Steroid Hormones Induce bcl-X Gene Expression through Direct Activation of Distal Promoter P4
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m312402200
ISSN1083-351X
AutoresLuciana Rocha-Viegas, Guillermo P. Vicent, J. Lino Barañao, Miguel Beato, Adalı́ Pecci,
Tópico(s)RNA Research and Splicing
ResumoBcl-X exists in at least five different isoforms with complex effects on programmed cell death. Glucocorticoids and progestins control bcl-X expression and influence the ratio between bcl-XL (antiapoptotic isoform) and bcl-XS (proapoptotic isoform) in different tissues. The 5′-UTR region of the mouse bcl-X gene contains at least five different promoters, which exhibit a tissue-specific pattern of promoter usage. Several mRNAs with different 5′-leading exons can be generated upon promoter activation. Here we explore the potential of the various bcl-X gene promoters to be regulated by glucocorticoids or progestins. We found that the region located immediately upstream of promoter 4 (P4) contains two hormone response element (HRE)-like sequences at positions –3040 (HRE I) and –3001 (HRE II) relative to the translation initiation codon. These HRE-like sequences confer hormone responsiveness to a core promoter and bind glucocorticoid or progesterone receptors in vitro. Point mutations of both HREs that prevent steroid receptor binding also eliminate hormonal inducibility. In cells treated with glucocorticoids, the hormone receptor is recruited to the P4 region containing the HREs. Analysis of the products of the endogenous bcl-X in epithelial mammary cells showed that only transcripts originating from P4 increased upon hormone treatment. This observation correlates with the induction of the bcl-XL mRNA, suggesting that P4 is one of the bcl-X promoters responsible for the generation of this antiapoptotic isoform. Bcl-X exists in at least five different isoforms with complex effects on programmed cell death. Glucocorticoids and progestins control bcl-X expression and influence the ratio between bcl-XL (antiapoptotic isoform) and bcl-XS (proapoptotic isoform) in different tissues. The 5′-UTR region of the mouse bcl-X gene contains at least five different promoters, which exhibit a tissue-specific pattern of promoter usage. Several mRNAs with different 5′-leading exons can be generated upon promoter activation. Here we explore the potential of the various bcl-X gene promoters to be regulated by glucocorticoids or progestins. We found that the region located immediately upstream of promoter 4 (P4) contains two hormone response element (HRE)-like sequences at positions –3040 (HRE I) and –3001 (HRE II) relative to the translation initiation codon. These HRE-like sequences confer hormone responsiveness to a core promoter and bind glucocorticoid or progesterone receptors in vitro. Point mutations of both HREs that prevent steroid receptor binding also eliminate hormonal inducibility. In cells treated with glucocorticoids, the hormone receptor is recruited to the P4 region containing the HREs. Analysis of the products of the endogenous bcl-X in epithelial mammary cells showed that only transcripts originating from P4 increased upon hormone treatment. This observation correlates with the induction of the bcl-XL mRNA, suggesting that P4 is one of the bcl-X promoters responsible for the generation of this antiapoptotic isoform. Correction: Steroid hormones induce bcl-X gene expression through direct activation of distal promoter P4Journal of Biological ChemistryVol. 296PreviewVOLUME 279 (2004) PAGES 9831–9839 Full-Text PDF Open Access Bcl-X is one of the members of Bcl-2 family proteins and plays a critical role in the control of apoptosis. At least five different isoforms produced by alternative splicing of a unique gene have been described previously (1Pecci A. Viegas L.R. Baranao J.L. Beato M. J. Biol. Chem. 2001; 276: 21062-21069Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Some of these isoforms exert opposite effects on programmed cell death (2Boise 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 (2933) Google Scholar, 3Fang W. Rivard J.J. Mueller D.L. Behrens T.W. J. Immunol. 