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• W2153289176 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 (this probe contains the 5-bp mutation at the –61 site shown in boldface); 5′-AACCACGCCCCTCCTCAAACCTGTGCCCCTCTTCC-3′, which corresponds to the –68 to –34 region of the CD11d promoter (this probe contains the 5-bp mutation at the –51 site shown in boldface). A more precise localization of TIEG1 binding was performed using a series of seven double-stranded oligonucleotide probes corresponding to the –66 to –40 region of the CD11d promoter. The seven probes contained a different 3-bp mutation (shown in boldface) and are as follows: WT, 5′-CCACGCCCCTCCTACCCACTGTGCCC-3′, which corresponds to the wild-type –66 to –40 region (the –61 and –51 sites that were mutated in the above probes are underlined for reference); mut1, 5′-AACCGCCCCTCCTACCCACTGTGCCC-3′; mut2, 5′-CCACGAAACTCCTACCCACTGTGCCCC-3′; mut3, 5′-CCACGCCCCTAAGACCCACTGTGCCCC-3′; mut4, 5′-CCACGCCCCTCCTAAAAACTGTGCCCC-3′; mut5, 5′-CCACGCCCCTCCTACCCACGTGGCCCC-3′; and mut6, 5′-CCACGCCCCTCCTACCCACTGTGAAAC-3′. The probes were end-labeled with [γ-32P]ATP to a specific activity of 2–4 × 108 cpm/μg. Each probe (1 × 104 cpm) was incubated for 30 min on ice with either 5 μg of nuclear extract, 100 ng of purified recombinant Sp1 protein, or 100–200 ng of purified GST·TIEG1 as previously described (18Noti J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar). Antibodies to Sp1 or TIEG1 were included in some incubation mixes for supershift analysis, and all reaction products were analyzed by polyacrylamide gel electrophoresis as described previously (18Noti J.D. Reinemann B.C. Petrus M.N. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar). Chromatin Immunoprecipitation—ChIP assays were performed using the ChIP assay kit essentially as described by the manufacturer (Upstate Biotechnology, Inc., Lake Placid, NY). Cells (1 × 108) were fixed in 1% formaldehyde for 10 min at 37 °C. To quench the cross-linking reaction, glycine was added to 125 mm final concentration. The cells were washed with cold PBS, lysed with SDS lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris-HCl, pH 8.1) containing protease inhibitors (1 mm PMSF, 1 μg/ml aprotinin, and 1 μg/ml pepstatin A), and the lysate was sonicated to shear the genomic DNA to ∼500 bp in length. The lysate was then diluted 10-fold in ChIP dilution buffer (0.01% SDS, 1% Triton X-100, 150 mm NaCl, 2 mm Tris-HCl, pH 8.1) containing the above protease inhibitor mix. An aliquot of the diluted lysate was incubated for 4 h at 65 °C to reverse the cross-links and used as input DNA. To reduce the nonspecific background, the remainder of the lysate was incubated with a salmon sperm DNA/protein A-agarose slurry for 30 min at 4 °C, followed by removal of the protein A-agarose by centrifugation. The supernatant fraction was incubated with anti-TIEG1 or anti-Sp1 antibodies, or no antibodies overnight at 4 °C. The anti-TIEG1·DNA·TIEG1 and anti-Sp1·DNA·Sp1 complexes were precipitated from the supernatant fractions following incubation with salmon sperm DNA·protein A-agarose for 1 h at 4 °C. The precipitated complexes were extensively washed with buffers from the manufacturer and resuspended in 250 μl of elution buffer (1% SDS, 0.1 m NaHCO3) for 15 min at room temperature to elute the DNA·TIEG1 and DNA·Sp1 complexes from the protein A-agarose. After removal of the protein A-agarose by centrifugation, 10 μl of 5 m NaCl was added to the supernatant, which was then heated for 4 h at 65 °C to reverse the cross-links. To purify the immunoprecipitated DNA, proteinase K was added (50 μg/ml final concentration) for 1 h at 45 °C, followed by phenol-chloroform extraction, ethanol precipitation, and resuspension of the DNA in 1 mm EDTA, 10 mm Tris-HCl, pH 8.0. The immunoprecipitated DNA was then amplified by the PCR using primers corresponding to regions of the CD11d promoter. The primers used to amplify the –200 to –20 region were: 5′-GCTCCTGAGGCCTGGGGGAGGGTGG-3′ (forward primer,–200 to –176) and 5′-GCCTCCACACAGCAGGAAGAGGGGC-3′ (reverse primer,–20 to –44). The primers used to amplify the –712 to –520 region were as follows: 5′-AACCACTCTTCTGATTTCTATCTTCG-3′ (forward primer,–712 to –688) and 5′-AGGCAACATAGCCAGACCCTGTC-3′ (–520 to –542). The PCR cycling parameters were as follows: 1 min at 95 °C, 30 s at 60 °C, 1 min at 72 °C, for 30 cycles. An aliquot of input genomic DNA from each cell line was amplified by the PCR along with aliquots of immunoprecipitated DNAs to assess the relative binding of each transcription factor. The PCR products were subjected to gel electrophoresis, stained with Vistra Green, and analyzed on a PhosphorImager (Amersham Biosciences). For analysis, the level of each immunoprecipitated DNA was compared with the level of the appropriate input DNA (detailed in the figure legends). To ensure that the PCRs were performed within the linear range of amplification, samples