Glucocorticoids Repress bcl-X Expression in Lymphoid Cells by Recruiting STAT5B to the P4 Promoter

The bcl-X gene plays a critical role in apoptosis. Six different isoforms generated by tissue-specific promoter usage and alternative splicing were described. Some of them exert opposite effects on cell death. In mammary epithelial cells glucocorticoids induce bcl-X expression and increase the ratio bcl-XL (antiapoptotic)/bcl-XS (apoptotic) by activating P4 promoter, which contains two hormone response elements. Here we show that, on mouse thymocytes and T lymphocyte derivative S49 cells, glucocorticoids inhibited transcription from P4 and decreased the ratio bcl-XL/bcl-XS favoring apoptosis. Upon hormonal treatment, glucocorticoid receptor (GR), steroid receptor coactivator-1, and RNA polymerase II were transiently recruited to P4 promoter, whereas STAT5B was also recruited but remained bound. Concomitant with the release of GR, silencing mediator for retinoic acid receptor and thyroid hormone receptor and histone deacetylase 3 were recruited, histone H3 was deacetylated, and RNA polymerase II left the promoter. Inhibition of STAT5 activity reverted glucocorticoid repression to activation of transcription and was accompanied by stable recruitment of GR and RNA polymerase II to P4. The bcl-X gene plays a critical role in apoptosis. Six different isoforms generated by tissue-specific promoter usage and alternative splicing were described. Some of them exert opposite effects on cell death. In mammary epithelial cells glucocorticoids induce bcl-X expression and increase the ratio bcl-XL (antiapoptotic)/bcl-XS (apoptotic) by activating P4 promoter, which contains two hormone response elements. Here we show that, on mouse thymocytes and T lymphocyte derivative S49 cells, glucocorticoids inhibited transcription from P4 and decreased the ratio bcl-XL/bcl-XS favoring apoptosis. Upon hormonal treatment, glucocorticoid receptor (GR), steroid receptor coactivator-1, and RNA polymerase II were transiently recruited to P4 promoter, whereas STAT5B was also recruited but remained bound. Concomitant with the release of GR, silencing mediator for retinoic acid receptor and thyroid hormone receptor and histone deacetylase 3 were recruited, histone H3 was deacetylated, and RNA polymerase II left the promoter. Inhibition of STAT5 activity reverted glucocorticoid repression to activation of transcription and was accompanied by stable recruitment of GR and RNA polymerase II to P4. Glucocorticoids have tissue-specific effects on apoptosis and cell survival. In monocytes, macrophages, and T lymphocytes they induce apoptosis (1Amsterdam A. Tajima K. Sasson R. Biochem. Pharmacol. 2002; 64: 843-850Crossref PubMed Scopus (138) Google Scholar), whereas they protect against apoptotic signals evoked by different stimuli in mammary epithelial cells (2Schorr K. Furth P.A. Cancer Res. 2000; 60: 5950-5953PubMed Google Scholar, 3Berg M.N. Dharmarajan A.M. Waddell B.J. Endocrinology. 2002; 143: 222-227Crossref PubMed Scopus (29) Google Scholar), endometrium (4Pecci A. Scholz A. Pelster D. Beato M. J. Biol. Chem. 1997; 272: 11791-11798Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), ovarian follicle (5Hillier S.G. Tetsuka M. J. Reprod. Immunol. 1998; 39: 21-27Crossref PubMed Scopus (83) Google Scholar), hepatocytes (6Yamamoto M. Fukuda K. Miura N. Suzuki R. Kido T. Komatsu Y. Hepatology. 1998; 27: 959-966Crossref PubMed Scopus (87) Google Scholar), and fibroblasts (7Gascoyne D.M. Kypta R.M. Vivanco M.M. J. Biol. Chem. 2003; 278: 18022-18029Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Several reports suggested that the opposite control of apoptosis by glucocorticoids is exerted by modulation of a few genes, such as bcl-2, bcl-X, bax, and NFκB, in a cell type-specific manner (1Amsterdam A. Tajima K. Sasson R. Biochem. Pharmacol. 