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• W2076249557 abstract "Steroidogenic factor-1 (SF-1) is an orphan nuclear receptor that binds DNA as a monomer and regulates the transcription of multiple target genes. A mutation in the proximal (P)-box of the first zinc finger of SF-1 (G35E) has been reported to cause complete XY sex reversal and adrenal insufficiency. Because this P-box region dictates DNA binding specificity, we investigated the effect of this mutation on DNA binding and regulation of target genes. Binding of the P-box mutant was markedly impaired for most native SF-1 response elements. However, mutant SF-1 bound to a subset of response elements containing a CCA AGGTCA motif. Mutagenesis studies of response elements revealed that the first nucleotide position in the 5′-flanking sequence triplet and the central part of the half-site dictate DNA binding specificity by the mutant SF-1. Further, introduction of a mutation into the SF-1 A-box, which has been proposed to bind to the 5′-flanking sequence triplet, eliminated binding by mutant SF-1 to all response elements tested. These data support the idea that the A-box stabilizes monomeric binding by nuclear receptors. This action may be particularly important when P-box binding affinity is compromised either by mutations in SF-1 or by sequence alterations in its binding site. Steroidogenic factor-1 (SF-1) is an orphan nuclear receptor that binds DNA as a monomer and regulates the transcription of multiple target genes. A mutation in the proximal (P)-box of the first zinc finger of SF-1 (G35E) has been reported to cause complete XY sex reversal and adrenal insufficiency. Because this P-box region dictates DNA binding specificity, we investigated the effect of this mutation on DNA binding and regulation of target genes. Binding of the P-box mutant was markedly impaired for most native SF-1 response elements. However, mutant SF-1 bound to a subset of response elements containing a CCA AGGTCA motif. Mutagenesis studies of response elements revealed that the first nucleotide position in the 5′-flanking sequence triplet and the central part of the half-site dictate DNA binding specificity by the mutant SF-1. Further, introduction of a mutation into the SF-1 A-box, which has been proposed to bind to the 5′-flanking sequence triplet, eliminated binding by mutant SF-1 to all response elements tested. These data support the idea that the A-box stabilizes monomeric binding by nuclear receptors. This action may be particularly important when P-box binding affinity is compromised either by mutations in SF-1 or by sequence alterations in its binding site. steroidogenic factor-1 side chain cleavage enzyme luteinizing hormone cAMP-response element-binding protein estrogen receptor thyroid hormone receptor glucocorticoid receptor proximal estrogen response element electrophoretic mobility shift assays Müllerian inhibiting substance Steroidogenic factor-1 (SF-1)1 (FTZF1) (1Lala D.S. Rice D.A. Parker K.L. Mol. Endocrinol. 1992; 6: 1249-1258Crossref PubMed Scopus (514) Google Scholar, 2Honda S. Morohashi K. Nomura M. Takeya H. Kitajima M. Omura T. J. Biol. Chem. 1993; 268: 7494-7502Abstract Full Text PDF PubMed Google Scholar) is an orphan nuclear receptor that plays an essential role in the development of the adrenal gland, testis, ovary, pituitary gonadotropes, and hypothalamus (3Luo X. Ikeda Y. Parker K.L. Cell. 1994; 77: 481-490Abstract Full Text PDF PubMed Scopus (1357) Google Scholar). SF-1 regulates the transcription of a variety of target genes involved in steroidogenesis and reproduction including DAX-1 (AHC) (4Yu R.N. Ito M. Jameson J.L. Mol. Endocrinol. 