The Role of the C-terminal Extension (CTE) of the Estrogen Receptor α and β DNA Binding Domain in DNA Binding and Interaction with HMGB
2004; Elsevier BV; Volume: 279; Issue: 15 Linguagem: Inglês
10.1074/jbc.m313335200
ISSN1083-351X
AutoresVida Senkus Melvin, J. Chuck Harrell, James S. Adelman, W. Lee Kraus, Mair E. A. Churchill, Dean P. Edwards,
Tópico(s)NF-κB Signaling Pathways
ResumoHMGB-1/-2 are coregulatory proteins that facilitate the DNA binding and transcriptional activity of steroid receptor members of the nuclear receptor family of transcription factors. We investigated the influence and mechanism of action of HMGB-1/-2 (formerly known as HMG-1/-2) on estrogen receptor α (ERα) and ERβ. Both ER subtypes were responsive to HMGB-1/-2 with respect to enhancement of receptor DNA binding affinity and transcriptional activity in cells. Responsiveness to HMGB-1/-2 was dependent on the C-terminal extension (CTE) region of the ER DNA binding domain (DBD) and correlated with a direct protein interaction between HMGB-1/-2 and the CTE. Thus the previously reported higher DNA binding affinity and transcription activity of ERα as compared with ERβ is not due to a lack of ERβ interaction with HMGB-1/-2. Using chimeric receptor DBDs, the higher intrinsic DNA binding affinity of ERα than ERβ was shown to be due to a unique property of the ERα CTE, independent of HMGB-1/-2. The CTE of both ER subtypes was also shown to be required for interaction with ERE half-sites. These studies reveal the importance of the CTE and HMGB-1/-2 for ERα and ERβ interaction with their cognate target DNAs. HMGB-1/-2 are coregulatory proteins that facilitate the DNA binding and transcriptional activity of steroid receptor members of the nuclear receptor family of transcription factors. We investigated the influence and mechanism of action of HMGB-1/-2 (formerly known as HMG-1/-2) on estrogen receptor α (ERα) and ERβ. Both ER subtypes were responsive to HMGB-1/-2 with respect to enhancement of receptor DNA binding affinity and transcriptional activity in cells. Responsiveness to HMGB-1/-2 was dependent on the C-terminal extension (CTE) region of the ER DNA binding domain (DBD) and correlated with a direct protein interaction between HMGB-1/-2 and the CTE. Thus the previously reported higher DNA binding affinity and transcription activity of ERα as compared with ERβ is not due to a lack of ERβ interaction with HMGB-1/-2. Using chimeric receptor DBDs, the higher intrinsic DNA binding affinity of ERα than ERβ was shown to be due to a unique property of the ERα CTE, independent of HMGB-1/-2. The CTE of both ER subtypes was also shown to be required for interaction with ERE half-sites. These studies reveal the importance of the CTE and HMGB-1/-2 for ERα and ERβ interaction with their cognate target DNAs. Nuclear hormone receptors comprise a superfamily of ligand-dependent transcription factors that regulate gene expression through interaction with specific hormone response elements (HREs) 1The abbreviations used are: HRE, hormone response element; GST, glutathione S-transferase; DTT, dithiothreitol; DBD, DNA binding domain; ER, estrogen receptor; CTE, C-terminal extension; EMSA, electrophoretic mobility shift assay. 