1994; 153: 4388-4398PubMed Google Scholar, 4Shiraiwa 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, 5Yang X.F. Weber G.F. Cantor H. Immunity. 1997; 7: 629-639Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), i.e. the ubiquous large isoform Bcl-XL and the tissue-specific isoform Bcl-Xγ protect cells against apoptosis (2Boise 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 (2933) Google Scholar, 5Yang X.F. Weber G.F. Cantor H. Immunity. 1997; 7: 629-639Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), whereas the short isoform, Bcl-XS, antagonizes cell death inhibition by interacting with Bcl-XL and Bcl-2 (2Boise 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 (2933) Google Scholar). Thus, the control of apoptosis in some cell types could involve changes in the relative proportions of various Bcl-X isoforms, suggesting an accurate regulation not only of transcription but also of splicing of bcl-X transcripts. Steroid hormones, in particular glucocorticoids and progestins, control programmed cell death in several tissues. Although the molecular mechanism of hormone-dependent apoptosis is still not completely known, a link between glucocorticoids and genes from the Bcl-2 family has been demonstrated in several systems (6Pecci A. Scholz A. Pelster D. Beato M. J. Biol. Chem. 1997; 272: 11791-11798Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 7Vicent G.P. Pecci A. Ghini A. Piwien-Pilipuk G. Galigniana M.D. Exp. Cell Res. 2002; 276: 142-154Crossref PubMed Scopus (21) Google Scholar, 8Yamamoto M. Fukuda K. Miura N. Suzuki R. Kido T. Komatsu Y. Hepatology. 1998; 27: 959-966Crossref PubMed Scopus (87) Google Scholar, 9Schorr K. Furth P.A. Cancer Res. 2000; 60: 5950-5953PubMed Google Scholar). bcl-X has been postulated as a key target gene on hormone-dependent apoptosis. In fact, the steroid hormones, glucocorticoids, and progesterone, have been shown to control bcl-X expression and to influence the ratio between bcl-XL (antiapoptotic isoform) and bcl-XS (proapoptotic isoform) in different cell types (6Pecci A. Scholz A. Pelster D. Beato M. J. Biol. Chem. 1997; 272: 11791-11798Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 7Vicent G.P. Pecci A. Ghini A. Piwien-Pilipuk G. Galigniana M.D. Exp. Cell Res. 2002; 276: 142-154Crossref PubMed Scopus (21) Google Scholar, 9Schorr K. Furth P.A. Cancer Res. 2000; 60: 5950-5953PubMed Google Scholar, 10Chang T.C. Hung M.W. Jiang S.Y. Chu J.T. Chu L.L. Tsai L.C. FEBS Lett. 1997; 415: 11-15Crossref PubMed Scopus (59) Google Scholar, 11Gascoyne D.M. Kypta R.M. Vivanco M.M. J. Biol. Chem. 2003; 278: 18022-18029Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The promoter region of the mouse bcl-X gene exhibits a complex structure. It contains at least five different promoters (P1–P5), 1The abbreviations used are: P, promoter; HRE, hormone response element; PR, progesterone receptor; GR, glucocorticoid receptor; cons, consensus; Dex, dexamethasone; EMSA, electrophoretic mobility shift assays; gapdh, glyceraldehyde-3-phosphate dehydrogenase; ChIP, chromatin immunoprecipitation. which are used in a tissue-specific manner (Fig. 1) (1Pecci A. Viegas L.R. Baranao J.L. Beato M. J. Biol. Chem. 2001; 276: 21062-21069Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Thus, several mRNAs differing in at least their 5′-leading exon can be generated upon alternative promoter usage. Although the physiological relevance of this mechanism is still not clear, it might be involved in the tissue-specific gene expression. Moreover, the activation of a particular promoter might generate specific splice isoforms. In this way, external signals like steroid hormones could influence the outcome of the splice process by regulating promoter choice by their activated hormone receptors. Steroids exert their action by interacting with their intracellular receptors, which are ligand-dependent