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The Glucocorticoid Receptor Is Tethered to DNA-bound Oct-1 at the Mouse Gonadotropin-releasing Hormone Distal Negative Glucocorticoid Response Element

1999; Elsevier BV; Volume: 274; Issue: 4 Linguagem: Inglês

10.1074/jbc.274.4.2372

1083-351X

Uma Chandran, Barbour S. Warren, Christopher T. Baumann, Gordon L. Hager, Donald Defranco,

Hypothalamic control of reproductive hormones

An element required for glucocorticoid repression of mouse gonadotropin-releasing hormone (GnRH) gene transcription, the distal negative glucocorticoid response element (nGRE), is not bound directly by glucocorticoid receptors (GRs) but is recognized by Oct-1 present in GT1–7 cell nuclear extracts or by Oct-1 purified from HeLa cells. Furthermore, purified full-length GRs interact directly with purified Oct-1 bound to the distal nGRE. Increasing the extent of distal nGRE match to an Oct-1 consensus site not only increases the affinity of Oct-1 binding, but also alters the conformation of DNA-bound Oct-1 and the pattern of protein DNA complexes formedin vitro with GT1–7 cell nuclear extracts. In addition, the interaction of purified GR with DNA-bound Oct-1 is altered when Oct-1 is bound to the consensus Oct-1 site. Mutation of the distal nGRE to a consensus Oct-1 site is also associated with reduced glucocorticoid repression in transfected GT1–7 cells. Furthermore, repression of GnRH gene transcription by 12-O-tetradecanoylphorbol-13-acetate, which utilizes sequences that overlap with the nGRE, is reversed by this distal nGRE mutation leading to activation of GnRH gene transcription. Thus, changes in the assembly of multi-protein complexes at the distal nGRE can influence the regulation of GnRH gene transcription. An element required for glucocorticoid repression of mouse gonadotropin-releasing hormone (GnRH) gene transcription, the distal negative glucocorticoid response element (nGRE), is not bound directly by glucocorticoid receptors (GRs) but is recognized by Oct-1 present in GT1–7 cell nuclear extracts or by Oct-1 purified from HeLa cells. Furthermore, purified full-length GRs interact directly with purified Oct-1 bound to the distal nGRE. Increasing the extent of distal nGRE match to an Oct-1 consensus site not only increases the affinity of Oct-1 binding, but also alters the conformation of DNA-bound Oct-1 and the pattern of protein DNA complexes formedin vitro with GT1–7 cell nuclear extracts. In addition, the interaction of purified GR with DNA-bound Oct-1 is altered when Oct-1 is bound to the consensus Oct-1 site. Mutation of the distal nGRE to a consensus Oct-1 site is also associated with reduced glucocorticoid repression in transfected GT1–7 cells. Furthermore, repression of GnRH gene transcription by 12-O-tetradecanoylphorbol-13-acetate, which utilizes sequences that overlap with the nGRE, is reversed by this distal nGRE mutation leading to activation of GnRH gene transcription. Thus, changes in the assembly of multi-protein complexes at the distal nGRE can influence the regulation of GnRH gene transcription. gonadotropin-releasing hormone glucocorticoid receptor negative glucocorticoid response element, EMSA, electrophoretic mobility shift assay 12-O-tetradecanoylphorbol-13-acetate Tris borate, EDTA bovine serum albumin mouse mammary tumor virus long terminal repeat Pit-1, Oct-1,unc-86. Gonadotropin-releasing hormone (GnRH)1 is secreted by neurons in the hypothalamus and is at the top of the endocrine axis that controls reproductive function. In recent years, molecular studies of GnRH gene regulation have been facilitated by the development of the immortalized GnRH-secreting GT1 cell lines (1Mellon P.L. Windle J.J. Goldsmith P.C. Padula P.C. Roberts J.L. Weiner R.I. Neuron. 1990; 5: 1-10Abstract Full Text PDF PubMed Scopus (898) Google Scholar). In these cell lines, GnRH expression is regulated by various neurotransmitters (2Martinez de la Escalera G. 