shorter binding site for Sp1. Transient overexpression of TIEG1 activated the CD11d promoter specifically in myeloid cells, whereas, down-regulation of TIEG1 with small interfering TIEG1 RNA also down-regulated expression of CD11d. In vivo, TIEG1 does not physically interact with Sp1. Cotransfection and electrophoretic mobility shift analyses of TIEG1, Sp1, and Sp3 revealed that TIEG1 competes with these Sp proteins for binding to overlapping sites in the CD11d promoter. Although TIEG1 and Sp1 are ubiquitously expressed in myeloid and non-myeloid cells, chromatin immunoprecipitation assays revealed differential occupancy of the CD11d promoter by these factors. In undifferentiated myeloid and non-myeloid cells, occupancy of the CD11d promoter by TIEG1 is similar. Upon differentiation of myeloid cells and subsequent up-regulation of CD11d expression, TIEG1 occupancy increases. In contrast, occupancy by TIEG1 remains low in non-myeloid cells exposed to phorbol ester. We propose that up-regulation of CD11d expression following differentiation of myeloid cells is mediated through increased binding of TIEG1 binding to the CD11d promoter. CD11d encodes the αD subunit for a leukocyte integrin that is expressed on myeloid cells. In this study we show that the –100 to –20 region of the CD11d promoter confers myeloid-specific activation of the CD11d promoter. Transforming growth factor β-inducible early gene-1 (TIEG1) was isolated in a yeast one-hybrid screen using the –100 to –20 region of the CD11d promoter as bait. Purified GST·TIEG1 protein was able to bind within the –61 to –45 region that overlaps a shorter binding site for Sp1. Transient overexpression of TIEG1 activated the CD11d promoter specifically in myeloid cells, whereas, down-regulation of TIEG1 with small interfering TIEG1 RNA also down-regulated expression of CD11d. In vivo, TIEG1 does not physically interact with Sp1. Cotransfection and electrophoretic mobility shift analyses of TIEG1, Sp1, and Sp3 revealed that TIEG1 competes with these Sp proteins for binding to overlapping sites in the CD11d promoter. Although TIEG1 and Sp1 are ubiquitously expressed in myeloid and non-myeloid cells, chromatin immunoprecipitation assays revealed differential occupancy of the CD11d promoter by these factors. In undifferentiated myeloid and non-myeloid cells, occupancy of the CD11d promoter by TIEG1 is similar. Upon differentiation of myeloid cells and subsequent up-regulation of CD11d expression, TIEG1 occupancy increases. In contrast, occupancy by TIEG1 remains low in non-myeloid cells exposed to phorbol ester. We propose that up-regulation of CD11d expression following differentiation of myeloid cells is mediated through increased binding of TIEG1 binding to the CD11d promoter. The β2-integrin family of membrane glycoproteins, also known as the leukocyte integrins, is composed of four distinct α-subunits that non-covalently associate with a common β-subunit and mediate a wide range of adhesion-dependent immunological responses (1Kishimoto T.K. Larson R.S. Corbi A.L. Dustin M.L. Staunton D.E. Springer T.A. Adv. Immunol. 1989; 46: 149-182Crossref PubMed Scopus (445) Google Scholar, 2Larson R.S. Springer T.A. Immunol. Rev. 1990; 114: 181-217Crossref PubMed Scopus (518) Google Scholar). The leukocyte integrins are essential for leukocyte migration, tumor cell lysis, phagocytosis, and the respiratory burst (3Harris E.S. McIntyre T.M. Prescott S.M. Zimmerman G.A. J. Biol. Chem. 2000; 275: 23409-23412Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 4Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6400) Google Scholar, 5Imhof B.A. Dunon D. Adv. Immunol. 1995; 58: 345-416Crossref PubMed Google Scholar, 6Sanchez-Madrid F. Corbi A.L. Semin. Cell Biol. 1992; 3: 199-210Crossref PubMed Scopus (35) Google Scholar) and are involved in a number of pathological conditions in the vascular system, including ischemic reperfusion injury, stroke, and atherosclerosis, and augment tissue damage in autoimmune diseases (7Noti J.D. Current Genomics. 