2002; 64: 843-850Crossref PubMed Scopus (138) Google Scholar, 4Pecci A. Scholz A. Pelster D. Beato M. J. Biol. Chem. 1997; 272: 11791-11798Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 8Lotem J. Sachs L. Cell Growth & Differ. 1995; 6: 647-653PubMed Google Scholar, 9Hoijman E. Rocha Viegas L. Keller Sarmiento M.I. Rosenstein R.E. Pecci A. Endocrinology. 2004; 145: 418-425Crossref PubMed Scopus (46) Google Scholar). These multiple effects could be achieved by composite regulatory cross-talk between GR 3The abbreviations used are: GR, glucocorticoid receptor; STAT5A, -B, signal transducer and activator of transcription 5 a/b; SRC-1, steroid receptor coactivator-1; SMRT, silencing mediator for retinoic acid receptor and thyroid hormone receptor; HDAC-3, histone deacetylase 3; NF-κB, nuclear factor kappa B; HRE, hormone response element; ChIP, chromatin immunoprecipitation; pol II, RNA polymerase II; Dex, dexamethasone; RU486, RU-38486; TSA, Tricostatin A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BRG-1, brahma-related gene 1; EMSA, electrophoretic mobility shift assay; JAK, Janus-associated kinase; NF1, nuclear factor 1; GILZ, glucocorticoid-induced leucine zipper; nt, nucleotide(s); wt, wild type. and a variety of nuclear modulators, which are selectively involved in the hormone-dependent expression of specific genes. One of the key genes mediating steroid hormone effects is bcl-X, a member of the Bcl-2 family, which plays a critical role in the control of programmed cell death. Six different Bcl-X isoforms can be generated by alternative splicing. Some of them exert opposite effects on apoptosis (10Boise L.H. Gonzalez-Garcia M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Nunez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2966) Google Scholar, 11Fang W. Rivard J.J. Mueller D.L. Behrens T.W. J. Immunol. 1994; 153: 4388-4398PubMed Google Scholar, 12Shiraiwa N. Inohara N. Okada S. Yuzaki M. Shoji S. Ohta S. J. Biol. Chem. 1996; 271: 13258-13265Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 13Yang X.F. Weber G.F. Cantor H. Immunity. 1997; 7: 629-639Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 14Schmitt E. Paquet C. Beauchemin M. Bertrand R. Oncogene. 2004; 23: 3915-3931Crossref PubMed Scopus (27) Google Scholar); i.e. the large isoform Bcl-XL protects cells against death (10Boise L.H. Gonzalez-Garcia M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Nunez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2966) Google Scholar), whereas the short isoform Bcl-XS antagonizes cell death inhibition by interacting with Bcl-XL and Bcl-2 (10Boise L.H. Gonzalez-Garcia M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Nunez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2966) Google Scholar). In previous works with mammary and endometrial epithelial cells, we have shown that glucocorticoids exert an antiapoptotic effect mediated by the induction of bcl-X expression and an increase in the ratio bcl-XL/bcl-XS (4Pecci A. Scholz A. Pelster D. Beato M. J. Biol. Chem. 1997; 272: 11791-11798Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 15Viegas L.R. Vicent G.P. Baranao J.L. Beato M. Pecci A. J. Biol. Chem. 2004; 279: 9831-9839Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). However, in thymocytes, glucocorticoids increase apoptosis by inhibiting bcl-X expression and decreasing the ratio bcl-XL/bcl-XS (16Vicent G.P. Pecci A. Ghini A. Piwien-Pilipuk G. Galigniana M.D. Exp. Cell Res. 2002; 276: 142-154Crossref PubMed Scopus (21) Google Scholar, 17Delfino D.V. Agostini M. Spinicelli S. Vito P. Riccardi C. Blood. 