1The abbreviations used are: HRE, hormone response element; GST, glutathione S-transferase; DTT, dithiothreitol; DBD, DNA binding domain; ER, estrogen receptor; CTE, C-terminal extension; EMSA, electrophoretic mobility shift assay. in target genes. The superfamily can be subdivided into: 1) classical steroid hormone receptors that typically interact with palindromic hexameric HREs as homodimers, 2) nonsteroidal or class II nuclear receptors for ligands such as thyroid hormone, retinoic acid, vitamin D, and fatty acids, that function primarily as heterodimers with RXR (retinoid X receptor) bound to direct repeat HREs, and 3) orphan receptors without known ligands that interact with HREs in various dimer and monomer configurations. The nuclear receptors are related through a common domain structure including conserved C-terminal ligand binding (LBD) and centrally located DNA binding domains (DBD), and a variable N-terminal domain that is required in many nuclear receptors for maximal transcription activity (Refs. 1Beato M. Klug J. Hum. Reprod. Update. 2000; 6: 225-236Crossref PubMed Scopus (481) Google Scholar and 2Mangelsdorf D. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6062) Google Scholar, reviews). The DBD consists of a highly conserved core with two asymmetric zinc fingers and an ∼30 amino acid segment, termed the C-terminal extension (CTE) (Fig. 1A). Within the core DBD, α-helix 1 extends between the two zinc fingers and makes base specific contacts in the major groove of the HRE DNA. The second α-helix (helix 2) does not contact DNA but is important for the overall folding of the core DBD (3Luisi B.F. Xu W.X. Otwinowski L.P. Freedman L.P. Yamamoto K.R. Sigler P.B. Nature. 1991; 352: 497-505Crossref PubMed Scopus (1228) Google Scholar, 4Schwabe J.W.R. Chapman L. Finch J.T. Rhodes D. Cell. 1993; 74: 57-578Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 5Zilliacus J. Wright A.P.H. Carlstedt-Duke J. Gustaffson J.-Å. Mol. Endocrinol. 1995; 9: 389-400Crossref PubMed Google Scholar). The CTE is not conserved and adopts different structural motifs dependent on the class of nuclear receptor (6Khorasanizadeh S. Rastinejad F. Trends Biochem. Sci. 2001; 26: 384-390Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 7Senkus Melvin V. Edwards D.P. Steroids. 1999; 64: 576-586Crossref PubMed Scopus (70) Google Scholar). Nonetheless, the CTE of different receptors does appear to share a functional role to stabilize the receptor-DNA complex by extending the protein-DNA interface beyond that of base-specific contacts made by the core DBD. The CTE of class II receptors (TR and VDR) forms an α-helix (helix 3) that projects across the minor groove between HRE half-sites, making extensive contacts along the phosphate backbone required for high affinity DNA binding and correct spacing with the RXR heterodimer (8Rastinejad F. Perlman T. Evans R. Sigler P. Nature. 1995; 375: 203-211Crossref PubMed Scopus (469) Google Scholar, 9Shaffer P.L. Gewirth D.T. EMBO J. 2002; 21: 2242-2252Crossref PubMed Scopus (107) Google Scholar). The CTE of orphan receptors forms an extended loop conformation that makes base-specific contacts in the minor groove immediately 5′ of the HRE. A short peptide motif termed a “GRIP-box” (RXGRZP where X is any amino acid and Z is a hydrophobic residue) mediates interaction of orphan receptor CTEs with the minor groove (10Zhao Q. Khorasanizadeh S. Miyoshi Y. Lazar M.A. Rastinejad F. Mol. Cell. 1998; 1: 849-861Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 11Gearhart M. Holmbeck S.M.A. Evans R.M. Dyson H.J. Wright P.E. J. Mol. Biol. 2003; 327: 819-832Crossref PubMed Scopus (75) Google Scholar). The CTE is also required for monomeric orphan receptor recognition of extended HRE half-sites through interaction with specific tri-nucleotide sequences in the minor groove just 5′ of the HRE (12Wilson T.E. Farner T.J. Milbrandt J. Mol. Cell. Biol. 1993; 13: 5794-5804Crossref PubMed Scopus (356) Google Scholar, 13Wilson T.E. Paulsen R.E. Padgett K.A. Milbrandt J. Science. 