2003; 4: 527-542Crossref Scopus (2) Google Scholar). Surface expression of the α-subunits, which are encoded by the CD11a (8Kurzinger K. Reynolds T. Germain R.N. Davignon D. Martz E. Springer T.A. J. Immunol. 1981; 127: 596-602PubMed Google Scholar), CD11b (9Springer T. Galfre G. Secher D.S. Milstein C. Eur. J. Immunol. 1979; 9: 301-306Crossref PubMed Scopus (870) Google Scholar), CD11c (10Springer T.A. Miller L.J. Anderson D.C. J. Immunol. 1986; 136: 240-245PubMed Google Scholar), and CD11d (11Van der Vieren M. Le Trong H. Wood C.L. Moore P.F. St John T. Staunton D.E. Gallatin W.M. Immunity. 1995; 3: 683-690Abstract Full Text PDF PubMed Scopus (231) Google Scholar) genes, and the β-subunit, encoded by the CD18 gene (12Law S.K. Gagnon J. Hildreth J.E. Wells C.E. Willis A.C. Wong A.J. EMBO J. 1987; 6: 915-919Crossref PubMed Scopus (119) Google Scholar), varies with the particular cell type. All leukocytes express CD18 (12Law S.K. Gagnon J. Hildreth J.E. Wells C.E. Willis A.C. Wong A.J. EMBO J. 1987; 6: 915-919Crossref PubMed Scopus (119) Google Scholar) and CD11a (8Kurzinger K. Reynolds T. Germain R.N. Davignon D. Martz E. Springer T.A. J. Immunol. 1981; 127: 596-602PubMed Google Scholar), whereas CD11b (9Springer T. Galfre G. Secher D.S. Milstein C. Eur. J. Immunol. 1979; 9: 301-306Crossref PubMed Scopus (870) Google Scholar), CD11c (10Springer T.A. Miller L.J. Anderson D.C. J. Immunol. 1986; 136: 240-245PubMed Google Scholar), and CD11d (11Van der Vieren M. Le Trong H. Wood C.L. Moore P.F. St John T. Staunton D.E. Gallatin W.M. Immunity. 1995; 3: 683-690Abstract Full Text PDF PubMed Scopus (231) Google Scholar) are expressed predominately on myeloid cells. The latest leukocyte integrin gene to be identified is CD11d, and as such, it is also the least understood with regards to its function and mode of regulation. CD11d is prominently expressed on macrophage foam cells and splenic red pulp macrophages, which suggests a role in the atherosclerotic process such as lipid scavenging and phagocytosis of pathogens and senescent erythrocytes (11Van der Vieren M. Le Trong H. Wood C.L. Moore P.F. St John T. Staunton D.E. Gallatin W.M. Immunity. 1995; 3: 683-690Abstract Full Text PDF PubMed Scopus (231) Google Scholar). Synovial macrophages and lung alveolar macrophages also highly express CD11d suggesting that this molecule might perpetuate the inflammatory reactions associated with rheumatoid arthritis and lung injury (13el-Gabalawy H. Canvin J. Ma G.M. Van der Vieren M. Hoffman P. Gallatin M. Wilkins J. Arthritis Rheum. 1996; 39: 1913-1921Crossref PubMed Scopus (32) Google Scholar, 14Shanley T.P. Warner R.L. Crouch L.D. Dietsch G.N. Clark D.L. O'Brien M.M. Gallatin W.M. Ward P.A. J. Immunol. 1998; 160: 1014-1020PubMed Google Scholar). To understand the molecular mechanisms responsible for expression of CD11d, we previously isolated a genomic clone containing the 5′-untranslated portion of CD11d and showed that cell-specific expression of this gene is mediated through both Sp1 and Sp3 (15Noti J.D. Johnson A.K. Dillon J.D. J. Biol. Chem. 2000; 275: 8959-8969Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Regulation by members of the Sp transcription factor family appears to be a common theme for expression of the leukocyte integrin genes as functional Sp1 sites are also found in the CD11a-c (16Lopez-Rodriguez C. Chen H.M. Tenen D.G. Corbi A.L. Eur. J. Immunol. 1995; 25: 3496-3503Crossref PubMed Scopus (37) Google Scholar, 17Chen 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, 18Noti J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar) and CD18 (19Rosmarin A.G. Luo M. Caprio D.G. Shang J. Simkevich C.P. J. Biol. Chem. 1998; 273: 13097-13103Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) genes. Because a number of transcription factors, including PU.1, GABPα, GABPβ, c-Jun, c-Fos, Ets, and c/EBP, were shown to functionally interact at sites adjacent to the Sp1 binding sites on the CD11a –c and CD18 genes (7Noti J.D. Current Genomics. 