2004; 104: 4134-4141Crossref PubMed Scopus (92) Google Scholar). The 5′ upstream region of the mouse bcl-X gene contains five different promoters, which exhibit a tissue-specific pattern of promoter usage (18Pecci A. Viegas L.R. Baranao J.L. Beato M. J. Biol. Chem. 2001; 276: 21062-21069Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Recently, we described two HREs located immediately upstream of P4, which bind GR and confer hormone responsiveness to the core promoter (15Viegas L.R. Vicent G.P. Baranao J.L. Beato M. Pecci A. J. Biol. Chem. 2004; 279: 9831-9839Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Only P4 is activated upon hormone treatment of the mammary epithelial HC11 cell line, and this effect correlates with an increased bcl-XL mRNA, suggesting that the antiapoptotic effect of glucocorticoids in this cellular context involves a direct induction of bcl-XL through the activation of P4 (15Viegas L.R. Vicent G.P. Baranao J.L. Beato M. Pecci A. J. Biol. Chem. 2004; 279: 9831-9839Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). To test whether a similar mechanism is involved in the hormonal regulation of cell types where glucocorticoids induce apoptosis, we performed in vivo analysis of bcl-X expression on thymocytes of mice treated with Dex for its physiological relevance, and on the T lymphocyte derivative S49 cell line, which is known to respond to glucocorticoids with programmed cell death (19Hyman R. J. Natl. Cancer Inst. 1973; 50: 415-422Crossref PubMed Scopus (52) Google Scholar, 20Gehring U. Ulrich J. Segnitz B. Mol. Cell Endocrinol. 1982; 28: 605-611Crossref PubMed Scopus (10) Google Scholar). It was previously shown that bcl-X expression decreases during the apoptotic process in thymocytes (8Lotem J. Sachs L. Cell Growth & Differ. 1995; 6: 647-653PubMed Google Scholar, 16Vicent G.P. Pecci A. Ghini A. Piwien-Pilipuk G. Galigniana M.D. Exp. Cell Res. 2002; 276: 142-154Crossref PubMed Scopus (21) Google Scholar, 17Delfino D.V. Agostini M. Spinicelli S. Vito P. Riccardi C. Blood. 2004; 104: 4134-4141Crossref PubMed Scopus (92) Google Scholar, 21Heermeier K. Benedict M. Li M. Furth P. Nunez G. Hennighausen L. Mech. Dev. 1996; 56: 197-207Crossref PubMed Scopus (120) Google Scholar). Only the levels of transcripts generated from P4, but not those derived from the other four promoters, were decreased in response to hormone in both the thymus and murine T lymphocyte S49 cell line. ChIP assays showed a transient loading of GR to the HREs accompanied by a stable recruitment of STAT5B at a potential binding site located between the HREs and the P4 TATA box. Concomitant with the release of GR, SMRT and HDAC3 were recruited, histone H3 was deacetylated, and pol II left the P4 region. Inhibition of STAT5 activity restored not only the increased occupancy by pol II, but also the stable binding of GR at the P4 region and the consequent induction of transcription. These results suggest that glucocorticoids repress bcl-X expression in thymocytes via cross-talk with STAT5B and contribute to the understanding of the molecular basis for tissue-specific hormonal effects on apoptosis. Steroids and Reagents—Dex (10 nm), RU486 (1 μm), and RPMI 1640 medium were purchased from Sigma. Dulbecco's modified Eagle's medium, horse serum, and fetal calf serum were purchased from Invitrogen. Fetal calf serum was previously charcoal-stripped to deplete it of steroid hormones (22Bottenstein J. Hayashi I. Hutchings S. Masui H. Mather J. McClure D.B. Ohasa S. Rizzino A. Sato G. Serrero G. Wolfe R. Wu R. Methods Enzymol. 