1992; 256: 107-110Crossref PubMed Scopus (279) Google Scholar). Although no structural information is available as yet, biochemical evidence indicates the CTE of steroid receptors also has a role in mediating high affinity DNA binding, by interacting with high mobility group proteins, HMGB-1 and HMGB-2, that function to facilitate receptor binding to cognate target DNA sites (14Senkus Melvin V. Roemer S.C. Churchill M.E.A. Edwards D.P. J. Biol. Chem. 2002; 277: 25115-25124Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). HMGB-1 and closely related HMGB-2 (formerly known as HMG-1 and -2) are members of a family of proteins that bind to duplex B-DNA with moderate affinity and little sequence specificity, but recognize and bind with high affinity to various distorted DNA structures. The nomenclature of HMGB-1 and -2 was adopted in 2001 to designate the canonical HMG “Box” DNA binding domains of these proteins (15Bustin M. Trends Biochem. Sci. 2001; 26: 152-153Abstract Full Text Full Text PDF PubMed Google Scholar). In addition to recognizing distorted DNA targets, the HMG Box also binds in the minor groove and induces sharp bends and distortions in linear duplex DNA. Thus by increasing the flexibility of DNA, HMGB-1/-2 are thought to have a general architectural role in the assembly of nucleoprotein complexes involved in regulation of transcription (see reviews in Refs. 16Thomas J.O. Travers A.A. Trends Biochem. Sci. 2001; 26: 167-174Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar, 17Bustin M. Mol. Cell. Biol. 1999; 19: 5237-5246Crossref PubMed Scopus (753) Google Scholar, 18Agresti A. Bianchi M.E. Curr. Opin. Genet. Dev. 2003; 13: 170-178Crossref PubMed Scopus (321) Google Scholar). Although HMGB-1/-2 enhance DNA binding and transcription activity of all steroid hormone receptors analyzed, including receptors for progesterone (PR), androgen (AR), glucocorticoids (GR), mineralocorticoids (MR), and estrogen (ER-α), they have no influence on class II nuclear receptors (19Boonyaratanakornkit V. Melvin V. Prendergast P. Altmann M. Ronfani L. Bianchi M.E. Taraseviciene L. Nordeen S.K. Allegretto E. Edwards D.P. Mol. Cell. Biol. 1998; 18: 4471-4487Crossref PubMed Scopus (304) Google Scholar, 20Verridjt G. Haelens A. Schoenmakers E. Rombauts W. Classens F. Biochem. J. 2002; 361: 97-103Crossref PubMed Google Scholar, 21Zhang C.C. Krieg S. Shapiro D.J. Mol. Endocrinol. 1999; 13: 632-643Crossref PubMed Google Scholar). This selective effect on the steroid class of nuclear receptors is dependent on the CTE and correlates with a direct protein interaction between HMGB-1/-2 and the CTE that does not occur with class II receptors (14Senkus Melvin V. Roemer S.C. Churchill M.E.A. Edwards D.P. J. Biol. Chem. 2002; 277: 25115-25124Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 20Verridjt G. Haelens A. Schoenmakers E. Rombauts W. Classens F. Biochem. J. 2002; 361: 97-103Crossref PubMed Google Scholar). HMGB-1/-2 also interacts with select groups of apparently unrelated sequence-specific transcription factors including p53 (22McKinney K. Prives C. Mol. Cell. Biol. 2002; 22: 6797-6808Crossref PubMed Scopus (125) Google Scholar), p73 (23Stros M. Ozaki T. Bacikova A. Kageyama H. Nakagawara A. J. Biol. Chem. 2002; 277: 7157-7164Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), Hox proteins (24Zappavigna V. Falciola L. Citterich M.H. Mavilio F. Bianchi M.E. EMBO J. 1996; 15: 4981-4991Crossref PubMed Scopus (216) Google Scholar), Oct proteins (25Zwilling S. Konig H. Wirth T. EMBO J. 1995; 14: 1198-1208Crossref PubMed Scopus (217) Google Scholar), Rel family members (26Agresti A. Lupo R. Bianchi M.E. Müller S. Biochem. Biophys. Res. Commun. 