were analyzed at 21, 24, 27, 30, 36, and 39 cycles. Small Interfering RNA Silencing of TIEG1 Expression—TIEG1 siRNA was prepared using the Silencer siRNA mixture kit (RNaseIII) essentially as described by the manufacturer (Ambion Inc., Austin, TX). TIEG1 cDNA corresponding to nucleotides 151–514 of the TIEG1 sequence (accession number NM005655) was prepared by PCR with the following primers containing the T7 promoter sequence at the 5′-ends: 5′-TAATACGACTCACTATAGGGCTGCGGAGGAAAGAATGG-3′ (forward primer, 151–172 region of TIEG1 in boldface) and 5′-TAATACGACTCACTATAGGGCAATGTGAGGTTTGGCAG-3′ (reverse primer, 497–514 region of TIEG1 in boldface). The PCR cycling parameters were as follows: 30 s at 95 °C, 1 min at 59 °C, for 6 cycles followed by 30 s at 95 °C, 1 min at 67 °C, for 30 cycles. The TIEG1 cDNA was used to prepare double strand RNA in a T7 transcription reaction. TIEG1 double strand RNA was incubated with DNase I and RNase I, purified on Ambion's Transcription Reaction Filter Cartridge, and then digested with RNase III to generate siRNA 12–15 bp in length. The size of the siRNA was confirmed on a polyacrylamide gel. HL60 and THP1 cells (1 × 107 cells in 200 μl of RPMI/0.1 mm dithiothreitol/10 mm dextrose) were electroporated at 300 V, 125 microfarads with 0.1–1.0 μg of TIEG1 siRNA. The cells were transferred to Petri dishes containing 15 ml of RPMI 1640/10% fetal calf serum. PMA was added (100 mm final concentration) after 1 h, and the cells were incubated for 48 h and harvested, and total RNA was isolated and analyzed by the PCR with primers specific to TIEG1, CD11c, CD11d, Sp1, and glyceraldehyde-phosphate dehydrogenase (GAPDH). The PCR products were subjected to gel electrophoresis, stained with Vistra Green, and analyzed on a PhosphorImager (Amersham Biosciences). Expression of TIEG1 and CD11d was determined relative to GAPDH (detailed in the figure legends). To ensure that the PCRs were performed within the linear range of amplification, samples were analyzed at 21, 24, 27, 30, 36, and 39 cycles. RT-PCR—Total RNA from untransfected and transfected cells was isolated using the RNeasy Mini kit (Qiagen Inc., Valencia, CA). Total RNA was reverse-transcribed using the Omniscript RT kit (Qiagen Inc.) and amplified using the Advantage 2 PCR kit (Clontech). The–100 to–20 Region of the CD11d Promoter Controls Myeloid Specificity—We had previously shown that Sp1 binds in vitro within the –63 to –40 region of the CD11d promoter and that both Sp1 and Sp3 activate expression of CD11d in myeloid but 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). Furthermore, in vivo genomic footprint analysis revealed that this region can bind one or more proteins, and it was presumed 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 ubiquitous, we reasoned that myeloid-specific expression of CD11d might be influenced by other transcription factors binding adjacent to the Sp binding site. To define the location of other transcription factor binding sites, site-directed mutagenesis of the region spanning the Sp1 binding site was performed. A series of 5-bp mutations was introduced throughout an extended region contained on a reporter construct CD11d(–173/+74)-luc (which contains the –173 to +74 region of the CD11d promoter fused to the luciferase gene) and analyzed in transient transfection assays (Fig. 1A). Three of five mutations (located at sites –69,–61, and –34) significantly reduced CD11d expression in the myeloid cell lines THP1 and HL60, but not in the B cell line IM9 or the T cell line Jurkat (Fig. 1B). A fourth mutation (located at site –91) significantly reduced expression in HL60, THP1, and Jurkat cells but had no effect on expression in IM9 cells. A fifth mutation (located at site –51) reduced CD11d expression in all cell lines. Further evidence that myeloid-specific and nonspecific sites are clustered near the Sp1 binding site was demonstrated by transient transfection analysis of a luciferase reporter construct containing four copies of the –100 to –20 region of the CD11d promoter fused upstream of the enhancerless, minimal SV40 promoter in pGL3-Promoter. The –100 to –20 region increased SV40 promoter activity ∼6- to 7-fold in HL60 and THP1 cells, whereas its activity in Jurkat and IM9 cells was increased ∼2-fold (Fig. 2). Identification of TIEG1 by Yeast One-hybrid Analysis—To identify the transcription factors that interact at the essential sites within the –100 to –20 region, a yeast one-hybrid screen was performed. Four copies of the –100 to –20 region were ligated upstream of the his3 and lacZ genes in pHISi-1 and pLacZi, respective" @default.
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• W2153289176 title "The Zinc Finger Transcription Factor Transforming Growth Factor β-Inducible Early Gene-1 Confers Myeloid-specific Activation of the Leukocyte Integrin CD11d Promoter" @default.
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