c-Myb and Members of the c-Ets Family of Transcription Factors Act as Molecular Switches to Mediate Opposite Steroid Regulation of the Human Glucocorticoid Receptor 1A Promoter
2005; Elsevier BV; Volume: 280; Issue: 52 Linguagem: Inglês
10.1074/jbc.m508245200
ISSN1083-351X
AutoresChuan-dong Geng, Wayne V. Vedeckis,
Tópico(s)NF-κB Signaling Pathways
ResumoSteroid auto-regulation of the human glucocorticoid receptor (hGR) 1A promoter in lymphoblast cells resides largely in two DNA elements (footprints 11 and 12). We show here that c-Myb and c-Ets family members (Ets-1/2, PU.1, and Spi-B) control hGR 1A promoter regulation in T- and B-lymphoblast cells. Two T-lymphoblast lines, CEM-C7 and Jurkat, contain high levels of c-Myb and low levels of PU.1, whereas the opposite is true in IM-9 B-lymphoblasts. In Jurkat cells, overexpression of c-Ets-1, c-Ets-2, or PU.1 effectively represses dexamethasone-mediated up-regulation of an hGR 1A promoter-luciferase reporter gene, as do dominant negative c-Myb (c-Myb DNA-binding domain) or Ets proteins (Ets-2 DNA-binding domain). Overexpression of c-Myb in IM-9 cells confers hormone-dependent up-regulation to the hGR 1A promoter reporter gene. Chromatin immunoprecipitation assays show that hormone treatment causes the recruitment of hGR and c-Myb to the hGR 1A promoter in CEM-C7 cells, whereas hGR and PU.1 are recruited to this promoter in IM-9 cells. These observations suggest that the specific transcription factor that binds to footprint 12, when hGR binds to the adjacent footprint 11, determines the direction of hGR 1A promoter auto-regulation. This leads to a "molecular switch" model for auto-regulation of the hGR 1A promoter. Steroid auto-regulation of the human glucocorticoid receptor (hGR) 1A promoter in lymphoblast cells resides largely in two DNA elements (footprints 11 and 12). We show here that c-Myb and c-Ets family members (Ets-1/2, PU.1, and Spi-B) control hGR 1A promoter regulation in T- and B-lymphoblast cells. Two T-lymphoblast lines, CEM-C7 and Jurkat, contain high levels of c-Myb and low levels of PU.1, whereas the opposite is true in IM-9 B-lymphoblasts. In Jurkat cells, overexpression of c-Ets-1, c-Ets-2, or PU.1 effectively represses dexamethasone-mediated up-regulation of an hGR 1A promoter-luciferase reporter gene, as do dominant negative c-Myb (c-Myb DNA-binding domain) or Ets proteins (Ets-2 DNA-binding domain). Overexpression of c-Myb in IM-9 cells confers hormone-dependent up-regulation to the hGR 1A promoter reporter gene. Chromatin immunoprecipitation assays show that hormone treatment causes the recruitment of hGR and c-Myb to the hGR 1A promoter in CEM-C7 cells, whereas hGR and PU.1 are recruited to this promoter in IM-9 cells. These observations suggest that the specific transcription factor that binds to footprint 12, when hGR binds to the adjacent footprint 11, determines the direction of hGR 1A promoter auto-regulation. This leads to a "molecular switch" model for auto-regulation of the hGR 1A promoter. Glucocorticoids (GCs) 2The abbreviations used are: GCglucocorticoidGRglucocorticoid receptorhGRhuman glucocorticoid receptorDEXdexamethasoneFPfootprintGREglucocorticoid response elementT-ALLT-cell acute lymphoblastic leukemiaβ-galβ-galactosidaseDBDDNA-binding domainFBSfetal bovine serumChIPchromatin immunoprecipitationPipes1,4-piperazinediethanesulfonic acidPGKphosphoglycerate kinaseCREBcAMP-responsive element-binding protein.2The abbreviations used are: GCglucocorticoidGRglucocorticoid receptorhGRhuman glucocorticoid receptorDEXdexamethasoneFPfootprintGREglucocorticoid response elementT-ALLT-cell acute lymphoblastic leukemiaβ-galβ-galactosidaseDBDDNA-binding domainFBSfetal bovine serumChIPchromatin immunoprecipitationPipes1,4-piperazinediethanesulfonic acidPGKphosphoglycerate kinaseCREBcAMP-responsive element-binding protein. specifically induce apoptosis in several types of leukemia and lymphoma, and they are used routinely in treating T-cell acute lymphoblastic leukemia (T-ALL) (1Harmon J.M. 