2003; 302: 421-426Crossref PubMed Scopus (87) Google Scholar), and EBV transcription factors Rta and Zebra (27Ellwood K.B. Yen Y-M. Johnson R.C. Carey M. Mol. Cell. Biol. 2000; 20: 4359-4370Crossref PubMed Scopus (81) Google Scholar, 28Mitsouras K. Wong B. Arayata C. Johnson R.C. Carey M. Mol. Cell. Biol. 2002; 22: 4390-4401Crossref PubMed Scopus (77) Google Scholar), to enhance their binding to cognate DNA sequences and transcription activity. The biological actions of estrogen are mediated by two estrogen receptor subtypes, ERα and ERβ, expressed from separate genes (29Kuiper G.G.J.M. Enmark E. Pelto-Huikko M. Nilsson S. Gustafsson J.-Å. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5925-5930Crossref PubMed Scopus (4209) Google Scholar). The precise physiological role of the two ER subtypes is not yet well defined. Genetic ablation experiments in mice suggest the effects of estrogen on development and differentiation of female reproductive target tissues are mediated predominantly by ERα, whereas ERβ is more important in development of the ovary and perhaps other non-reproductive tissues (Ref. 30Crouse J.F. Korach K.S. Endocrine Rev. 1999; 20: 358-417Crossref PubMed Scopus (1771) Google Scholar for review). ERα is a more potent transcriptional activator than ERβ in cell culture based assays (31McInerney E.M. Weis K.E. Sun J. Mosselman S. Katzenellenbogen B.S. Endocrinology. 1998; 139: 4513-4522Crossref PubMed Scopus (234) Google Scholar) and in cell-free transcription assays with chromatin templates containing multiple EREs (32Cheung E. Schwabish M.A. Kraus W.L. EMBO J. 2003; 22: 600-611Crossref PubMed Scopus (40) Google Scholar). Interestingly, ERα and ERβ exhibit similar ligand-dependent transcriptional activities with naked DNA templates in vitro, indicating a role for chromatin in distinguishing the intrinsic activities of the two ERs (32Cheung E. Schwabish M.A. Kraus W.L. EMBO J. 2003; 22: 600-611Crossref PubMed Scopus (40) Google Scholar). Swapping experiments with the N-terminal regions of ERα and ERβ showed that this differential transcription potency both in vivo and in vitro is largely attributable to the N-terminal domain (31McInerney E.M. Weis K.E. Sun J. Mosselman S. Katzenellenbogen B.S. Endocrinology. 1998; 139: 4513-4522Crossref PubMed Scopus (234) Google Scholar, 32Cheung E. Schwabish M.A. Kraus W.L. EMBO J. 2003; 22: 600-611Crossref PubMed Scopus (40) Google Scholar). In tissues that express both ERs, a role for ERβ as an attenuator of ERα activity has been suggested (33Hall J.M. McDonnell D.P. Endocrinology. 1999; 140: 5566-5578Crossref PubMed Google Scholar, 34Lindberg M.K. Movérare S. Skrtic S. Gao H. Dahlman-Wright K. Gustafsson J.-Å. Ohlsson C. Mol. Endocrinol. 2003; 17: 203-208Crossref PubMed Scopus (415) Google Scholar). Despite the fact that the core DBDs of ERα and ERβ are nearly identical with a 96% amino acid identity, ERα has been reported to have a higher affinity for estrogen response elements (EREs) than ERβ (35Cowley S.M. Hoare S. Mosselman S. Parker M.G. J. Biol. Chem. 1997; 272: 19858-19862Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 36Loven M.A. Wood J.R. Nardulli A.M. Mol. Cell. Endocrinol. 2001; 181: 151-163Crossref PubMed Scopus (101) Google Scholar, 37Hyder S.M. Chiapetta C. Stancel G.M. Biochem. Pharmacol. 1999; 57: 597-601Crossref PubMed Scopus (86) Google Scholar), and only ERα is capable of inducing a directed bend in ERE target DNA (38Schultz J.R. Loven M.A. Senkus Melvin V. Edwards D.P. Nardulli A.M. J. Biol. Chem. 2002; 277: 8702-8707Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The differential ability to induce a directed bend in DNA has been attributed to the CTEs of ERα and ERβ (38Schultz J.R. Loven M.A. Senkus Melvin V. Edwards D.P. Nardulli A.M. J. Biol. Chem. 2002; 277: 8702-8707Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The mechanism for the higher DNA affinity of ERα is not known, but is likely due to differences in the CTE or other regions