1979; 58: 94-109Crossref PubMed Scopus (274) Google Scholar). The inhibitor AG490 (100 μm) was purchased from Calbiochem. Plasmid Constructions—The vector pP4-extended (expressing luciferase under the control of bcl-X P4 promoter), pNM1–9SalI, pNM1–9SacI-EcoRI and pNM1–9EagI were described before (15Viegas L.R. Vicent G.P. Baranao J.L. Beato M. Pecci A. J. Biol. Chem. 2004; 279: 9831-9839Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). pP4ΔSTAT5 was generated by mutating (by PCR) the nucleotides located at –240, –239, –238, and –234 to adenines relative to the P4 transcription start site. The plasmid pBKSP4 was generated by amplifying from pNM1–9SalI plasmid, a fragment of 143 bp with the oligonucleotide 5′-exonD: 5′-CCAGGATCTGAGTTCCACTCTTGAACAGAATAACGC-3′ corresponding to the nucleotides –2694 to –2658 and the oligonucleotide 3′-exonD:5′-AAATGAGCTATAACTCAGTTTTTCAA-3′ corresponding to the nucleotides –2551 to –2577 as forward and reverse primers, respectively. The PCR product was blunted, cut with SpeI, and then cloned into SpeI and EcoRV sites of pBluescript KS–. Animal Treatment—Male CF-1 mice of 20-g body weight were injected with Dex (0.5 mg/100 g body weight in 0.2 ml of vehicle: ethanol:propyleneglycol:NaCl (0.9%), 3:3:34) intraperitoneally. This dose is able to induce internucleosomal DNA fragmentation (16Vicent G.P. Pecci A. Ghini A. Piwien-Pilipuk G. Galigniana M.D. Exp. Cell Res. 2002; 276: 142-154Crossref PubMed Scopus (21) Google Scholar). RU486 (0.5 mg/100 g body weight in 0.2 ml of corn oil) was injected subcutaneously 45 min before Dex. Mice injected with vehicle alone were used as control. All animals were treated and cared for in accordance with standard international animal care protocols (23CCACGuide to the Care and Use of Experimental Animals. Canadian Council of Animal Care, Ottawa1980Google Scholar). Animals were sacrificed by cervical dislocation at different times, according to the subsequent analysis, and thymuses were excised. The thymocyte isolation was performed according to Hoijman et al. (9Hoijman E. Rocha Viegas L. Keller Sarmiento M.I. Rosenstein R.E. Pecci A. Endocrinology. 2004; 145: 418-425Crossref PubMed Scopus (46) Google Scholar) and Vicent et al. (16Vicent G.P. Pecci A. Ghini A. Piwien-Pilipuk G. Galigniana M.D. Exp. Cell Res. 2002; 276: 142-154Crossref PubMed Scopus (21) Google Scholar). Cell Cultures and Transfection Assays—Cells were cultured at 37 °C under humidified atmosphere with 5% CO2 in p100 plates. COS-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum containing penicillin (100 IU/ml), streptomycin (100 μg/ml), and glutamine (2 mm). S49 cells from mouse T lymphoma were grown in Dulbecco's modified Eagle's medium supplemented with 10% horse serum and 1% penicillin/streptomycin. For transient transfections 5 × 105 cells plated in 60-mm plates were transfected with Lipofectin 2000 (Invitrogen) following the instructions of the manufacturer. 3 μg of pP4-extended reporter vector (15Viegas L.R. Vicent G.P. Baranao J.L. Beato M. Pecci A. J. Biol. Chem. 2004; 279: 9831-9839Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) or pP4ΔSTAT5 (P4 reporter vector with four point mutations on the STAT5 site) and 1 μg of pGR (24Godowski P.J. Rusconi S. Miesfeld R. Yamamoto K.R. Nature. 1987; 325: 365-368Crossref PubMed Scopus (320) Google Scholar) were co-transfected into COS-1 cells with increasing amounts of constitutively active mutants of STAT5B (pSTAT5B-N642H) and STAT5A (pSTAT5A-N642H) vectors (kindly provided by T. Kitamura (25Ariyoshi K. Nosaka T. Yamada K. Onishi M. Oka Y. Miyajima A. Kitamura T. J. Biol. Chem. 2000; 275: 24407-24413Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar)). 