transcription factors (12Beato M. Herrlich P. Schutz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1639) Google Scholar). After hormone binding, the hormone receptor complex regulates the expression of target genes by binding to specific DNA sequences in chromatin called hormone response elements (HREs). In particular, progesterone and glucocorticoid receptors (hereafter denoted PR and GR, respectively) bound to the same palindromic consensus sequence TG-TYCTXXXACARGA (12Beato M. Herrlich P. Schutz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1639) Google Scholar) located in the promoter or enhancer regions of target genes. To explore the possibility of a direct effect of steroid hormones on the bcl-X gene expression, we performed transient transfection assays of expression vectors containing the luciferase gene under the control of different bcl-X promoter regions. In several cell lines, mainly P4 responds selectively to the synthetic glucocorticoid dexamethasone or to the progestin agonist R5020. The screening of the region around this promoter revealed the presence of two HRE-like sequences located at positions –3040 (HRE I) and –3001 (HRE II) relative to the translation initiation site. Both HREs differ from the consensus HRE (cons-HRE) by three mismatches. Here we show that recombinant GR and PR bind specifically to a bcl-X oligonucleotide containing HRE I and II sequences. An analysis of the endogenous gene expression showed that only those transcripts generated by the activation of P4 increased their levels upon steroid treatment in mouse epithelial mammary cells. This observation was confirmed by chromatin immunoprecipitation assays, which demonstrated the loading of GR and increased occupancy by the RNA polymerase II at the P4 region after the addition of dexamethasone. P4 activation correlates with the induction of the bcl-XL mRNA, suggesting that P4 may be one of the bcl-X promoters responsible for the generation of this antiapoptotic isoform. These results contribute to the understanding of the molecular basis of hormone-dependent apoptosis. Steroids and Reagents—R5020 was purchased from PerkinElmer Life Sciences. Dexamethasone (Dex) and RU 38486 (RU) from Sigma were used for all of the hormonal treatments. Hormones were dissolved in absolute ethanol. For in vitro assays, 1000× solutions were prepared. Dulbecco's modified Eagle medium and fetal calf serum were purchased from Invitrogen. RPMI 1640 medium was purchased from Sigma. Fetal calf serum was previously charcoal-stripped to deplete it of steroid hormones (13Bottenstein 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 (273) Google Scholar). Expression Vectors—The vector pGAW (kindly provided by Dr. Guntram Suske, IMT, Philipps Universität, Marburg, Germany) is a derivative plasmid from pGL3-basic (Promega). This vector was used to subclone the bcl-X promoter fragments upstream of the luciferase gene. The expression vector P1 contained a genomic sequence of mouse bcl-X gene from nucleotides –594 to –95 relative to the translation initiation codon. It was generated by subcloning a HindIII-BamHI fragment from pNM1–9 SalI plasmid (1Pecci A. Viegas L.R. Baranao J.L. Beato M. J. Biol. Chem. 2001; 276: 21062-21069Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) into pGAW. Vector P5-P2 contained a genomic sequence from nucleotides –3420 to –537. This fragment includes from P5 to P2 a promoter region. This vector was generated by digesting a 2884-bp fragment of pNM1–9EagI (1Pecci A. Viegas L.R. Baranao J.L. Beato M. J. Biol. Chem. 2001; 276: 21062-21069Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) with SacI enzyme and cloning it into SacI site of pGAW. The vector P2 contained the P2 promoter region from nucleotides –1000 to –537, and it was generated by digestion of P5-P2 with EcoRI enzyme followed by religation. P3 vector was generated by amplifying from pNM1–9 SalI plasmid, a fragment of 497 bp with the oligonucleotides 5′-CATGGAATTCGGTCATCCTATGCTACATAG-3′ and 5′-CGAGCTCGAGCTAGTTCCTTCATCCATCTG-3′ as forward and reverse primers, respectively. The PCR product was cloned into the EcoRI and XhoI sites of pGAW. Vector P4 "core" was generated by digesting P4-P3 vector with XhoI enzyme followed by Klenow treatment and then by digestion with SspI enzyme followed by religation. Vector P4-P3 was generated by subcloning an EcoRI-XhoI fragment from P5-P2 into pGAW. Vector P4 "extended" was generated by digestion of P5-P2 with BamHI enzyme followed by religation. The obtained vector was then digested with XhoI enzyme followed by Klenow treatment. This vector then was cut with SspI enzyme and religated. Vector P4-extended ΔHRE was generated by cutting from P4 extended, a fragment of 95 bp between the nucleotides –3045 and –2950 with XmnI and MscI enzymes followed by Klenow treatment and religation. Cell Cultures and Transfection Assay—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 medium supplemented with 10% fetal calf serum containing penicillin (100 IU/ml), streptomycin (100 μg/ml), and glutamine (2 mm). Mammary epithelial T47D and HC11 (the latter one was kindly provided by Dr. Nancy Hynes, Basel, Switzerland) were grown in RPMI 1640 medium plus 10% fetal calf serum and 1% penicillin/streptomycin. 5 μg/ml insulin were added to HC11 cell cultures. For transient transfections, 5 × 105 cells plated in 60-mm plates were transfected with Lipofectin 2000 (Invitrogen) following the instructions of the manufacturer. 5 pmols of each expression vector expressing luciferase under the control of bcl-X promoters were transfected in HC11, T47D, or COS-1 cells. 1 μg of PR (expressing human progesterone isoform B receptor) (14Kastner P. Bocquel M.T. Turcotte B. Garnier J.M. Horwitz K.B. Chambon P. Gronemeyer H. J. Biol. Chem. 1990; 265: 12163-12167Abstract Full Text PDF PubMed Google Scholar) or GR (expressing human glucocorticoid receptor) (15Godowski P.J. Rusconi S. Miesfeld R. Yamamoto K.R. Nature. 1987; 325: 365-368Crossref PubMed Scopus (283) Google Scholar) was cotransfected into COS-1 cells. 3 μg of pCMV-LacZ were also introduced as control of transfection. The plasmids were diluted in 100-μl medium and added dropwise to an equal volume of medium containing 4 μl of Lipofectin 2000. 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 fetal calf serum and the antibiotics described above and incubated overnight at 37 °C in 5% CO2 atmosphere. The cells were then incubated with the corresponding steroids for 36 h. After incubations, cells were harvested in lysis buffer (Promega, catalog number E3971) and luciferase activity was measured with luciferase kit according to manufacturer protocol (Promega, catalog number E1501). β-Galactosidase activity was measured as described previously (16Truss M. Bartsch J. Schelbert A. Hache R.J. Beato M. EMBO J. 1995; 14: 1737-1751Crossref PubMed Scopus (265) Google Scholar). In Silico Analysis—Screening for the potential HREs was performed using MatInspector software (17Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2427) Google Scholar). Electrophoretic Mobility Shift Assays (EMSA)—EMSAs were performed with the synthetic oligonucleotides: cons-HRE (5′-TCGGAGTGCCTAGAGAACAAACTGTTCTGACTCAAC-3′) (18Chalepakis G. Schauer M. Cao X.A. Beato M. DNA Cell Biol. 1990; 9: 355-368Crossref PubMed Scopus (62) Google Scholar); bcl-X HRE (5′-GAGTTTGAAACAATTCTGGTGTGTCTGTTCCCACATGGGCTCAGCTCTCCAGCACACACCAAATTTCAGTCAAGGGG-3′); and mutated bcl-X HRE(mut) (5′-GAGTTTGAAACAATTCTAGTATGTCTATTTCCACATGGGCTCAACTTTCCAACATACACCAAATTTCATCAAGGGG-3′) as described previously (19Di Croce L. Koop R. Venditti P. Westphal H.M. Nightingale K.P. Corona D.F. Becker P.B. Beato M. Mol. Cell. 1999; 4: 45-54Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The complementary strands were annealed in equimolar amounts (100 nm each) in annealing buffer (10 mm Tris-HCl, pH 8, 1 mm EDTA, 30 mm KCl) by denaturation at 95 °C for 5 min and cooling down to room temperature. Double-stranded oligonucleotides were radiolabeled