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Glucocorticoids have been implicated in physiological regulation of GnRH, as stress-related reproductive disorders and high cortisol levels in women have been associated with reductions in circulating luteinizing hormone (15Berga S.L. Mortola J.F. Girton L. Suh B. Laughlin G. Pham P. Yen S.S.C. J. Clin. Endocrinol. Metab. 1989; 68: 301-308Crossref PubMed Scopus (282) Google Scholar, 16Reame N.E. Sander S.E. Case G.D. Kelch R.P. Marshall J.C. J. Clin. Endocrinol. Metab. 1985; 61: 301-308Crossref Scopus (164) Google Scholar, 17Suh B.Y. Liu J.H. Berg S.L. Quigley M.E. Laughlin G.A. Yen S.S.C. J. Clin. Endocrinol. Metab. 1988; 66: 733-739Crossref PubMed Scopus (137) Google Scholar). Glucocorticoids can directly suppress gonadotropin secretion from the pituitary (18Padmanabhan V. Keech C. Convey E.M. Endocrinology. 1983; 112: 1782-1787Crossref PubMed Scopus (93) Google Scholar), but the possibility that they also act at a hypothalamic level, i.e.directly on GnRH neurons, has been suggested in a few physiological studies (19Dubey A.K. Plant T.M. Biol. Reprod. 1985; 33: 423-431Crossref PubMed Scopus (184) Google Scholar, 20Brann D.W. Mahesh V.B. Biol. Reprod. 1991; 44: 1005-1015Crossref PubMed Scopus (33) Google Scholar). The detection of glucocorticoid receptors (GRs), a member of the steroid/thyroid hormone family of nuclear receptors (21Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6324) Google Scholar, 22Green S. Chambon P. Trends Genet. 1988; 4: 309-312Abstract Full Text PDF PubMed Scopus (831) Google Scholar, 23Beato M. Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2850) Google Scholar), within a subset of GnRH neurons (24Ahima R.S. Harlan R.E. Neuroendocrinology. 1992; 56: 845-850Crossref PubMed Scopus (78) Google Scholar), provides additional support for the notion that glucocorticoids exert direct effects on GnRH gene expression. The GT1–7 cell line also contains functional GRs that, in the presence of glucocorticoid agonist dexamethasone, repress GnRH promoter activity (7Chandran U.R. Attardi B. Friedman R. Dong K.W. Roberts J.L. DeFranco D.B. Endocrinology. 1994; 134: 1467-1474Crossref PubMed Scopus (75) Google Scholar). Two regions of the mouse GnRH promoter, the distal and proximal glucocorticoid response elements (nGREs), mediate glucocorticoid repression of transcription (25Chandran U.R. Attardi B. Friedman R. Zheng Z. Roberts J.L. DeFranco D.B. J. Biol. Chem. 1996; 271: 20412-20420Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). GRs do not bind directly to either of these nGREs, but at the distal nGRE they are part of a multi-protein complex that also includes Oct-1 (25Chandran U.R. Attardi B. Friedman R. Zheng Z. Roberts J.L. DeFranco D.B. J. Biol. Chem. 1996; 271: 20412-20420Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), a member of the POU domain family of transcription factors (26Sturm R. Baumruker T. Franza B.R. Herr W. Genes Dev. 1987; 1: 1147-1160Crossref PubMed Scopus (132) Google Scholar, 27Herr W. Cleary M.A. Genes Dev. 1995; 9: 1679-1693Crossref PubMed Scopus (346) Google Scholar, 28Wegner M. Drolet D.W. Rosenfeld M.G. Curr. Opin. Cell Biol. 1993; 5: 488-498Crossref PubMed Scopus (233) Google Scholar, 29Kutoh E. Strömstedt P.-E. Poellinger L. Mol. Cell. Biol. 1992; 12: 4960-4969Crossref PubMed Scopus (89) Google Scholar, 30Ryan A.K. Rosenfeld M.G. Genes Dev. 1997; 11: 1207-1225Crossref PubMed Scopus (444) Google Scholar). The fact that GR and Oct-1 co-occupy the distal nGRE (25Chandran U.R. Attardi B. Friedman R. Zheng Z. Roberts J.L. DeFranco D.B. J. Biol. Chem. 1996; 271: 20412-20420Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) of the mouse GnRH promoter suggests that functionally relevant interactions between GR and Oct-1 may not be limited to solution as observed in other studies. We therefore have examined whether GR interacts directly with DNA-bound Oct-1. We show here that purified GR indeed can associate with Oct-1 bound to the GnRH distal nGRE. This provides the