3 μg of pCMV-LacZ were also used as control of transfection. The plasmids were diluted in 100 μl of medium and added dropwise to an equal volume of medium containing 4 μl of Lipofectin 2000 (Invitrogen). After 20 min, the transfection mixture was added dropwise to the cells. 6 h later, the medium was replaced by medium containing 10% charcoal-stripped FCS and the antibiotics described above, and the mixture was incubated overnight at 37 °C in a 5% CO2 atmosphere. The cells were then incubated with Dex for 36 h. After incubations, cells were harvested in lysis buffer (Promega, Madison, WI, cat. no. E3971), and luciferase activity was measured with a luciferase kit according to the manufacturer protocol (Promega, cat. no. E1501). β-Galactosidase activity was measured as described before (26Truss M. Bartsch J. Schelbert A. Hache R.J. Beato M. EMBO J. 1995; 14: 1737-1751Crossref PubMed Scopus (265) Google Scholar). The JAK inhibitor AG490 (100 μm) was added 2 h before Dex, and the HDAC inhibitor TSA (16 μm) was added 30 min before Dex. RNA Analysis—Animals were injected with vehicle or Dex for 2 h, sacrificed, and thymuses were excised. In addition, S49 cells were treated with or without Dex during 5 h. RNA was extracted by the single step method (27Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (65696) Google Scholar). RNase protection analysis was performed as described before (28Zinn K. DiMaio D. Maniatis T. Cell. 1983; 34: 865-879Abstract Full Text PDF PubMed Scopus (719) Google Scholar). The bcl-X coding region probe was prepared from plasmid pGLD3 (kindly provided by J. M. Hardwick, The Johns Hopkins Hospital, Baltimore, MD), which was digested with HinfI and transcribed by T3 RNA polymerase. The full-length transcript size of the bcl-X Riboprobe was 294 nt, and the protected fragments for bcl-XL and bcl-XS were 237 and 155 nt long, respectively. For preparing the P1 Riboprobe, plasmid pNM1–9SalI (15Viegas L.R. Vicent G.P. Baranao J.L. Beato M. Pecci A. J. Biol. Chem. 2004; 279: 9831-9839Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) was digested with MaeIII and transcribed by T3 RNA polymerase. The full-length transcribed Riboprobe was 558 nt, and the protected fragment was 176 nt. For preparing the P2 Riboprobe, plasmid pNM1–9EagI (15Viegas L.R. Vicent G.P. Baranao J.L. Beato M. Pecci A. J. Biol. Chem. 2004; 279: 9831-9839Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) was digested with EcoRI and transcribed by T7 RNA polymerase; the full-length transcribed Riboprobe was 495 nt, and the protected fragment was 124 nt long. P4 Riboprobe was prepared by cutting the plasmid pBKSP4 with SpeI and transcribed with T3 RNA polymerase. The full-length transcribed Riboprobe was 207 nt, and the protected fragment was 147 nt long. For preparing the P5 Riboprobe plasmid, pNM1–9SacI-EcoRI was digested with SacI and transcribed with T3 RNA polymerase; the full-length Riboprobe was 670 nt long, and the protected fragment was 248 nt long. The GAPDH template pTRIGAPDH (Ambion, Austin, TX) was digested with BglII and transcribed with T3 RNA polymerase as described previously (4Pecci A. Scholz A. Pelster D. Beato M. J. Biol. Chem. 1997; 272: 11791-11798Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The probe length was 359 nt, and the size of the protected fragment was 316 nt. [α-32P]CTP (Amersham Biosciences) radiolabeled RNA probes were prepared using a kit according to the instructions of the manufacturer (Promega). The probes were co-precipitated with RNA samples and dissolved in hybridization buffer, denatured at 95 °C for 10 min, and hybridized at 52 °C for 18 h. After digestion with RNases A and T1, followed by