2003; 4: 527-542Crossref Scopus (2) Google Scholar), we reasoned that the CD11d promoter may also be regulated by transcription factors binding near the Sp1/Sp3 site. In this study we show that transforming growth factor-β-inducible early gene-1 (TIEG1), 1The abbreviations used are: TIEG1, transforming growth factor-β-inducible early gene-1; AD, activation domain; BD, binding domain; 3-AT, 3-amino-1,2,4-triazole; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift analysis; RT, reverse transcriptase; ChIP, chromatin immunoprecipitation; PMA, phorbol 12-myristate 13-acetate; Rb, human retinoblastoma protein; HRP, horseradish peroxidase; GAPDH, glyceraldehyde-phosphate dehydrogenase; HA, hemagglutinin; PMSF, phenylmethylsulfonyl fluoride; CMV, cytomegalovirus; siRNA, small interference RNA; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; TGF, transforming growth factor. a member of the Kruppel-like factor family (20Subramaniam M. Harris S.A. Oursler M.J. Rasmussen K. Riggs B.L. Spelsberg T.C. Nucleic Acids Res. 1995; 23: 4907-4912Crossref PubMed Scopus (226) Google Scholar, 21Blok L.J. Grossmann M.E. Perry J.E. Tindall D.J. Mol. Endocrinol. 1995; 9: 1610-1620Crossref PubMed Google Scholar), binds to a site overlapping the Sp1 site and activates the CD11d gene specifically in myeloid cells. In contrast, TIEG1 down-regulates CD11d expression in non-myeloid cells. We also provide evidence that up-regulation of CD11d by prolonged exposure to a high concentration of phorbol ester specifically in myeloid cells is accompanied by increased binding of TIEG1 to the CD11d promoter in vivo. Cell Culture—THP1 (acute monocytic leukemia, ATCC TIB-202), HL60 (promyelocytic leukemia, ATCC CCL-240), IM9 (B-cell multiple myeloma, ATCC CCL-159), Jurkat (T-cell acute leukemia, ATCC TIB-152), and K562 (highly undifferentiated progenitors of erythrocytes, granulocytes, and monocytes, ATT CCL-243) cells were grown in RPMI 1640 medium containing 10% fetal calf serum (IM9 cells were grown in 20% fetal calf serum). 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 medium contained 100 units/ml each of streptomycin and penicillin. For certain experimental procedures, cells were stimulated with 10 mm phorbol 12-myristate 13-acetate (PMA) for 24 h, or 100 nm PMA for 48 h. Yeast One-hybrid Analysis—Following the protocol outlined in the MATCHMAKER One-Hybrid System (Clontech, Palo Alto, CA), four copies of the –100 to –20 region of the CD11d promoter were ligated into the SmaI site of yeast reporter pHisi-1 and into the XhoI site of yeast reporter pLacZi to create reporters pHisi-1-CD11d(–100/–20) and pLacZi-CD11d(–100/–20). A yeast dual reporter strain was prepared by first integrating pHis-1-CD11d(–100/–20) followed by pLacZi-CD11d(–100/–20) into the genome of yeast strain YM4271. The dual reporter strain was selected and maintained on SD minimal medium lacking histidine and uracil. A human spleen cDNA library (Clontech, cat. #HL4054AH), prepared in the plasmid pACT2 to generate fusions of spleen cDNA with the GAL4 Activation Domain (AD), was transformed into the yeast dual reporter strain. Yeast transformants were selected on SD/–Leu/–His/–Ura medium containing 30 or 45 mm 3-amino-1,2,4-triazole (3-AT). The spleen cDNA/Gal4 fusion plasmids were recovered from the transformants and re-transformed into the dual reporter strain and plated onto SD/–Leu/–His/–Ura/45 mm 3-AT medium containing 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). All cDNA/Gal4AD fusion plasmids that retested positive also expressed robust β-galactosidase activity from the integrated LacZ reporter. The recovered plasmids were sequenced and analyzed by a BLAST search of GenBank™. Full-length TIEG1 cDNA corresponding to the partial cDNAs in the recovered plasmids were obtained as detailed below. Plasmids—Four copies of the –100 to –20 region of the CD11d promoter were placed upstream of the minimal SV40 promoter in pGL3-Promoter (Promega, Madison, WI). The –173 to +74 region of the CD11d promoter was fused to the luciferase gene in pGL3-Basic (Promega) to create reporter plasmid CD11d(–173/+74)-luc (15Noti J.D. Johnson A.K. Dillon J.D. J. Biol. Chem. 2000; 275: 8959-8969Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 22Noti J.D. J. Biol. Chem. 1997; 272: 24038-24045Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). 