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Biochemistry. 2003; 42: 10978-10990Crossref PubMed Scopus (58) Google Scholar, 27Pedersen K.B. Geng C.-D. Vedeckis W.V. Biochemistry. 2004; 43: 10851-10858Crossref PubMed Scopus (34) Google Scholar, 28Littlefield B.A. Hoagland H.C. Greipp P.R. Cancer Res. 1989; 46: 3945-3950Google Scholar). Conversely, an auto-down-regulation of GR levels is frequently observed in cells resistant to hormone-mediated apoptosis, such as the pre-B-lymphoblast cell line, IM-9, and the lowered GR concentration actually dampens GC signaling (9Breslin M.B. Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2001; 15: 1381-1395Crossref PubMed Scopus (127) Google Scholar, 19Rosewicz S. McDonald A.R. Maddux B.A. Goldfine I.D. Miesfeld R.L. Logsdon C.D. J. Biol. Chem. 1988; 263: 2581-2584Abstract Full Text PDF PubMed Google Scholar, 26Pedersen K.B. Vedeckis W.V. Biochemistry. 2003; 42: 10978-10990Crossref PubMed Scopus (58) Google Scholar). Thus, steroid auto-up-regulation of GR gene expression in vivo could provide a sensitive indicator of hormonal sensitivity of hematologic malignancies. Human GR gene expression is controlled by at least three promoters, 1A, 1B, and 1C (9Breslin M.B. Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2001; 15: 1381-1395Crossref PubMed Scopus (127) Google Scholar, 10Nunez B.S. Vedeckis W.V. Mol. Cell Endocrinol. 2002; 189: 191-199Crossref PubMed Scopus (50) Google Scholar, 29Encio I.J. Detera-Wadleigh S.D. J. Biol. Chem. 1991; 266: 7182-7188Abstract Full Text PDF PubMed Google Scholar). Promoters 1B and 1C are ubiquitously expressed, whereas the 1A promoter is selectively expressed in hematopoietic cells (9Breslin M.B. Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2001; 15: 1381-1395Crossref PubMed Scopus (127) Google Scholar, 10Nunez B.S. Vedeckis W.V. Mol. Cell Endocrinol. 2002; 189: 191-199Crossref PubMed Scopus (50) Google Scholar). The transcripts emanating from all three promoters are auto-up-regulated in response to DEX treatment in hormone-sensitive, CEM-C7 cell, T-lymphoblasts, and down-regulated in IM-9 cells that are resistant to hormone-mediated apoptosis (9Breslin M.B. Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2001; 15: 1381-1395Crossref PubMed Scopus (127) Google Scholar, 26Pedersen K.B. Vedeckis W.V. Biochemistry. 2003; 42: 10978-10990Crossref PubMed Scopus (58) Google Scholar). Because no consensus GREs are present in any of these promoters, the molecular mechanism for auto-regulation of the 1A, 1B, and 1C promoters was unknown (9Breslin M.B. Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2001; 15: 1381-1395Crossref PubMed Scopus (127) Google Scholar, 10Nunez B.S. Vedeckis W.V. Mol. Cell Endocrinol. 2002; 189: 191-199Crossref PubMed Scopus (50) Google Scholar, 30Breslin M.B. Vedeckis W.V. J. Steroid Biochem. Mol. Biol. 1998; 67: 369-381Crossref PubMed Scopus (57) Google Scholar). Recently, we have identified the DNA sequence that mediates up-regulation of 1A promoter activity (9Breslin M.B. Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2001; 15: 1381-1395Crossref PubMed Scopus (127) Google Scholar, 31Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2004; 18: 912-924Crossref PubMed Scopus (33) Google Scholar). This DNA sequence contains a half GRE (footprint 11 (FP11)) and a sequence (FP12) containing overlapping consensus binding sites for c-Myb or c-Ets proteins. Both FP11 and FP12 are required for full hormone responsiveness of the hGR 1A promoter. In the present study we have further investigated the roles of c-Myb and c-Ets protein members in steroid-mediated auto-regulation of the hGR 1A promoter. Our data show that c-Myb and Ets family members (Ets-1, Ets-2, PU.1, and Spi-B) can affect hGR 1A promoter activity. Western blot and functional studies indicate that c-Myb and PU.1 are the most likely candidates in mediating the opposite hormonal response of the hGR 1A promoter in T- and B-lymphoblasts. Chromatin immunoprecipitation analyses show that, in response to DEX treatment, GR and c-Myb are recruited by the hGR 1A promoter in CEM-C7 T-cells, whereas GR and PU.1 are recruited in IM-9 B-cells. These results suggest a "molecular switch" model for hGR 1A promoter regulation, in which GR binding to the hGR 1A promoter recruits cell type-specific transcription factors to an adjacent DNA sequence. c-Myb is recruited in T-lymphoblasts, and this results in GR up-regulation and apoptosis. PU.1 is recruited in B-cells that do not undergo hormone-mediated apoptosis, and the GR is down-regulated in these cells. These findings may lead to novel clinical therapies that could increase the response rate and the magnitude of the response to steroid treatment in certain hematologic malignancies. Cell Culture—The human Jurkat (T-ALL) and IM-9 B-lymphoblastic cell lines (both from the American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 plus 10% fetal bovine serum (FBS; Invitrogen). The human, CEM-C7, acute lymphoblastic leukemia cell line was a kind gift from Dr. E Brad Thompson (University of Texas Medical Branch, Galveston, TX), and it was maintained in RPMI 1640 supplemented with 10% dialyzed FBS (Invitrogen). All of the cells were grown in 5% CO2 at 37 °C. To treat the cells, 1 μm dexamethasone (Sigma) in ethanol was added to the culture medium, and the same final concentration of ethanol vehicle (0.01%) was used in the controls. DNA Constructs—The human 1A GR promoter-luciferase reporter constructs pXP1-1A -964/+269 and pXP1-1A +41/+269-Luc (and its FP11/12 deletions) were described previously (9Breslin M.B. Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2001; 15: 1381-1395Crossref PubMed Scopus (127) Google Scholar, 31Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2004; 18: 912-924Crossref PubMed Scopus (33) Google Scholar). PCR-directed mutagenesis was performed to make the FP11 deletion in promoter 1A -964/+269, as previously described (9Breslin M.B. Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2001; 15: 1381-1395Crossref PubMed Scopus (127) Google Scholar, 31Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2004; 18: 912-924Crossref PubMed Scopus (33) Google Scholar). The primers used for this deletion were: forward, 5′-CAAGCCCTGCAGGACGTGTCCAACGGAAGC-3′, and reverse, 5′-GCTTCCGTTGGACACGTCCTGCAGGGCTTG-3′. For the FP12 and FP11/12 deletions in pXP1-1A -964/+269, PCR-amplified fragments -964/+253 (FP12 deleted) and -964/+243 (FP11/12 deleted) containing engineered BamHI (5′) and HindIII (3′) restriction sites were inserted into the pXP1 vector digested with the same restriction enzymes. The primers used to construct these deletions were: forward primer, 5′-CCAAACTCATCAATGTATCT-3′; FP12del reverse (FP12 deletion), 5′-GAGAAGCTTGCGCATTTTACGGTCCTG-3′; and FP11/12del reverse (FP11/12 deletion), 5′-CACAAGCTTTACGGTCCTGCAGGGCTTGAA-3′. All of the constructs were confirmed by DNA sequencing. The