with T4 polynucleotide kinase and [γ-32P]ATP. Recombinant PR and GR were expressed in baculovirus and purified as described previously (19Di Croce L. Koop R. Venditti P. Westphal H.M. Nightingale K.P. Corona D.F. Becker P.B. Beato M. Mol. Cell. 1999; 4: 45-54Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Nuclear extracts containing PR were prepared from T47D cells treated for 2 h with 10 nm R5020 as described previously (20Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 192499Crossref PubMed Scopus (2214) Google Scholar). Binding reactions were carried out in 30-μl reaction buffer containing 10 mm Tris-HCl, pH 8.0, 0.5 mm EDTA, 5% glycerol, 0.5 mm 2-mercaptoethanol, 90 mm NaCl, 1 μg of poly(dI-dC), 50 ng of radiolabeled probe, 100 ng of calf thymus DNA, and 3 μg/μl bovine serum albumin. 1–8-μl aliquots of the nuclear extract or 15–120 ng of recombinant PR/GR were added to the binding reaction and incubated for 30 min at room temperature. Specific competition assays were performed by adding 10–500-fold molar excess of unlabeled bcl-X HRE. For the detection of the complexes, the reaction mixture was subjected to electrophoresis for 3 h on 3.5% acrylamide, 20% glycerol, 0.5% agarose, 0.3× Tris borate-EDTA gel. Results were visualized by autoradiography of the dried gel and analyzed using a PhosphorImager (Fuji FLA 3000G) and quantification software (Image Gauge, version 3.1). The monoclonal GR antibody (BuGR, Affinity Bioreagents, Golden, CO) was included in the incubation mixture for supershift experiments. Dimethyl Sulfate Methylation Assay—Methylation was carried out by the addition of 1 μl of 10% dimethyl sulfate to 20 μl of binding buffer (see above) containing 50 ng of end-labeled DNA, 250 ng of poly(dI-dC), 3 μg/μl bovine serum albumin, and 15–75 ng of recombinant PR. After 1 min at room temperature, the reaction was stopped by the addition of 2 μl of 250 mm dithiothreitol. After extraction with phenol-chloroform and ethanol precipitation, the samples were treated with 1 m piperidine during 30 min at 90 °C, dried under vacuum, and analyzed in 6.5% sequencing gels. RNA Analysis—Cells were resuspended in denaturing solution (4 m guanidinium thiocyanate, 25 mm sodium citrate, pH 7, 0.1 m β-mercaptoethanol, and 0.5% sarcosyl), and total RNA was extracted by the single step method (21Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). For reverse transcription, 4 μg of total RNA were used. The first cDNA strand was synthesized with Superscript reverse transcriptase (Invitrogen) 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 and the oligonucleotide 3′-exonD (5′-AAATGAGCTATAACTCAGTTTTTCAA-3′) corresponding to the nucleotides –2551 to –2577 were used as forward and reverse primers, respectively. The reaction yielded a 143-bp length cDNA fragment. For the amplification of the cDNAs generated by the activation of P1 the oligonucleotides, bcl-X P1 (5′-CCTGAAGCTCTCTCTCTCTCTTCA-3′) corresponding to the nucleotides –137 to –114 and exonStop (5′-CCCGTAGAGATCCACAAAAGTGTC-3′), which hybridized with the 5′-region of the second coding exon of bcl-XL or bcl-XS, were used as forward and reverse primers, respectively. The reaction yielded a 730-bp length cDNA fragment. PCR amplification of the cDNAs containing exon B were performed with the oligonucleotides bcl-X P2 (5′-GACTAGTCCAGGTTGTGAGGGGGCAGGTTCCTAAGCTTCGCAATTCCTCT-3′) corresponding to the nucleotides –619 to –577 and exonStop (described above). The reaction yielded a 908- and 726-bp length cDNA fragment corresponding to bcl-XL and bcl-XS isoforms respectively. All of the PCRs were normalized against gapdh expression. Primers gapdh-for (5′-TCATCAACGGGAAGC CCATCACCATCTTC-3′) and gapdh-rev (5′-GTCTTCTGGTTGGCAGTAATGGCATGGACT-3′), which specifically hybridize with gapdh mRNA, were used. The reaction yielded a 357-bp length cDNA fragment. 