digestion with proteinase K, the samples were precipitated, denatured, and subjected to electrophoresis on a 5% denaturing acrylamide gel. For reverse transcription, 4 μg of total RNA were used. The first cDNA strand was synthesized with Superscript reverse transcriptase and 25 ng/μl oligo(dT) (Invitrogen) as reverse complementary primer. For PCR amplification of the P4 5′-leading exon, the oligonucleotide 5′-exonD:(5′-CCAGGATCTGAGTTCCACTCTTGAACAGAATTAACGC-3′) corresponding to the nucleotides –2694 to –2658 from ATG and the oligonucleotide 3′-exonD: (5′-AAATGAGCTATAACTCAGTTTTTCAA-3′) corresponding to the nucleotides –2551 to –2577 from ATG were used as forward and reverse primers, respectively. The reaction yielded a 143-bp length cDNA fragment. PCRs were normalized against GAPDH expression. Primers GAPDH-for (5′-TCATCAACGGGAAGCCCATCACCATCTTC-3′) and GAPDH-rev (5′-GTCTTCTGGTTGGCAGTAATGGCATGGACT-3′), which specifically hybridize with GAPDH mRNA, were used. The reaction yielded a 357-bp length cDNA fragment. PCRs were normalized against GAPDH expression. Primers GAPDH-for (5′-TCATCAACGGGAAGCCCATCACCATCTTC-3′) and GAPDH-rev (5′-GTCTTCTGGTTGGCAGTAATGGCATGGACT-3′), which specifically hybridize with GAPDH mRNA, were used. To achieve semiquantitative conditions, reverse transcriptase-PCRs were terminated and the products were quantified when all of the samples were in the linear range of amplification. The cDNA pool (2 μl), 1.25 units of Thermus aquaticus Taq polymerase (Invitrogen), and amplification primers (20 pmol of each) in 50 μl of PCR mixture (1 μl of polymerase buffer, 2 mm MgCl2, 200 μm each dNTP) denatured 3 min at 96 °C followed by 8, 15, 25, and 30 cycles of amplification by using a step program (96 °C for 40 s; 65 °C (for GAPDH) and 60 °C (for exonD) for 30 s; and 72 °C for 1 min) and a final extension at 72 °C for 10 min. PCR products were analyzed by electrophoresis in 1.6% agarose gels and visualized under UV light. Quantification was performed with a phosphorimaging device (Fuji FLA 3000G) using Image-Gauge software. In all cases the quantification was normalized against the GAPDH signal. ChIP Assays—S49 cells were untreated or incubated for different times (from 0 to 30 min) with 10 nm Dex, and ChIP assays were performed as described previously (29Strutt H. Paro R. Methods Mol. Biol. 1999; 119: 455-467PubMed Google Scholar, 30Eberhardy S.R. D'Cunha C.A. Farnham P.J. J. Biol. Chem. 2000; 275: 33798-33805Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In addition, animals were injected with vehicle or Dex for 30 min, sacrificed, and thymuses were excised. Thymocytes were dispersed in 2 ml of phosphate saline buffer; then 220 μl of cross-linking solution (50 mm HEPES, pH 8; 0.1 m NaCl; 1 mm EDTA; 0.5 mm EGTA; 11% formaldehyde) was added to the samples, and they were incubated for 10 min at 37 °C. ChIP assays were then performed as above. The following antibodies were used: mouse monoclonal α-GR (BuGR, Affinity Bioreagents, Golden, CO), mouse monoclonal α-pol II (8WG16, Berkeley Antibody Co.), mouse monoclonal α-STAT5B (G-2, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal α-STAT5A (L-20, Santa Cruz Biotechnology), mouse monoclonal α-SRC1 (Upstate Biotechnologies), rabbit polyclonal α-SMRT (Santa Cruz Biotechnology), rabbit polyclonal α-BRG-1 (Santa Cruz Biotechnology), rabbit polyclonal α-HDAC3 (Abcam), and rabbit polyclonal α-acetylhistone H3 (Upstate Biotechnologies). Control for nonspecific interaction of DNA was performed by using as nonspecific antibody normal rabbit and mouse IgG (data not shown). For each experiment, PCRs were performed with different numbers of cycles or with dilution series of input DNA to