pCYGR construct was provided by Dr. John A. Cidlowski (National Institute of Environmental Health Sciences, Research Triangle Park, NC). The human c-Myb expression construct, pcDNA3-c-MybHA, and the c-Myb DNA-binding domain (DBD) expression construct, pcDNA3-c-Myb DBD, were provided by Dr. Giuseppe Raschellà (Ente Nuove Tecnologie Energia Ambiente, Rome, Italy) (32Tanno B. Negroni A. Vitali R. Pirozzoli M.C. Cesi V. Mancini C. Calabretta B. Raschella G. J. Biol. Chem. 2002; 277: 23172-23180Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The mouse c-Myb expression plasmid, pC75, and the C-terminal negative regulatory domain truncated c-Myb expression plasmid, pCt, were gifts from Dr. E. Premkumar Reddy and Dr. Ramana V. Tantravahi (Temple University, Philadelphia, PA) (33Dudek H. Tantravahi R.V. Rao V.N. Reddy E.S. Reddy E.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1291-1295Crossref PubMed Scopus (96) Google Scholar). pFN Ets-1, pFN Ets-2, and pFN Ets-2 DBD were supplied by Dr. Craig A. Hauser (The Burnham Institute, La Jolla, CA) (34Foos G. Garcia-Ramirez J.J. Galang C.K. Hauser C.A. J. Biol. Chem. 1998; 273: 18871-18880Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The PU.1 and Spi-B expression constructs were described previously (35Ray D. Bosselut R. Ghysdael J. Mattei M.G. Tavitian A. Moreau-Gachelin F. Mol. Cell Biol. 1992; 12: 4297-4304Crossref PubMed Scopus (145) Google Scholar). Transient Transfection and Luciferase Reporter Gene Assays—Superfect transfection reagent (Qiagen) was used to transfect Jurkat cells, according to the manufacturer's directions. The cells were treated (with EtOH or DEX) 24 h after transfection and were collected for analysis after an additional 24 h of incubation. The collected cells were lysed and measured for firefly luciferase and β-galactosidase activity on an Ascent Luminoskan (Labsystems, Franklin, MA) as previously described (9Breslin M.B. Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2001; 15: 1381-1395Crossref PubMed Scopus (127) Google Scholar, 31Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2004; 18: 912-924Crossref PubMed Scopus (33) Google Scholar). Electroporation—IM-9 cells were electroporated using a method modified from the laboratory of Dr. Jeffrey M. Harmon (Uniformed Services University of the Health Sciences, Bethesda, MD). 3J. M. Harmon, unpublished observation. The cells in log phase growth were counted and harvested by centrifugation at 800 × g for 10 min (4 °C). After two washes with RPMI 1640 medium (without FBS), the cells were resuspended in cold RPMI 1640 (without FBS and containing 10 mm HEPES, pH 7.2) at 2.5 × 107 cells/ml. 200 μl of cell suspension and 10 μg of DNA were combined in a Bio-Rad 0.4-cm electroporation cuvette, and the mixture was incubated on ice for 10 min. Electroporation was done with a Gene Pulser II (Bio-Rad) at 340 V/960 microfarad. Electroporated cells were then incubated on ice for 10 min before being transferred to 10 ml of culture medium (with FBS) at room temperature. The electroporated cells were allowed to grow in 5% CO2 at 37 °C for 24 h before DEX or ethanol treatment. Western Blotting—The cells were lysed with 1× Laemmli sample buffer with a protease inhibitor mixture (Sigma). The proteins resolved on SDS-PAGE (8%) were transferred to Immobilon-nitrocellulose membranes (Millipore, Bedford, MA). The membranes were blocked with 5% nonfat milk and developed using ECL (catalog number RPN2106; Amersham Biosciences). Rabbit polyclonal actin, tubulin, hGR (H-300), c-Myb, Spi-B, and PU.1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Chromatin Immunoprecipitation