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 Thermus aquaticus Taq polymerase (Invitrogen), and amplification primers (20 pmol each) in 50 μl of PCR mixture (1× 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), 60 °C (for exonD), 58 °C (for bcl-X P1 and bcl-X P2) for 30 s; and 72 °C for 1 min) and a final extension at 72 °C for 10 min. 10 μl of PCR products were analyzed by electrophoresis in 1.5% agarose gels and visualized under UV light. The negative was scanned, and the density was quantified with ImageQuant software (Molecular Dynamics, Amersham Biosciences). RNase protection analysis was performed as described previously (22Zinn K. DiMaio D. Maniatis T. Cell. 1983; 34: 865-879Abstract Full Text PDF PubMed Scopus (590) Google Scholar). For preparing the bcl-X probe, plasmid pGLD3 was digested with HinfI and transcribed by T3 RNA polymerase. The full-length transcript size of the bcl-X riboprobe was 294 nucleotides, and the protected fragments for bcl-XL and bcl-XS were 237- and 155-bp long, respectively. The gapdh template pTRIGAPDH (Ambion, Austin, TX) was digested with BglII and transcribed with T3 RNA polymerase. The probe length was 359 nucleotides, and the size of the protected fragment was 316 bp. [α-32P]CTP (Amersham Biosciences) radiolabeled RNA probes were prepared using a kit according to the instructions of the manufacturer (Promega). The probes were coprecipitated 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. Quantification was performed with a PhosphorImager (Fuji FLA 3000G) using ImageGauge software. In all of the cases, the quantitation was normalized against gapdh signal. Chromatin Immunoprecipitation (ChIP)—HC11 cells were untreated or incubated for 30 min with 10 nm Dex, and ChIP assays were performed as described previously (23Strutt H. Paro R. Methods Mol. Biol. 1999; 119: 455-467PubMed Google Scholar, 24Eberhardy 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) by using the following monoclonal antibodies, α-GR (BuGR, Affinity Bioreagents, Golden, CO) and α-Pol-II (8WG16, Berkeley Antibody Company). For each experiment, PCRs were performed with different numbers of cycles or with dilution series of input DNA to determine the linear range of the amplification. All of the results shown fall within this range. Primer sequences are available on request. bcl-X P4 Promoter Region Contains HRE-like Sequences—To check the hormone responsiveness of the bcl-X promoters, expression vectors containing the luciferase reporter gene under the control of different promoter regions of the mouse bcl-X gene (Fig. 2A) were cotransfected with GR (Fig. 2B) or PR (Fig. 2C) in COS-1 cells. With a fragment containing all of the five promoters, we observed around a 2-fold induction by the synthetic glucocorticoid, dexamethasone (Fig. 2B, lane 2), or by the progestin agonist R5020 (Fig. 2C, lane 2). Of the five promoter fragments tested, only the one containing an extended P4 region from –3288 to –2652 relative to the translation initiation codon exhibited a robust response to dexamethasone (Fig. 2B, lane 5) and to R5020 (Fig. 2C, lane 5). In all of the cases, the coincubation with the antagonist RU38486 completely abolished the hormone effects (Fig. 2, B and C, lanes 3 and 6). Vectors with P1, P2, or core P4 did not respond either to glucocorticoid or to progestin treatment (Fig. 2, B and C, lanes 8, 14, and 17, respectively). However, whereas treatment with dexamethasone had no effect (Fig. 2B, lane 11) in cells transfected with the P3 vector, treatment with R5020 increased luciferase expression 100-fold molar excess of the mutated form was required to reduce by 50% binding of PR to the labeled cons-HRE (Fig. 6C). These experiments suggest that PR binds to the bcl-X mutHREs with approximately five times less affinity compared with the bcl-X HREs. bcl-X HREs Are Necessary to Confer Steroid Responsiveness to P4 —To test the functional relevance of HRE I and II, we performed transient transfection assays with the extended P4ΔHRE expression vector, which contains a deletion of 95 nucleotides including both putative HREs (Fig. 7A). This construct did not respond to either R5020 (Fig. 