outside the highly conserved core DBDs. The only member of the steroid class of nuclear receptors that has not been analyzed for interaction with HMGB-1/-2 is ERβ. Therefore, in the present study, we sought to further examine the role of the steroid receptor CTE in DNA binding and response to HMGB-1/-2 by performing a comparative analysis of ERα and ERβ. HMGB-1/-2 stimulated DNA binding and transcription activity of both ERα and ERβ and the CTE was required for physical interaction with and functional responsiveness to HMGB-1/-2. The CTE was also found to be responsible for the different intrinsic DNA binding affinities of ERα and ERβ independent of HMGB-1/-2. Unexpectedly, the CTE was also required for ERα and ERβ interaction with half-site EREs. Expression and Purification of GST Fusion Proteins—The expression vectors for GST-HMGB-1 and the ERα DBD (amino acids 198–288) have been described previously (14Senkus Melvin V. Roemer S.C. Churchill M.E.A. Edwards D.P. J. Biol. Chem. 2002; 277: 25115-25124Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). All other DNA-binding domain (DBD) vectors were constructed similarly by subcloning into pGEX2T (Amersham Biosciences), with an in-frame glutathione S-transferase (GST) for expression as a GST fusion protein. Chimeric DBDs were subcloned using “splicing by PCR overlap extension” as previously described (14Senkus Melvin V. Roemer S.C. Churchill M.E.A. Edwards D.P. J. Biol. Chem. 2002; 277: 25115-25124Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and also inserted into pGEX2T (Amersham Biosciences). DBD-GST fusion proteins were expressed in Bl21 bacterial cells and purified by a three-step procedure including affinity chromatography on glutathione-Sepharose resins and thrombin cleavage to remove the GST moiety, DNA cellulose, and FPLC gel filtration by Superdex-30 chromatography. The final product was concentrated by an Amicon stirred cell concentrator (39Melvin V.S. Edwards D.P. Steroid Receptor Methods: Protocols and Assays. Humana Press, Totawa, NJ2001: 39-54Google Scholar). Purified DBD concentrations were determined by comparison to known concentrations of lysozyme on silver stain SDS gel electrophoresis. Relative DNA binding affinity differences were verified using independently purified DBDs, and protein concentrations were determined independently by Lowry assay and UV spectrophotometric quantitation methods using extinction coefficients of 13,260 for both ERα and ERβ DBDs. Production and Purification of Baculovirus-expressed Recombinant Proteins—Full-length human PR-A and HMGB-2 with N-terminal 6× histidine tags, and human ERα and ERβ with N-terminal FLAG sequences (DYCDDDDK) were expressed from baculovirus vectors in Sf9 insect cells as described (19Boonyaratanakornkit V. Melvin V. Prendergast P. Altmann M. Ronfani L. Bianchi M.E. Taraseviciene L. Nordeen S.K. Allegretto E. Edwards D.P. Mol. Cell. Biol. 1998; 18: 4471-4487Crossref PubMed Scopus (304) Google Scholar, 40Kraus W.L. Kadonaga J.T. Genes Dev. 1998; 12: 331-342Crossref PubMed Scopus (289) Google Scholar). For ERα and ERβ, estradiol 17β (200 nm) was added to Sf9 cell cultures to activate receptors during expression in vivo. His-tagged proteins were purified by nickel affinity resins as previously described for PR except that HMGB-2 was dialyzed against 20 mm Tris, pH 8.0, 100 mm NaCl, 10% glycerol, and 1 mm DTT to exchange the β-mercaptoethanol for DTT and prevent oxidation (14Senkus Melvin V. Roemer S.C. Churchill M.E.A. Edwards D.P. J. Biol. Chem. 2002; 277: 25115-25124Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 16Thomas J.O. Travers A.A. Trends Biochem. Sci. 2001; 26: 167-174Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar, 17Bustin