5-bp mutations (changing Gs to As and Cs to Ts) were introduced into CD11d(–173/+74)-luc using the QuikChange site-directed mutagenesis system (Stratagene, La Jolla, CA). Expression plasmids pPacSp1 and pPacSp3, which express Sp1 and Sp3 from the Drosophila actin promoter, and the empty cassette plasmid pPacO, were generously provided by Dr. R. Tjian. Expression plasmids pCMV4-Sp1/flu and pCMV4-Sp3/flu, which express HA-tagged Sp1 and Sp3 from the cytomegalovirus early promoter, were generously provided by Dr. J. M. Horowitz (23Udvadia A.J. Templeton D.J. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3953-3957Crossref PubMed Scopus (198) Google Scholar, 24Udvadia A.J. Rogers K.T. Higgins P.D. 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). Full-length TIEG1 cDNA (accession number NM005655) was amplified from human spleen Quick-Clone cDNA (Clontech) by PCR. XhoI sites were incorporated at the 5′-ends of the amplification primers to facilitate cloning of full-length TIEG1 into the mammalian expression plasmid pCMV-HA (Clontech) and the GST fusion plasmid pET42a (Novagen, Madison, WI). Transfection and Reporter Assays—Human cells were transfected by electroporation as previously described (15Noti J.D. Johnson A.K. Dillon J.D. J. Biol. Chem. 2000; 275: 8959-8969Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and analyzed by the Dual-Luciferase Reporter Assay System (Promega). Approximately 1 × 107 cells were cotransfected with 10 μg of each firefly luciferase reporter plasmid, 5 μg of each expression plasmid when used (see figure legends for specific details), and 2 μg of Renilla luciferase plasmid pRL-SV40 (Promega). The total concentration of transfected DNA was adjusted to 20 μg with pCMV-HA. Luciferase activity in cells 24 h post-transfection was measured in a LB96V-2 Wallac Berthold plate luminometer and normalized against Renilla luciferase activity or against the total protein concentration in the cellular extract. The assays were performed in triplicate and repeated three to four times to ensure reproducibility. Statistical analysis was performed by using Microsoft Excel (Microsoft, Roselle, IL), and pooled data from individual experiments were expressed as means ± S.D. GST·TIEG1 Preparation—Full-length cDNA for TIEG1 was cloned in-frame with the GST portion of the bacterial expression plasmid pET42a. The pET42a-GST·TIEG1 plasmid was transformed into the Escherichia coli strain BL21(DE3), and a single isolated colony was grown in a 500-ml culture and induced with 0.4 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h as per the manufacturer's instructions (Novagen). The induced cells were harvested, resuspended in a wash/bind buffer containing Triton X-100 and lysozyme, and lysed by sonication following the protocol outlined in the Bugbuster GST-Bind purification kit (Novagen). The lysate was poured through a column loaded with GST-Bind resin, and bound GST·TIEG1 protein was subsequently eluted off the resin with reduced glutathione. Western blot analyses of the GST·TIEG1 protein with anti-GST monoclonal antibodies (Novagen) and anti-TIEG1 antibodies generously supplied by Dr. T. C. Spelsberg (20Subramaniam M. Harris S.A. Oursler M.J. Rasmussen K. Riggs B.L. Spelsberg T.C. Nucleic Acids Res. 1995; 23: 4907-4912Crossref PubMed Scopus (226) Google Scholar, 25Johnsen S.A. Subramaniam M. Janknecht R. Spelsberg T.C. Oncogene. 