7B, compare lane 5 with lane 2) in T47D cells or dexamethasone in HC11 cells (Fig. 7C, compare lane 5 with lane 2). These results suggest that HRE I and HRE II are bona fide functional HREs, which confer hormone responsiveness to P4. P4 Is Activated by Steroid Hormones in Vivo—Transcriptional activation of P4 by hormones in vivo was confirmed by RNase protection assay and reverse transcriptase-PCR in HC11 cells. As previously shown (6Pecci A. Scholz A. Pelster D. Beato M. J. Biol. Chem. 1997; 272: 11791-11798Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), hormonal treatment lead to accumulation of transcripts for both bcl-XL and bcl-XS isoforms but the proportion of bcl-XL mRNA increased more markedly (Fig. 8A, lane 2). No hormonal effect was observed when we analyzed the PCR products obtained from transcripts generated by the proximal promoters P1 or P2 (Fig. 8B, lanes 2 and 5, respectively). However, using specific primers for the amplification of the P4 5′-leading exon, an increase of the product was observed in samples obtained from cells treated with dexamethasone (Fig. 8C, upper panel, lane 2). This hormone effect was abolished by the coincubation with RU38486 (Fig. 8C, upper panel, lane 3). As a control, no change was detected in gapdh RNA levels after hormone treatment (Fig. 8C, lower panel). ChIPs experiments demonstrated that, 30 min after treatment of HC11 cells with dexamethasone, GR was recruited to the P4 promoter region of bcl-X containing the putative HREs (Fig. 9A, upper panel, lane 2). No differences in the binding of GR were observed in the bcl-X proximal promoter region encompassing P1 or P2 (Fig. 9A, middle panel, lane 2). We next tested whether the bcl-X P4 promoter region was transcriptionally active after the hormone treatment. ChIP experiments using a monoclonal RNA-polymerase II (RNApol II) antibody demonstrated a specific recruitment of the enzyme to this region after incubating HC11 cells with dexamethasone (Fig. 9B, upper panel, lane 4). Hormonal treatment did not influence the binding of RNApol II to the bcl-X proximal promoter region (Fig. 9B, middle panel, lane 4). In all of the cases, ChIPs of the gapdh gene were used as control (Fig. 9, A and B, lower panel). Thus, the specific binding of GR to the bcl-X P4 promoter region correlates with an increase in the binding of the RNApol II to this region, suggesting that GR would mediate in vivo activation of P4. The results demonstrate that glucocorticoid treatment induces the transcription of endogenous bcl-X gene, mainly through the activation of P4 in mammary epithelial cells. Among other genes, bcl-X has been postulated to be crucial in hormone-dependent apoptosis. In many cases, the patterns of Bcl-XL or Bcl-XS expression are different from or opposite to those reported for Bcl-2, suggesting that Bcl-XL and Bcl-2 regulate cell survival and death at different stages of cell differentiation through tissue-specific control of their expression (27Krajewski S. Bodrug S. Gascoyne R. Berean K. Krajewska M. Reed J.C. Am. J. Pathol. 1994; 145: 515-525PubMed Google Scholar). In mammary gland, Bcl-XL is the most abundant cell survival member from the Bcl-2 family. However, it has been demonstrated that this protein is not essential during mammopoiesis but is critical for controlled apoptosis during the first phase of involution (28Walton K.D. Wagner K.U. Rucker III, E.B. Shillingford J.M. Miyoshi K. Hennighausen L. Mech. Dev. 2001; 109: 281-293Crossref PubMed Scopus (70) Google Scholar). Although the ratio of bcl-XS/bcl-XL remained stable in the virgin, pregnant, and lactating gland, it increased 6-fold during the first 2 days of involution (29Heermeier K. Benedict M. Li M. Furth P. Nunez G. Hennighausen L. Mech. Dev. 1996; 56: 197-207Crossref PubMed Scopus (120) Google Scholar). To ensure a correct control of apoptosis, the ratio between antiapoptotic and proapoptotic proteins (i.e. Bcl-2/Bax or Bcl-XL/Bcl-XS) must be precisely regulated. Thus, the regulation of cell death requires