M. Mol. Cell. Biol. 1999; 19: 5237-5246Crossref PubMed Scopus (753) Google Scholar, 18Agresti A. Bianchi M.E. Curr. Opin. Genet. Dev. 2003; 13: 170-178Crossref PubMed Scopus (321) Google Scholar, 19Boonyaratanakornkit V. Melvin V. Prendergast P. Altmann M. Ronfani L. Bianchi M.E. Taraseviciene L. Nordeen S.K. Allegretto E. Edwards D.P. Mol. Cell. Biol. 1998; 18: 4471-4487Crossref PubMed Scopus (304) Google Scholar). FLAG-tagged ERα and ERβ were purified as described (40Kraus W.L. Kadonaga J.T. Genes Dev. 1998; 12: 331-342Crossref PubMed Scopus (289) Google Scholar) except that the receptors were eluted from anti-FLAG-affinity resins in a buffer containing 20 mm Tris, pH 7.5, 150 mm NaCl, 20% glycerol, 50 μm ZnCl2, 0.2 mm EDTA, 2 mm DTT, 0.1% Nonidet P-40, 0.1 mg/ml FLAG peptide, and 0.5 mg/ml insulin. The eluates were then dialyzed against elution buffer with higher Nonidet P-40 (0.2%) and lacking the FLAG peptide. Purified proteins were analyzed by silver stain or Coomassie Blue-stained SDS gel electrophoresis and judged to be at ≥90% pure. Electrophoretic Mobility Shift Assays (EMSAs)—EMSAs for full-length ERα and ERβ were performed as described previously (14Senkus Melvin V. Roemer S.C. Churchill M.E.A. Edwards D.P. J. Biol. Chem. 2002; 277: 25115-25124Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 19Boonyaratanakornkit V. Melvin V. Prendergast P. Altmann M. Ronfani L. Bianchi M.E. Taraseviciene L. Nordeen S.K. Allegretto E. Edwards D.P. Mol. Cell. Biol. 1998; 18: 4471-4487Crossref PubMed Scopus (304) Google Scholar) in a DNA binding reaction containing 10 mm HEPES, pH 7.8, 100 mm KCl, 20% glycerol, 4 mm MgCl2, 1 mm DTT 0.1 μg poly(dI-dC), 5 μg of ovalbumin, and 0.6 nm32P-labeled duplex DNA fragments formed from the appropriate oligonucleotides. Binding reactions were carried out for 30 min at 4 °C and samples were then electrophoresed at 4 °C on non-denaturing 5% polyacrylamide gels (40:1 acrylamide/bisacrylamide ratio) containing 2.5% glycerol impregnated in the gels, and 0.25× TBE (0.02 m Tris, pH 8.0, 0.02 m boric acid, 0.5 mm EDTA) as running buffer. Isolated DBDs used a binding reaction containing 10 mm Tris, pH 8.0, 50 mm KCl, 6% glycerol, 1 mm DTT, 100 ng of poly(dI-dC), and 0.1% Nonidet P-40 or Igepal CA630 (Sigma), and samples were electrophoresed on 8% polyacrylamide gels. Gels were dried, autoradiographedm and free [32P]DNA and [32P]DNA-protein complexes were quantitated by direct scanning of gels for radioactivity by a series 400 Molecular Dynamics PhosphorImager. Data were expressed graphically as the normalized fraction of DNA bound versus DBD concentration. For EMSAs that exhibited two mobility ER-complexes, bound DNA was taken as the sum of both complexes. The fraction bound DNA was calculated as 1-(free DNA/(bound DNA+free DNA)). The data were normalized by setting the fraction of DNA bound at saturation to 1.0 and all other values were a fraction of 1.0 (41Hoopes B. LeBlanc J. Hawley D. J. Biol. Chem. 1992; 267: 11539-11547Abstract Full Text PDF PubMed Google Scholar). Saturation typically occurred at fraction bound values of 0.7 to greater than 0.9. All DNA binding curves were best fit to the following equation: y = ((1/Kd)·(xn))/(1 + ((1/Kd)·(xn)), where y is the normalized fraction DNA bound, x is the total DBD concentration, and n is the Hill cooperativity coefficient. The Hill cooperativity coefficient varied from n = 1 to 2, and the curves shown are the best fit of the data by Kaleidagraph using an R value = 1. The apparent dissociation constant (Kdapp) was determined as the DBD concentration at which y = 0.5 from the average curve of at least three independent experiments. Double-stranded DNA fragments were end-labeled by Klenow fill-in of 5′-overhanging