first evidence of glucocorticoid repression of transcription that is mediated by the tethering of GR to a DNA-bound transcription factor. We furthermore show that the nature of Oct-1 interactions at the distal nGRE influences transcriptional repression, not only by glucocorticoids, but also by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Thus, alternative conformational states of Oct-1, which are dictated by precise DNA contacts made at different binding sites, may influence the assembly of multi-protein complexes that impart unique regulatory properties upon linked promoters. DNA fragments containing 3446 base pairs of the mouse GnRH promoter (25Chandran U.R. Attardi B. Friedman R. Zheng Z. Roberts J.L. DeFranco D.B. J. Biol. Chem. 1996; 271: 20412-20420Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) were mutagenized to create a consensus Oct-1 binding site at the distal nGRE using the protocol supplied in the in vitro mutagenesis kit (CLONTECH). The primer used for the mutagenesis, 5′-GCTAAGATTTGCATGACCAGG-3′, contained nucleotide changes at base pairs −206 and −207 of the mouse GnRH promoter, to generate a consensus Oct-1 binding site at the mouse GnRH distal nGRE. The GnRH nGRE mutation was confirmed by dideoxy sequencing (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). The mouse GnRH promoter containing either the wild type GnRH distal nGRE or the consensus Oct-1 binding site linked to the luciferase reporter gene (25Chandran U.R. Attardi B. Friedman R. Zheng Z. Roberts J.L. DeFranco D.B. J. Biol. Chem. 1996; 271: 20412-20420Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) was digested with HindIII andBstXI to generate two fragments, one from base pairs −3446 to −471, and the other containing the GnRH promoter up to base pair −471 linked to the luciferase reporter gene. The GnRH promoter deletion to base pair −471 was then blunt-ended and ligated to generate the −471mGnRH-Luc. GT1–7 cells were grown as described previously (25Chandran U.R. Attardi B. Friedman R. Zheng Z. Roberts J.L. DeFranco D.B. J. Biol. Chem. 1996; 271: 20412-20420Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). 5 μg of plasmid DNA along with 5 μg of herring sperm DNA carrier were transfected into GT1–7 cells on 60-mm dishes using the calcium phosphate precipitation method as described previously (25Chandran U.R. Attardi B. Friedman R. Zheng Z. Roberts J.L. DeFranco D.B. J. Biol. Chem. 1996; 271: 20412-20420Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The precipitate was allowed to sit on the cells for 12–16 h, after which the cells were washed with Tris-buffered saline and refed with fresh medium. The cells were then treated with either 10−6m dexamethasone or 100 ng/ml TPA (Sigma) where indicated. Following a 24-h incubation, cells were harvested and assayed for luciferase activity (Luciferase kit, Promega, Madison, WI) using the same amount of total protein from each of the plates. The mean luciferase activity for each construct was determined from at least five independent experiments. The luciferase activity between controls and after dexamethasone or TPA treatment was compared using the Wilcoxon signed rank test. The percent change in luciferase activity generated by wild type and mutant reporters was compared using the Mann-Whitney rank-sum test. Either commercially available BuGR2 monoclonal antibody or clone 57 polyclonal antibody (Affinity BioReagents, Neshanic Station, NJ) was used to detect GR in protein-DNA complexes. The antibody against Oct-1 was obtained from Santa Cruz Biologicals (Santa Cruz, CA). The sequences of L7, L73M, and L7M oligonucleotides are shown in Fig. 1. Single-stranded oligonucleotides were 32P-labeled with polynucleotide kinase (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar) and then hybridized to their complementary strands to generate double-stranded probes. Nuclear extracts from GT1–7 cells were prepared as described previously (25Chandran U.R. Attardi B. Friedman R. Zheng Z. Roberts J.L. DeFranco D.B. J. Biol. Chem. 1996; 271: 20412-20420Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). 