not only an accurate control of transcription but also control of splicing of bcl-X gene. The complex structure of mouse bcl-X gene with at least four different exons located upstream of the unique open reading frame suggests that promoter choice and alternative splicing may provide tissue-specific response to different stimuli. The experiments summarized in this paper support the notion that steroid hormones modulate bcl-X gene expression through the activation of a distal promoter. We identified two novel HREs immediately upstream of the bcl-X promoter P4 that specifically bind GR and PR in vitro with an affinity only 2-fold lower than a canonical HRE. Deletion of both bcl-X HREs contacted by the hormone receptors in vitro eliminates hormone induction of the P4 reporter gene in transfection assays, supporting a role of the HREs in the in vivo response of the bcl-X gene. Following hormone treatment of HC11 cells, in vivo binding of GR to the bcl-X P4 promoter region was detected. This recruitment of the hormone receptor correlates with an increased binding of RNApol II with accumulation of mRNA transcripts generated from the P4 promoter and with an increase in the ratio of bcl-XL isoform to bcl-XS isoform. These results support the hypothesis that GR mediates in vivo activation of P4 in mouse mammary epithelial cells. Because P4 is the only known bcl-X promoter responding to steroid hormones in this cell line, our results are congruent with the notion that P4 activation by hormones generates mainly the antiapoptotic bcl-XL isoform, although the mechanism of this differential activation is still unknown. After completion of the experimental part of this work, Gascoyne et al. (11Gascoyne D.M. Kypta R.M. Vivanco M.M. J. Biol. Chem. 2003; 278: 18022-18029Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) describe the existence of other HRE-like sequences on the mouse bcl-X gene and show an in vitro interaction of these elements with nuclear extract of dexamethasone-treated fibrosarcoma cells (11Gascoyne D.M. Kypta R.M. Vivanco M.M. J. Biol. Chem. 2003; 278: 18022-18029Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). However, these HREs are different from those we found and their studies do not address selective promoter usage controlled by glucocorticoids. The ChIP assays performed in our study did not discriminate whether GR binds in vivo to the HREs described here or to those described by Gascoyne et al. (11Gascoyne D.M. Kypta R.M. Vivanco M.M. J. Biol. Chem. 2003; 278: 18022-18029Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). However, according to our transient transfection assays, only HRE I and HRE II, as described here, confer steroid responsiveness to P4 in mammary epithelial cells. In fact, the vector P4ΔHRE, which contains two of the HRE-like sequences described by Gascoyne et al. (11Gascoyne D.M. Kypta R.M. Vivanco M.M. J. Biol. Chem. 2003; 278: 18022-18029Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), did not respond to dexamethasone or to the progestagen R5020 in HC11 and T47D cells, respectively. It is possible that the cell context determines GR or PR binding to different HREs in the proximity of P4. As suggested in a previous study (30Nordeen S.K. Suh B.J. Kuhnel B. Hutchison C.D. Mol. Endocrinol. 1990; 4: 1866-1873Crossref PubMed Scopus (169) Google Scholar), the cooperativity among several HRE sequences may contribute to the inducibility of the promoter. Thus, the degree of occupancy of the different HREs may mediate recruitment of different tissue-specific or ubiquitous factors, which consequently determine hormone-dependent bcl-X expression. In this sense, the presence of several HREs within a short DNA region may provide the basis for specifying context-dependent hormonal regulation of programmed cell death. We thank Bernhard Gross for the preparation of recombinant PR, Dr. Damian G. Romero for P3 vector construction, and Dr. Nancy Hynes for HC11 cells.
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