Assay (ChIP Assay)—Formaldehyde cross-linking and chromatin immunoprecipitation assays of tissue culture cells were performed as described (36Boyd K.E. Farnham P.J. Mol. Cell Biol. 1999; 19: 8393-8399Crossref PubMed Scopus (147) Google Scholar, 37Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar) with some modifications. IM-9 and CEM-C7 cells (1 × 107) were treated for 24 h with/or without 1 μm DEX. The cells were incubated in 1% formaldehyde (Sigma) for 10 min at room temperature. Cross-linking was stopped by adding glycine to a final concentration of 125 mm. The cells were spun down and washed three times with ice-cold phosphate-buffered saline before resuspending in 200 μl of cell lysis buffer (5 mm Pipes-KOH, pH 8.0, 85 mm KCl, 0.5% Nonidet P-40) containing protease inhibitors (1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride). After incubating for 10 min on ice, the nuclei were pelleted, resuspended in 200 μl of nuclear lysis buffer (50 mm Tris-HCl, pH 8.1, 10 mm EDTA, 1% SDS with protease inhibitors), and incubated on ice for 10 min. The chromatin released from the nuclei was sonicated at 4 °C with a Branson sonifier to obtain DNA lengths of 0.1-1.5 kilobase pairs. The sonicated cell lysate was clarified by centrifugation at 13,000 × g for 10 min at 4 °C. The supernatant containing the sheared chromatin was used for the immunoprecipitation assay. 40 μl of the supernatant (sonicated chromatin) was diluted 5-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA, 16.7 mm Tris-HCl, pH 8.1, 167 mm NaCl plus protease inhibitors) and precleared with 6 μg of rabbit normal IgG (Santa Cruz Biotechnology), followed by the addition of a salmon sperm DNA/protein A-agarose slurry (Upstate Biotechnology, Inc.). Six μg of antibodies (normal rabbit IgG, hGR H-300, or c-Myb) (Santa Cruz Biotechnology) were added to precleared chromatin and incubated overnight at 4 °C. The immune complexes formed were collected using salmon sperm DNA/protein A-agarose beads and washed as described by others (38Boyd K.E. Farnham P.J. Mol. Cell Biol. 1997; 17: 2529-2537Crossref PubMed Scopus (139) Google Scholar). The beads were washed once in low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.1, 150 mm NaCl), once in high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.1, 500 mm NaCl), once in LiCl wash buffer (0.25 m LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mm EDTA, 10 mm Tris-HCl, pH 8.0), and twice in TE buffer (10 mm Tris-HCl, pH 8.0, 1 mm EDTA). The chromatin DNA-protein-antibody complexes were eluted using elution buffer (1% SDS, 0.1 m NaHCO3), and the DNA-protein formaldehyde cross-links were reversed by incubating at 65 °C with 0.3 m NaCl overnight. For PCR amplification, the DNA was purified with a Qiaquick PCR purification kit (Qiagen) and eluted in 50 μl of Tris-HCl elution buffer. The PCR mixtures included 1 μl of purified DNA template, 0.04 μm of each primer, 2 mm MgCl2, 0.25 mm dNTP (A, T, G, and C), 1× PCR Buffer II (no Mg2+), and 1 unit of AmpliTaq Gold (Applied Biosystems, Foster City, CA) in a total volume of 25 μl. The primers used for PCR amplification were: 1) hGR 1A far upstream (control), spanning -3436/-3642 (3.7 kilobase pairs upstream of hGR 1A FP12 (9Breslin M.B. Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2001; 15: 1381-1395Crossref PubMed Scopus (127) Google Scholar, 31Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2004; 18: 912-924Crossref PubMed Scopus (33) Google Scholar)), hGR1A -3436 AS (reverse, 5′-CCTCGTTGGCACTAATTC-3′) and hGR 1A -3642/-3616 S (forward, 5′-CTTTGACATGCTTGGAGTGTGCCCTCT-3′); 2) PGK gene (heterologous gene control), PGK AS (reverse, 5′-GGGTGACTTCGGGTGCTTTC-3′) and PGK S (forward, 5′-GGGTGTGGGGCGGTAGTGT-3′); and 3) 1A promoter/exon +146/+316 (FP5-FP12), which contains binding sites for GR and c-Myb/Ets (31Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2004; 18: 912-924Crossref PubMed Scopus (33) Google Scholar), hGR1A +294/+317 AS (reverse, 5′-CTCTTACCCTCTTTCTGTTTCTA-3′) and hGR1A 146 (hGR 1A+55/+77, forward, 5′-CTTGCTCCCTCTCGCCCTCATTC-3′). PCR mixtures were resolved on 5% PAGE and visualized by EtBr staining after 28-35 cycles of amplification. Hormone Responsiveness of the hGR 1A Promoter Depends upon Footprints 11 and 12—We previously identified a hGR 1A promoter that can be significantly auto-up-regulated in T-lymphoblasts (9Breslin M.B. Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2001; 15: 1381-1395Crossref PubMed Scopus (127) Google Scholar, 26Pedersen K.B. Vedeckis W.V. Biochemistry. 2003; 42: 10978-10990Crossref PubMed Scopus (58) Google Scholar), and it is much more sensitive than the other two major hGR promoters, 1B and 1C (9Breslin M.B. Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2001; 15: 1381-1395Crossref PubMed Scopus (127) Google Scholar, 26Pedersen K.B. Vedeckis W.V. Biochemistry. 2003; 42: 10978-10990Crossref PubMed Scopus (58) Google Scholar, 31Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2004; 18: 912-924Crossref PubMed Scopus (33) Google Scholar). Deletion analysis and in vitro DNase I footprinting using nuclear extracts from DEX-treated CEM-C7 cells identified the DNA sequences that mediate the hormone responsiveness in T-cells: FP11, a nonconsensus GRE half-site, and FP12. Computer analysis of FP12 revealed perfectly overlapping c-Myb and c-Ets binding sequences (Fig. 1A), and our previous analysis confirmed that these proteins do bind to this sequence (31Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2004; 18: 912-924Crossref PubMed Scopus (33) Google Scholar). To more fully characterize the role of these DNA elements for both basal promoter activity and in hormone responsiveness, we performed internal deletions of FP11 and FP12, singly or in combination. Deletion of either of these two footprints causes nonresponsiveness of the 1A promoter to DEX, whether a full-length promoter (-964/+269; Fig. 1B) or a shorter promoter that retained about 60% of the basal promoter activity (+41/+269; Fig. 1C) is used. The cause of the apparent down-regulation of promoter activity upon DEX treatment of the full-length promoter in which FP12 has been deleted (Fig. 1B) is unknown, but this may result from the influence of other upstream sequences that are lacking in the shorter promoter construct. These data clearly show that FP11 and FP12 are the critical and perhaps sole sequences required for the hormone-induced up-regulation of hGR 1A promoter activity in the Jurkat T-lymphoblast line. Besides being critical for hormonal responsiveness, these two DNA elements also contribute much to the basal promoter activity, because their deletion causes a dramatic decrease in transcriptional activity (Fig. 1, B and C). In fact, when a minimal hGR 1A promoter containing only FP11 and FP12 (+242/+269) was cloned into pXP1, it retained about 70% percent of the basal activity of the full-length promoter and was completely hormone-responsive (data not shown). Thus, it appears that nearly all of the protein factors that control basal promoter activity and hormonal responsiveness can be recruited to this small DNA element (FP11/FP12) in the hGR 1A promoter. Ets Family Members Are Selectively Expressed in Lymphoid Cell Lineages—Based upon sequence analysis of FP12, we previously showed that c-Myb and two c-Ets protein family members, c-Ets-1 and c-Ets-2, are able to bind to FP12 in vitro (31Geng C.