ends with [α-32P]dATP and [α-32P]dCTP: EREpal 5′-gatcGATTTGTCAAGGTCACTGTGACCTTGACACAGT-3′; TCA-EREhalf 5′-gatcGATTTGTCAAGGTCAGATGGCGGGCACAGTCTAG-3′; and TAG-EREhalf 5′-gatcGATTTGTAGAGGTCAGATGGCGGGCACAGTCTAG-3′. Mammalian Cell Transfection—CV-1 cells were transfected by an adenovirus-mediated method as previously described (14Senkus Melvin V. Roemer S.C. Churchill M.E.A. Edwards D.P. J. Biol. Chem. 2002; 277: 25115-25124Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 19Boonyaratanakornkit V. Melvin V. Prendergast P. Altmann M. Ronfani L. Bianchi M.E. Taraseviciene L. Nordeen S.K. Allegretto E. Edwards D.P. Mol. Cell. Biol. 1998; 18: 4471-4487Crossref PubMed Scopus (304) Google Scholar), and at 24-h post-transfection, cells were treated with or without estradiol 17β (10 nm) for 24 h at 37 °C. The total amount of plasmid DNA in each transfection was equalized with empty vector (pBlueScript) such that each well received equimolar amounts of plasmid DNA. A pRSV-βGal plasmid was included in each transfection as an internal control for well-to-well variation in transfection efficiency. Cell lysates were assayed for luciferase and β-galactosidase activities with a Monolight 2010 luminometer as described previously (14Senkus Melvin V. Roemer S.C. Churchill M.E.A. Edwards D.P. J. Biol. Chem. 2002; 277: 25115-25124Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 19Boonyaratanakornkit V. Melvin V. Prendergast P. Altmann M. Ronfani L. Bianchi M.E. Taraseviciene L. Nordeen S.K. Allegretto E. Edwards D.P. Mol. Cell. Biol. 1998; 18: 4471-4487Crossref PubMed Scopus (304) Google Scholar). Luciferase values were corrected for variations in transfection efficiency by calculating luciferase/β-galactosidase ratios (LUC/βGAL). GST Pull-down Assays—Bacterial cell lysates expressing GST or GST-HMGB-1 were incubated in suspension with 25 μl of glutathione Sepharose beads in 500 μl lysis buffer (20 mm Tris-HCl, pH 7.5, 1 m NaCl, 5 mm DTT, 1 mm EDTA, 50 μm ZnCl2, 10% glycerol, and protease inhibitors) for 1h at 4 °C. Beads were washed three times in lysis buffer and followed by washing in binding buffer (10 mm Tris, pH 7.8, 50 mm NaCl, 2 mm MgCl2, 1 mm EDTA, 1 mm DTT, and 10% glycerol). Receptor DBDs (1 μg), were added and incubated in suspension for 1 h (250 μl) at 4 °C. Beads were then pelleted, washed in binding buffer, and protein was eluted in SDS sample buffer and analyzed by Western immunoblot with a rabbit polyclonal antisera raised against peptide sequences in the ER DBDs (42Smith D.F. Lubahn D.B. McCormick D.J. Wilson E.M. Toft D.O. Endocrinology. 1988; 122: 2816-2825Crossref PubMed Scopus (38) Google Scholar). HMGB Enhances the DNA Binding Affinity and Transcriptional Activity of ERβ—Although ERα and ERβ can activate many of the same target genes in response to estrogen, ERβ has been reported to exhibit a 3–4-fold lower affinity for EREs and as much as a 10-fold weaker transcriptional activity than ERα (31McInerney E.M. Weis K.E. Sun J. Mosselman S. Katzenellenbogen B.S. Endocrinology. 1998; 139: 4513-4522Crossref PubMed Scopus (234) Google Scholar, 32Cheung E. Schwabish M.A. Kraus W.L. EMBO J. 2003; 22: 600-611Crossref PubMed Scopus (40) Google Scholar, 33Hall J.M. McDonnell D.P. Endocrinology. 1999; 140: 5566-5578Crossref PubMed Google Scholar, 34Lindberg M.K. Movérare S. Skrtic S. Gao H. Dahlman-Wright K. Gustafsson J.-Å. Ohlsson C. Mol. Endocrinol. 2003; 17: 203-208Crossref PubMed Scopus (415) Google Scholar, 35Cowley S.M. Hoare S. Mosselman S. Parker M.G. J. Biol. Chem. 1997; 272: 19858-19862Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 36Loven M.A. Wood J.R. Nardulli A.M. Mol. Cell. Endocrinol. 2001; 181: 151-163Crossref PubMed Scopus (101) Google Scholar, 37Hyder S.M. Chiapetta C. Stancel G.M. Biochem. Pharmacol. 