2002; 21: 5783-5790Crossref PubMed Scopus (124) Google Scholar) were performed as previously described (22Noti J.D. J. Biol. Chem. 1997; 272: 24038-24045Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). GST Pull-Down Assays—Varying amounts (50 ng to 2 μg) of purified GST·TIEG1 or unfused GST protein were mixed with 200 μl of GST-Bind resin (Novagen) and incubated for 30 min at 25 °C. The resin was washed with phosphate-buffered saline (PBS) and mixed with 50 ng of purified recombinant Sp1 protein (Promega) in 20 mm Tris-HCl, pH 8.0, 1 mm MgCl2, 2 mm ZnCl2, 0.1% dithiothreitol, 10% glycerol, 1 mm PMSF, 1 μg/ml aprotinin, and 1 μg/ml pepstatin A and incubated at 4 °C for 60 min. The resin was collected by centrifugation, washed with PBS, resuspended in SDS sample buffer, and analyzed by Western blot (22Noti J.D. J. Biol. Chem. 1997; 272: 24038-24045Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) with anti-Sp1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) in the primary reaction and horseradish peroxidase (HRP)-labeled anti-goat immunoglobulins (Amersham Biosciences) in the secondary incubation reaction. In Vivo Coimmunoprecipitation—Approximately 1 × 107 THP1 or HL60 cells were transfected with 5 μg of pCMV4-Sp1/flu, pCMV-HA-TIEG1, or CMV-Rb (human retinoblastoma protein) (24Udvadia A.J. Rogers K.T. Higgins P.D. 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) or combinations of each (see figure legend for details). The transfected cells were harvested 24 h later, washed in cold PBS, and resuspended in 1 ml of lysis buffer (1% Nonidet P-40, 150 mm NaCl, 1 mm PMSF, 1 μg/ml aprotinin, 1 μg/ml pepstatin A, and 50 mm Tris-HCl, pH 8.0). The cell lysate was incubated on ice for 20 min and clarified by centrifugation, anti-Sp1 antibodies were added to achieve a 5 μg/ml final concentration, and incubation was continued for 1 h at 4 °C. To precipitate the immune complexes, 50 μl of Protein G-Sepharose 4 Fast Flow (Amersham Biosciences) was added to the lysate with incubation for 1 h at 4 °C. The immune complexes were pelleted by centrifugation, washed extensively with lysis buffer and a final wash with 50 mm Tris-HCl, pH 8.0, and resuspended in SDS sample buffer for analysis by Western blot. To detect proteins in the immune complexes, rabbit anti-HA polyclonal antibodies (to detect HA-TIEG1 and HA-Sp1) and rabbit anti-Rb (to detect CMV-Rb) were added in the primary reaction followed by the addition of goat anti-rabbit HRP in the secondary reaction. Electrophoretic Mobility Shift Analysis—EMSA was performed as previously described (26Noti J.D. Reinemann C. Petrus M.N. Mol. Immunol. 1996; 33: 115-127Crossref PubMed Scopus (21) Google Scholar) using nuclear extracts that were either purchased (Active Motif, Carlsbad, CA) or prepared as previously described (26Noti J.D. Reinemann C. Petrus M.N. Mol. Immunol. 1996; 33: 115-127Crossref PubMed Scopus (21) Google Scholar). To initially localize the binding site of TIEG1, the following double-stranded oligonucleotide probes were used: 5′-TGTTCCATAATTAACCACGCCCCTCCTACCCACTGTGCCCCTCTTCCTGC-3′, which corresponds to the –80 to –31 region of the CD11d promoter; 5′-TTCCATAATTAACCAATAAACTCCTACCCACTGTG-3′, which corresponds to the –78 to –44 region of the CD11d promoter the 5-bp at the –61 site shown in which corresponds to the to region of the CD11d promoter the 5-bp at the site shown in A of TIEG1 binding was performed using a of double-stranded oligonucleotide probes corresponding to the to region of the CD11d promoter. The probes contained a in and are as which corresponds to the to region –61 and sites that were in the probes are for and The probes were with to a specific activity of × × was incubated for 30 min on ice with either 5 μg of nuclear 100 ng of purified recombinant Sp1 or ng of purified GST·TIEG1 as previously described J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar). to Sp1 or TIEG1 were in incubation for and reaction were analyzed by as described previously J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar). assays were performed using the kit as described by the Biotechnology, × were in for 10 min at °C. To the was added to mm final The cells were washed with cold PBS, lysed with SDS lysis buffer (1% 10 mm 50 mm Tris-HCl, pH containing mm PMSF, 1 μg/ml aprotinin, and 1 μg/ml pepstatin and the lysate was to the genomic DNA to in The lysate was in buffer Triton 150 