determine the linear range of amplification; all results shown fall within this range. Primer sequences are available on request. The density of bands was quantified with Image J software (version 1.35s, 2005). In Silico Analysis—Screening for potential transcription factor binding sites was performed using MatInspector software (31Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2432) Google Scholar). Protein Analysis—Protein extracts were prepared by lysing cells as described before (9Hoijman E. Rocha Viegas L. Keller Sarmiento M.I. Rosenstein R.E. Pecci A. Endocrinology. 2004; 145: 418-425Crossref PubMed Scopus (46) Google Scholar). Protein concentration was measured by Bradford assay (Bio-Rad). For co-immunoprecipitation assays mouse polyclonal α-STAT5B (Santa Cruz Biotechnology) was used. Protein extract (0.6 mg) was incubated overnight with 2 μg of antibody at 4 °C on a vertical rotator. Protein A/G-Sepharose beads (Santa Cruz Biotechnology) were added, and the mixture was incubation continued for 1 h. Samples were washed three times with lysis buffer and resuspended in 2× sample buffer (250 mm Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 2% β-mercaptoethanol, 0.006% bromphenol blue). For Western blots, samples were boiled for 5 min and applied on a 15% SDS-polyacrylamide gel, and electrophoresis was performed at 25 mA for 2 h. The resolved proteins were transferred to a Hybond ECL membrane (Amersham Biosciences) by electro-blotting. Antibody incubation was performed in blocking buffer (1% skim milk, 0.5% Tween) in Tris-buffered saline at 4 °C. As primary antibodies, the rabbit polyclonal α-STAT5A, the mouse monoclonal α-STAT5B described before, the rabbit polyclonal α-Bcl-XL/S, and the mouse monoclonal α-phosphorylated tyrosine were applied (Santa Cruz Biotechnology). For tubulin expression the rabbit polyclonal γ-tubulin antibody (Santa Cruz Biotechnology) was used. As secondary antibody, a peroxidase-labeled α-rabbit or α-mouse antibody, was used (Amersham Biosciences). Proteins bands were detected using the ECL kit (Amersham Biosciences). Electrophoretic Mobility Shift Assay—EMSAs were performed with the 303-bp oligonucleotide bclX-P4 wild type or mutated, generated by digesting pP4-extended or pP4ΔSTAT5 vector, between the nucleotides –3133 and –2829, with AseI and BglII enzymes. Nuclear extracts from S49 cells treated for 30 min with Dex (10 nm) were prepared as previously described (32Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2244) Google Scholar). Binding reactions were carried out according to our previous description (15Viegas L.R. Vicent G.P. Baranao J.L. Beato M. Pecci A. J. Biol. Chem. 2004; 279: 9831-9839Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Results were visualized by autoradiography of the dried gel and analyzed using a phosphorimaging device (Fuji FLA 3000G) and quantification software (Image-Gauge version 3.1). The monoclonal α-GR antibody (BuGR, Affinity Bioreagents) and the polyclonal α-STAT5A and monoclonal α-STAT5B antibodies (Santa Cruz Biotechnology) were included in the incubation mixture for supershift assays. Statistical Analysis—Statistical analysis was performed by using the two-way analysis of variance followed by Turkey's test using GraphPad Instat software (version 3.01, 1998). Dex Decreases the Levels of bcl-X Transcripts in Thymocytes through the Inhibition of P4 Activity—Lymphoid cells, especially thymocytes, undergo apoptosis in response to glucocorticoids. It was previously shown that the relative abundance of bcl-X isoforms changes during the apoptotic process (8Lotem J. Sachs L. Cell Growth & Differ. 