0.05 μm32P-labeled double-stranded oligonucleotide (L7 or L7M) was incubated with 1× gel shift buffer (100 mm Tris-HCl, pH 8.0, 50% glycerol, 10 mmEDTA, 10 mm dithiothreitol) and 1 μg/ml poly(dI-dC) in a 15-μl reaction volume with 5 μg of GT1–7 cell nuclear extract for 15 min at room temperature. To resolve protein-DNA complexes, the reaction mix was run on native 10% polyacrylamide (75:1) gels in 1× TBE (Tris borate, EDTA) buffer. Gels were electrophoresed in 1× TBE at 180 V for approximately 30 min. For competition assays, unlabeled double-stranded competitor DNA was incubated with GT1–7 nuclear extract for 10 min prior to the addition of radiolabeled probe. For supershift assays with BuGR2 monoclonal GR antibody, 1 μl of BuGR2 was first incubated with GT1–7 extract for 10 min at room temperature, after which the radiolabeled probe was added. The incubation was then continued for another 15 min. For supershift assays with the Oct-1 polyclonal antibody, probe and nuclear extract were incubated for 15 min at room temperature, after which the antibody was added. The incubation at room temperature was continued for an additional 30 min. For EMSAs with purified Oct-1, 0.05 μm32P-labeled double-stranded oligonucleotide was incubated with 1× Oct-1 gel shift buffer (4% glycerol, 20 mmHepes-KOH 7.9, 60 mm KCl, 2 mm dithiothreitol, and 1 mg/ml BSA), 1 μg of poly(dI-dC), 1 μl of 10 mg/ml BSA (New England Biolabs, Beverly, MA). and 2 μl of purified Oct-1 for 15 min at room temperature. For supershift assays. 2 μl of Oct-1 antibody was added and the incubation continued for an additional 15 min. Protein-DNA complexes were resolved as described above. For EMSAs with purified Oct-1 and purified rat GR (32Warren B.S. Kusk P. Wolford R.G. Hager G.L. J. Biol. Chem. 1996; 271: 11434-11440Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), 0.05 μm32P-labeled double-stranded oligonucleotide was incubated in 1× Oct-1 gel shift buffer (see above), 1 μg of poly(dI-dC), 1 μl of 10 mg/ml BSA, 2 μl of purified Oct-1, and 0.5 μl of purified GR. The reaction was incubated for 15 min at room temperature and then 5 min on ice. Protein-DNA complexes were resolved as described above. 0.05 μm32P-labeled double-stranded oligonucleotide (L7 or L7M) was incubated with 1× Oct-1 gel shift buffer in a 15 μl reaction volume with 5 μg of GT1–7 cell nuclear extract for 15 min at room temperature. Sequencing grade trypsin (Promega, Madison, WI) was then added at increasing concentrations (1.25–5 ng/ml) and the reaction incubated for another 10 min on ice. To resolve protein-DNA complexes, the reaction mix was run on native 10% polyacrylamide (75:1) gels in 1× TBE buffer. Gels were electrophoresed in 1× TBE at 180 V for approximately 30 min. Oct-1 was either purified from HeLa cells using established methods (33Murphy S. Pierani A. Scheidereit C. Melli M. Roeder R.G. Cell. 1989; 59: 1071-1080Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 34Jackson S.P. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1781-1785Crossref PubMed Scopus (141) Google Scholar) or from aDrosophila cell (S2) expression system (Invitrogen Corp., Carlsbad, CA). In the latter case, FLAG epitope-tagged full-length human Oct-1 was purified as described (35Chiang C.-M. Roeder R.G. Peptide Res. 1993; 6: 62-64PubMed Google Scholar). Briefly, cells were lysed by sonication, and the supernatant from a 11000 × gcentrifugation incubated with anti-FLAG M2 affinity gel (Sigma) at 4 °C overnight. The M2 agarose was washed five times and the bound protein eluted by incubation with the FLAG peptide (Sigma) for 1 h at 4 °C. The eluate was concentrated on a Centricon-30 centrifugal concentrator (Amicon Inc., Beverly, MA), and aliquots were frozen at −140 °C. Previous results from our laboratory showed that Oct-1 present in GT1–7 cell nuclear extracts binds to the mouse GnRH distal nGRE in vitro (25Chandran U.R. Attardi B. Friedman R. Zheng Z. Roberts J.L. DeFranco D.B. J. Biol. Chem. 1996; 271: 20412-20420Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). As the Oct-1-containing complex formed on this nGRE appeared to contain multiple proteins, we wished to determine whether Oct-1 binding to this site required GT1–7 cell-specific nuclear factors. Oct-1 was purified from HeLa cell nuclear extract by a combination of wheat germ agglutinin chromatography and DNA affinity chromatography as described previously (33Murphy S. Pierani A. Scheidereit C. Melli M. Roeder R.G. Cell. 1989; 59: 1071-1080Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 34Jackson S.P. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1781-1785Crossref PubMed Scopus (141) Google Scholar). The purity of the Oct-1 preparation was confirmed by silver staining, which showed a single protein band (data not shown). The DNA binding activity of the purified Oct-1 protein was tested using a consensus Oct-1 binding site (29Kutoh E. Strömstedt P.-E. Poellinger L. Mol. Cell. Biol. 1992; 12: 4960-4969Crossref PubMed Scopus (89) Google Scholar) oligonucleotide (i.e. L73M) that was derived from the GnRH distal nGRE (Fig.1). The 23-base pair L73M and 55-base pair L7M oligonucleotides (hereafter designated “consensus Oct-1 site”) contain two nucleotide substitutions at the Oct-1 homology sequence in the GnRH distal nGRE. As shown in Fig.2 A (lane 2), purified Oct-1 binds strongly to the consensus Oct-1 site. Oct-1 binding to this sequence is not competed effectively by the distal GnRH nGRE (Fig. 2 A, lane 3), suggesting that Oct-1 binds to the consensus Oct-1 site more strongly than to the GnRH distal nGRE. Oct-1 binding to the consensus site is competed by excess cold consensus site oligonucleotide (data not shown) and is completely inhibited by the inclusion of an Oct-1 antibody in the binding reaction (Fig. 2 A, lane 4). The inability of the GnRH distal nGRE to compete with the consensus Oct-1 site for Oct-1 binding was not surprising, given the limited homology of the Oct-1 sequence in this nGRE to a consensus Oct-1 site (Fig. 1). Specifically, the Oct-1 binding site in this element has only a 5/8 match on the bottom strand and 6/8 match on the top strand to a consensus Oct-1 binding site. Nonetheless, as shown in Fig.2 B (lane 2), purified Oct-1 also binds the distal GnRH nGRE oligonucleotide L7, although with lower affinity than to the nGRE mutant containing a consensus Oct-1 site. A 250-fold molar excess of the consensus Oct-1 site effectively competes for binding with the distal nGRE (Fig. 2 B, lane 3). As shown previously, a 250-fold molar excess of the distal nGRE is not able to compete for binding with the consensus Oct-1 site (Fig. 2 A, lane 3). The fact that the protein-DNA complex formed on the distal nGRE contains Oct-1 and not a contaminant in our Oct-1 preparation was confirmed by the ability of an Oct-1 antibody to prevent the formation of an Oct-1·nGRE complex (Fig. 2 B, lane 4). Thus, Oct-1 has the capacity to interact directly with the GnRH distal nGREin vitro, i.e. in the absence of other GT1–7 nuclear proteins. The binding of purified Oct-1 to the GnRH distal nGRE generates a single protein-DNA complex, which is indistinguishable in its electrophoretic mobility from the complex formed on the consensus Oct-1 site. When crude GT1–7 cell nuclear extract was used, multiple distinct protein-DNA complexes formed on the distal nGRE (Fig.3 A, lane 3). Previously, we showed that one multi-protein DNA complex formed on this nGRE (i.e. complex C1-L7) contains Oct-1 and GR (25Chandran U.R. Attardi B. Friedman R. Zheng Z. Roberts J.L. DeFranco D.B. J. Biol. Chem. 1996; 271: 20412-20420Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Interestingly, when the consensus Oct-1 site oligonucleotide was incubated with GT1–7 cell nuclear extract, only a single protein-DNA complex was formed (Fig. 3 A, lane 4). As expected from our previous studies with purified