mm NaCl, 2 mm Tris-HCl, pH containing the of the lysate was incubated for 4 h at °C to reverse the and used as To the the of the lysate was incubated with a for 30 min at 4 followed by of the protein by The was incubated with anti-TIEG1 or anti-Sp1 or antibodies at 4 °C. The and complexes were from the following incubation with for 1 h at 4 °C. The complexes were extensively washed with from the and resuspended in μl of buffer (1% for min at to the and complexes from the protein of the protein by centrifugation, 10 μl of 5 was added to the which was for 4 h at °C to reverse the To the was added (50 μg/ml final for 1 h at 45 followed by and of the DNA in 1 mm 10 mm Tris-HCl, pH The DNA was amplified by the using primers corresponding to of the CD11d promoter. The primers used to the to –20 region to and to The primers used to the to region were as to and to The were as 1 min at 30 at 60 1 min at for 30 of genomic DNA from each cell was amplified by the with of to the binding of each transcription factor. The were to with and analyzed on a (Amersham For the of each DNA was with the of the DNA in the figure To ensure that the were performed within the range of were analyzed at and RNA of TIEG1 was prepared using the kit as described by the TIEG1 cDNA corresponding to of the TIEG1 (accession number NM005655) was prepared by with the following primers containing the promoter at the region of TIEG1 in and region of TIEG1 in The were as 30 at 1 min at for followed by 30 at 1 min at for 30 The TIEG1 cDNA was used to RNA in a transcription reaction. TIEG1 RNA was incubated with and purified on and with to generate in The of the was on a HL60 and THP1 cells × 107 cells in 200 μl of mm mm were at with μg of TIEG1 The cells were to containing ml of RPMI fetal calf PMA was added mm final 1 h, and the cells were incubated for 48 h and harvested, and total RNA was isolated and analyzed by the with primers specific to TIEG1, CD11d, Sp1, and glyceraldehyde-phosphate The were to with and analyzed on a (Amersham Expression of TIEG1 and CD11d was to in the figure To ensure that the were performed within the range of were analyzed at and RNA from and transfected cells was isolated using the kit CA). RNA was using the kit and amplified using the 2 kit of the CD11d previously shown that Sp1 binds in within the to region of the CD11d promoter and that both Sp1 and Sp3 expression of CD11d in myeloid not non-myeloid cells (15Noti J.D. Johnson A.K. Dillon J.D. J. Biol. Chem. 2000; 275: 8959-8969Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). in genomic analysis revealed that this region bind or and it was that Sp proteins were bound (15Noti J.D. Johnson A.K. Dillon J.D. J. Biol. Chem. 2000; 275: 8959-8969Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Because Sp proteins are we reasoned that myeloid-specific expression of CD11d might be by transcription factors binding adjacent to the Sp binding site. To the of transcription factor binding site-directed mutagenesis of the region the Sp1 binding site was A of 5-bp mutations was introduced region contained on a reporter CD11d(–173/+74)-luc the –173 to +74 region of the CD11d promoter fused to the luciferase and analyzed in assays of mutations at sites and reduced CD11d expression in the myeloid cell THP1 and not in the cell IM9 or the cell Jurkat A at site reduced expression in and Jurkat cells on expression in IM9 cells. A at site reduced CD11d expression in cell evidence that myeloid-specific and sites are near the Sp1 binding site was by analysis of a luciferase reporter containing four copies of the –100 to –20 region of the CD11d promoter fused upstream of the minimal SV40 promoter in The –100 to –20 region increased SV40 promoter activity to in HL60 and THP1 cells, whereas its activity in Jurkat and IM9 cells was increased of TIEG1 by Yeast One-hybrid the transcription factors that interact at the essential sites within the –100 to –20 a yeast one-hybrid screen was copies of the –100 to –20 region were ligated upstream of the and genes in and