-D. Vedeckis W.V. Mol. Endocrinol. 2004; 18: 912-924Crossref PubMed Scopus (33) Google Scholar). Thus, we wished to determine which c-Ets protein family members are indeed expressed in the three lymphoid cell lines used in our studies. Although there are over 20 different Ets family protein members, various c-Ets proteins are selectively expressed in certain hematopoietic lineages (39Anderson M.K. Hernandez-Hoyos G. Diamond R.A. Rothenberg E.V. Development. 1999; 126: 3131-3148Crossref PubMed Google Scholar). This allowed us to focus on the most likely candidates that might be present in the three cell lines. In particular, Spi-B and PU.1 are two Ets proteins that are expressed during, and are important for, differentiation of lymphoblasts. Spi-B is selectively expressed during T-cell development, and its level drops in mature cells, and PU.1 is restricted to B-cells and macrophages (39Anderson M.K. Hernandez-Hoyos G. Diamond R.A. Rothenberg E.V. Development. 1999; 126: 3131-3148Crossref PubMed Google Scholar). Using Western blotting, we analyzed the relative levels of four Ets family members that were possibly expressed in our T-cell (CEM-C7 and Jurkat cells) and B-cell (IM-9 cells) model systems (Fig. 2). We found that: 1) c-Ets-1/2 are expressed at comparable levels in all three cell lines; 2) Spi-B is present in the three lines, but it is expressed at much higher levels in CEM-C7 and Jurkat T-cells than in the B-cells; and 3) PU.1 is expressed at a high level in IM-9 B-cells, whereas it is undetectable in T-cells. Electrophoretic mobility supershift assays using T- and B-cell lymphoblast nuclear extracts demonstrated that Spi-B and PU.1 can bind to FP12 in vitro (data not shown). Thus, it seemed feasible that Spi-B and PU.1 are the Ets-related proteins that may be involved in cell type-specific regulation of hGR 1A promoter expression in lymphoblasts. In particular, because PU.1 is only present at high levels in IM-9 Pre-B-cells, it seemed likely that this transcription factor is responsible for DEX-mediated down-regulation of the hGR 1A promoter in these cells. In addition, because the c-Ets and c-Myb binding sites in FP12 overlap (Fig. 1A), we postulated that c-Myb and certain Ets members (depending on the cell type) antagonize each other at this binding site to oppositely regulate the response of the hGR 1A promoter to hormone in different lymphoid cell types. Regulation of the Hormonal Response of the hGR 1A Promoter in T-lymphoblasts by c-Myb and Ets Proteins—To determine whether c-Myb and members of the c-Ets family of transcription factors can indeed affect the response of the hGR 1A promoter to DEX, we performed transient cotransfection experiments with an hGR 1A promoter/luciferase reporter gene. The overexpression of c-Myb in Jurkat cells did not greatly affect the responsiveness of the hGR 1A promoter to DEX induction (Fig. 3A). Although this might suggest that the c-Myb site in FP12 is not functional in vivo, this is not the case. The C-terminal truncated c-Myb variant (c-Myb(Ct)) lacking the negative regulatory domain can functionally stimulate the promoter activity by 3-4-fold (Fig. 3A), which means that the c-Myb-binding site is specific and capable of recruiting c-Myb protein in vivo. Thus, the inability of transfected full-length c-Myb to further increase the hormonal response (Fig. 3A) may indicate that sufficient endogenous c-Myb is already present in Jurkat cells. This is supported by the fact that overex
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