1995; 6: 647-653PubMed Google Scholar, 16Vicent G.P. Pecci A. Ghini A. Piwien-Pilipuk G. Galigniana M.D. Exp. Cell Res. 2002; 276: 142-154Crossref PubMed Scopus (21) Google Scholar, 17Delfino D.V. Agostini M. Spinicelli S. Vito P. Riccardi C. Blood. 2004; 104: 4134-4141Crossref PubMed Scopus (92) Google Scholar, 21Heermeier K. Benedict M. Li M. Furth P. Nunez G. Hennighausen L. Mech. Dev. 1996; 56: 197-207Crossref PubMed Scopus (120) Google Scholar). We first examined by RNase protection assays the expression levels of the bcl-XL and bcl-XS isoforms in thymocytes of animals treated for 2 h with Dex. In thymus from untreated mice both isoforms are expressed at the ratio bcl-XL/bcl-XS = 2.8 ± 0.1 (Fig. 1A, lane 2). The level of bcl-XL was drastically decreased upon hormonal treatment, which had little effect on the levels of bcl-XS transcripts being the ratio bcl-XL/bcl-XS = 1.2 ± 0.1 (Fig. 1A, lane 3). This effect was likely mediated by GR, because it was inhibited in the presence of the GR antagonist, RU486, which restored the bcl-XL/bcl-XS ratio to 2.3 ± 0.2 (Fig. 1A, lane 5). Treatment with RU486 had neither significant effect on transcript levels nor on the ratio of both isoforms (Fig. 1A, lane 4). Similar results were previously observed in rat thymocyte primary cultures treated with glucocorticoids (16Vicent G.P. Pecci A. Ghini A. Piwien-Pilipuk G. Galigniana M.D. Exp. Cell Res. 2002; 276: 142-154Crossref PubMed Scopus (21) Google Scholar). The levels of Bcl-XL protein also decreased after 8 h of Dex injection (Fig. 1B, lane 2). The co-injection of the antagonist RU486 abolished this effect (Fig. 1B, lane 3). We conclude that glucocorticoids affect bcl-XL transcript and Bcl-XL protein levels in thymocytes in a way compatible with their proapoptotic effect in these cells. To determine which of the various bcl-X promoters was responsible for the hormonal decrease in bcl-X transcripts, we performed RNase protection assays using specific Riboprobes for the different 5′-leading exons. Hormone treatment had no effect on transcripts generated from promoters P1, P2, or P5 (Fig. 1, C, D, and F, respectively). In contrast, the level of transcripts containing the 5′-leading exon of P4 decreased 2.6 ± 0.2-fold versus control (ratio bcl-X P4/GAPDH = 1.0 ± 0.1), in thymus samples obtained from animals treated with Dex (ratio bcl-X P4/GAPDH = 0.4 ± 0.1) (Fig. 1E, upper panel, lanes 1 and 2, respectively). The hormone effect was abolished by the co-injection of RU486 (ratio bcl-X P4/GAPDH = 0.9 ± 0.1) (Fig. 1E, upper panel, lane 3). As control, no change was detected in GAPDH RNA levels after hormone treatment (Fig. 1E, lower panel). Transcripts generated from P3 were not analyzed, because this promoter is completely inactive in thymocytes (18Pecci A. Viegas L.R. Baranao J.L. Beato M. J. Biol. Chem. 2001; 276: 21062-21069Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). We conclude that the changes in bcl-X expression reflect an effect of the hormone on the accumulation of transcripts derived from P4. These changes in bcl-X transcript levels could result from a decrease in the transcription rate or in the stability of the transcripts. We therefore tested whether the bcl-X P4 region was occupied by the transcriptional machinery after hormone treatment. ChIP assays using a monoclonal pol II antibody demonstrated the presence of the enzyme bound to the P4 region in thymocytes obtained from untreated mice (2.1% DNA bound) (Fig. 2A, upper panel, lane 1), as expected given the basal activity of the promoter. After 30 min, the hormonal treatment reduced the levels of pol II bound to P4 region (0.84% DNA bound) (Fig. 2A, upper panel, lane 2). These findings suggest that the glucocorticoid-induced