Oct-1 (see Fig. 2), the distal nGRE did not effectively compete with the consensus Oct-1 containing oligonucleotide for binding of Oct-1 present in the GT1–7 cell nuclear extract (data not shown). A comparison of the competitive strength of the wild type nGREversus the consensus Oct-1 sequence for GT1–7 nuclear extract binding revealed that the apparent affinity of GT1–7 nuclear extract for the consensus Oct-1 sequence is approximately 10-fold higher than for the GnRH distal nGRE (data not shown). Furthermore, the Oct-1 containing C1-L7M protein-DNA complex, formed on the consensus Oct-1 site had a slightly increased electrophoretic mobility as compared with the Oct-1-containing complex, C1-L7, formed on the distal nGRE (Fig. 3 A, compare lanes 4 and3). This apparent migration difference, although small, was noted in multiple EMSAs performed with these oligonucleotides using different GT1–7 cell nuclear extract preparations (data not shown). These large complexes migrate slowly through the 10% polyacrylamide gels and are difficult to resolve. However, we were able to resolve the distal nGRE-protein complex, C1-L7, and the consensus Oct-1 site-protein complex, C1-L7M, on a lower percentage gel (Fig. 3 B, lanes 1 and 2). As will be shown below, this differential migration most likely reflects the fact that GR is present within the multi-protein C1-L7 complex, but not the C1-L7M complex (see Fig. 3 C). In addition to Oct-1, one multi-protein complex formed on the distal nGRE when using GT1–7 cell nuclear extracts also includes GR (25Chandran U.R. Attardi B. Friedman R. Zheng Z. Roberts J.L. DeFranco D.B. J. Biol. Chem. 1996; 271: 20412-20420Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). To determine whether GR is also present in the complex formed at the mutant nGRE possessing a consensus Oct-1 binding site, we added the BuGR2 anti-GR antibody to a binding reaction containing the mutant nGRE and GT1–7 cell nuclear extract. As shown in Fig. 3 C(lanes 1 and 2), BuGR2 does not supershift the complex formed on the consensus Oct-1 binding site. Thus, increasing the affinity of Oct-1 binding site by mutation of the GnRH distal nGRE alters the recruitment of GR into a multi-protein complex formed on this site with GT1–7 cell nuclear proteins. GRs contained within a multi-protein complex on the distal nGRE do not bind DNA directly at this site (25Chandran U.R. Attardi B. Friedman R. Zheng Z. Roberts J.L. DeFranco D.B. J. Biol. Chem. 1996; 271: 20412-20420Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The only GT1–7 cell nuclear protein that we have established is directly bound to the distal nGRE is Oct-1 (25Chandran U.R. Attardi B. Friedman R. Zheng Z. Roberts J.L. DeFranco D.B. J. Biol. Chem. 1996; 271: 20412-20420Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Does GR bind Oct-1 in this complex and does GR distinguish between Oct-1 bound at either a consensus site Oct-1 site or the distal nGRE? GR and Oct-1 have been shown to interact in vitro in the absence of DNA. In fact, it has been postulated that repression of histone H2b promoter activity by glucocorticoids is brought about by the sequestration of Oct-1 by GR in solution (29Kutoh E. Strömstedt P.-E. Poellinger L. Mol. Cell. Biol. 1992; 12: 4960-4969Crossref PubMed Scopus (89) Google Scholar). Furthermore, although GR and Oct-1 can bind simultaneously in vitro to two distinct sites on the mouse mammary tumor virus long terminal repeat (MMTV LTR; Ref. 23Beato M. Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2850) Google Scholar), GR and Oct-1 have never definitively been shown to interact with each other while DNA-bound. In order to reveal whether GR and Oct-1 can co-occupy the GnRH distal nGRE in vitro, we performed EMSAs with this nGRE using purified preparations of rat GR (32Warren B.S. Kusk P. Wolford R.G. Hager G.L. J. Biol. Chem. 1996; 271: 11434-11440Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) and Oct-1. As shown in Fig.4 (lanes 1 and2), an Oct-1·nGRE complex (i.e. complex C1-L7) formed in vitro was supershifted (i.e. complex S1-L7) by the addition of purified GR to the binding reaction. The supershift occurs only when purified GR is added to Oct-1 and not when another protein fraction from the GR purification procedure is incubated with Oct-1 (data not shown). We also tested the ability of purified GR and Oct-1 to bind to the mutant nGRE possessing a consensus Oct-1 binding site. The binding of Oct-1 to the consensus Oct-1 binding site oligonucleotide (complex C1-L7M) is also supershifted by the addition of GR (Fig. 4, lanes 3 and4), but this supershift (complex S1-L7M) is much less pronounced than the GR supershift of the Oct-1·wild type nGRE complex (Fig. 4, compare lanes 2 and 4). Therefore, GR appears to interact differently with Oct-1 depending upon the context of the Oct-1 binding site. It is surprising that purified GR interacts with Oct-1 at the consensus site since GR was not part of the protein complex formed at the consensus Oct-1 binding site in EMSAs with GT1–7 nuclear extract (see Fig. 3 C). This may be due to differences in the relative ratios of GR and Oct-1 in EMSAs with purified proteins versus GT1–7 nuclear extract. Thus, GR can interact with DNA-bound Oct-1 in vitro, particularly at the wild type GnRH distal nGRE. Altering the sequence of the Oct-1 recognition site within the nGRE may affect GR interactions with DNA-bound Oct-1, but additional characterization of these ternary Oct-1·GR·DNA complexes is required to fully address this issue. POU domain proteins such as Oct-1 exhibit some flexibility in their interaction with target DNA sites (36Huang C.C. Herr W. Mol. Cell. Biol. 1996; 16: 2967-2976Crossref PubMed Google Scholar, 37Misra V. Walker S. Yang P. Hayes S. O'Hare P. Mol. Cell. Biol. 1996; 16: 4404-4413Crossref PubMed Scopus (39) Google Scholar). Such diversity in sequence recognition allows for the recruitment by Oct-1 of unique transcriptional coactivators (37Misra V. Walker S. Yang P. Hayes S. O'Hare P. Mol. Cell. Biol. 1996; 16: 4404-4413Crossref PubMed Scopus (39) Google Scholar). This selectivity is dictated, in part, by the ability of Oct-1 to adopt different conformations at different binding sites (37Misra V. Walker S. Yang P. Hayes S. O'Hare P. Mol. Cell. Biol. 1996; 16: 4404-4413Crossref PubMed Scopus (39) Google Scholar). Since GR interactions with Oct-1 in vitro were altered upon conversion of the distal GnRH nGRE to a consensus Oct-1 site, we set out to examine whether Oct-1 conformation was indeed distinguishable at these two sites. Purified Oct-1 bound to either an nGRE or a consensus Oct-1 site was subjected to a limited trypsin digestion (38Kusk P. John S. Fragoso G. Michelotti J. Hager G.L. J. Biol. Chem. 1996; 271: 31269-31276Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 39Wood J.R. Greene G.L. Nardulli A.M. Mol. Cell. Biol. 1998; 18: 1927-1934Crossref PubMed Scopus (108) Google Scholar) and resulting protein-DNA complexes examined by EMSAs. As shown in Fig.5 A, treatment with increasing amounts of trypsin resulted in the appearance of trypsin-resistant fragments on both the L7 and L7M probes. However, Oct-1 bound at these different DNA elements exhibited a differential sensitivity to trypsin. Complexes labeled 1 and 2 represent intact Oct-1 bound to the L7 and L7M probes (Fig. 5 A, lanes 2 and 8). When bound to the consensus Oct-1 site, predominant trypsin-resistant Oct-1-DNA complexes, i.e.designated a–e (Fig. 5 A, lanes 9–12) were observed, while at the nGRE, a different set of trypsin-resistant Oct-1·DNA complexes, i.e. Aand B (Fig. 5 A, lanes 5 and6), were noted. Complexes A and B on the distal nGRE exhibit different electrophoretic mobilities than complexes d and e on the consensus Oct-1 oligonucleotide. The difference in mobility is particularly evident when lanes 5 and 11 are placed next to each other (Fig. 5 B, lanes 1 and2). The GnRH distal nGRE contributes to glucocorticoid repression of GnRH promoter activity (25