1999; 57: 597-601Crossref PubMed Scopus (86) Google Scholar). Therefore, we sought to determine whether a difference in interaction with HMGB-1/-2 contributes to these distinct activities of ERα and ERβ. We previously found that HMGB-1 and -2 are functionally interchangeable with respect to stimulating the DNA binding affinity of steroid receptors in vitro and enhancing transcriptional activity in mammalian cells (19Boonyaratanakornkit V. Melvin V. Prendergast P. Altmann M. Ronfani L. Bianchi M.E. Taraseviciene L. Nordeen S.K. Allegretto E. Edwards D.P. Mol. Cell. Biol. 1998; 18: 4471-4487Crossref PubMed Scopus (304) Google Scholar). Therefore, HMGB-1 and -2 were used interchangeably and are collectively referred to as HMGB in the remainder of this article. Receptor DNA binding was analyzed by electrophoretic gel mobility shift assay (EMSA) by varying the concentration of receptors in the presence of a constant amount of 32P-labeled DNA probe. The DNA duplex fragment contains a consensus palindromic ERE based on the well characterized ERE in the vitellogenin gene (36Loven M.A. Wood J.R. Nardulli A.M. Mol. Cell. Endocrinol. 2001; 181: 151-163Crossref PubMed Scopus (101) Google Scholar) that is recognized by human and other species of ER. As shown in Fig. 2 A and B, purified full-length ERα and ERβ bound to EREpal in a dose-dependent, saturable manner. Based on apparent dissociation constants (Kdapp) estimated from the ER concentration at half-maximal DNA binding, ERα bound to the EREpal with a 4-fold higher affinity than ERβ (Fig. 2B). This affinity difference is consistent with previous reports that ERα has a higher affinity for EREpal than ERβ (35Cowley S.M. Hoare S. Mosselman S. Parker M.G. J. Biol. Chem. 1997; 272: 19858-19862Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 36Loven M.A. Wood J.R. Nardulli A.M. Mol. Cell. Endocrinol. 2001; 181: 151-163Crossref PubMed Scopus (101) Google Scholar, 37Hyder S.M. Chiapetta C. Stancel G.M. Biochem. Pharmacol. 1999; 57: 597-601Crossref PubMed Scopus (86) Google Scholar). For both ERs, addition of HMGB significantly left-shifted the DNA binding curves (Fig. 2B), indicating an increase in binding affinity for EREpal. In the absence of HMGB, ERα and ERβ produced two protein-DNA complexes and both contained ERα or ERβ, respectively, as demonstrated by supershifts with ERα- or ERβ-specific antibodies (Fig. 2A). The exact nature of the two mobility ER complexes is not known. Both complexes were obtained in the absence of HMGB, indicating that they do not represent ER-complexes containing and lacking HMGB. Because it is well accepted that ER preferentially binds to EREpal as a dimer, suggests the faster mobility complex contains an ER dimer, whereas the slower mobility complex contains a higher order ER oligomer (Fig. 2A). At low concentrations of ER (α and β), addition of HMGB stimulated the formation of both mobility complexes, but at high receptor concentrations, HMGB predominantly stimulated the slower mobility complex concomitant with a decrease of the faster mobility complex (Fig. 2A). Multiple mobility ER complexes have been reported previously, but the relevance of the different complexes to ER function is not known (36Loven M.A. Wood J.R. Nardulli A.M. Mol. Cell. Endocrinol. 2001; 181: 151-163Crossref PubMed Scopus (101) Google Scholar). HMGB was not retained as a stable component of the stimulated complexes, as they have the same mobility as the unstimulated complexes (Fig. 2A) and are not supershifted by HMGB antibodies (data not shown). Ternary HMGB complexes by EMSAs have not typically been detected with steroid receptors or other transcription factors, suggesting HMGB acts as
Referência(s)