1998; 12: 1010-1022Crossref PubMed Scopus (111) Google Scholar), steroidogenic acute regulatory protein (StAR) (5Sugawara T. Holt J.A. Kiriakidou M. Strauss J.F.R. Biochemistry. 1996; 35: 9052-9059Crossref PubMed Scopus (236) Google Scholar), cholesterol side chain cleavage enzyme (SCC, Cyp11a) (6Clemens J.W. Lala D.S. Parker K.L. Richards J.S. Endocrinology. 1994; 134: 1499-1508Crossref PubMed Scopus (152) Google Scholar), aromatase (Cyp19) (7Fitzpatrick S.L. Richards J.S. Mol. Endocrinol. 1993; 7: 341-354PubMed Google Scholar,8Lynch J.P. Lala D.S. Peluso J.J. Luo W. Parker K.L. White B.A. Mol. Endocrinol. 1993; 7: 776-786Crossref PubMed Scopus (213) Google Scholar), Müllerian inhibiting substance (M1S, AMH) (9Shen W.H. Moore C.C. Ikeda Y. Parker K.L. Ingraham H.A. Cell. 1994; 77: 651-661Abstract Full Text PDF PubMed Scopus (482) Google Scholar), and luteinizing hormone (LH) β subunit (10Halvorson L.M. Ito M. Jameson J.L. Chin W.W. J. Biol. Chem. 1998; 273: 14712-14720Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar).SF-1 belongs to the nuclear receptor superfamily (NR5A1) and shares several well conserved domains with other family members. These regions include an amino-terminal, two zinc finger DNA-binding domain, a putative ligand-binding/dimerization domain, and a carboxyl-terminal AF2 transactivation domain (see Fig. 1 A) (11Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6026) Google Scholar, 12Ito M., Yu, R.N. Jameson J.L. Mol. Endocrinol. 1998; 12: 290-301Crossref PubMed Scopus (123) Google Scholar). SF-1 regulates gene transcription through its interactions with nuclear receptor coactivators such as steroid receptor coactivator-1 and CREB-binding protein (12Ito M., Yu, R.N. Jameson J.L. Mol. Endocrinol. 1998; 12: 290-301Crossref PubMed Scopus (123) Google Scholar, 13Monte D. DeWitte F. Hum D.W. J. Biol. Chem. 1998; 273: 4585-4591Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) as well as by direct interactions with other transcription factors such as DAX-1, Egr-1, SOX9, WT-1, and CREB (10Halvorson L.M. Ito M. Jameson J.L. Chin W.W. J. Biol. Chem. 1998; 273: 14712-14720Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 14Ito M., Yu, R. Jameson J.L. Mol. Cell. Biol. 1997; 17: 1476-1483Crossref PubMed Scopus (390) Google Scholar, 15De Santa Barbara P. Bonneaud N. Boizet B. Desclozeaux M. Moniot B. Sudbeck P. Scherer G. Poulat F. Berta P. Mol. Cell. Biol. 1998; 18: 6653-6665Crossref PubMed Scopus (505) Google Scholar, 16Nachtigal M.W. Hirokawa Y. Enyeart-VanHouten D.L. Flanagan J.N. Hammer G.D. Ingraham H.A. Cell. 1998; 93: 445-454Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 17Ito M. Park Y. Weck J. Mayo K.E. Jameson J.L. Mol. Endocrinol. 2000; 14: 66-81PubMed Google Scholar).Figure 5Effect of A-box mutations on G35E P-box mutant binding to the wild-type aromatase SF-1 response element. A, mutations were introduced into the A-box of wild-type SF-1 (R92Q, K94Q) or the G35E mutant (G35E/R92Q, G35E/K94Q).B, EMSAs were performed using in vitro translated products of wild-type SF-1 and the mutants described above inA (3 μl) and radiolabeled probe (20 fmol) corresponding to the wild-type aromatase response element. WT, wild-type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1DNA binding of the G35E P-box mutant to various SF-1 response elements. A, schematic representation of the human P-box mutation (30Achermann J.C. Ito M. Ito M. Hindmarsh P.C. Jameson J.L. Nat. Genet. 1999; 22: 125-126Crossref PubMed Scopus (541) Google Scholar) is shown. Glycine, at the last position of the P-box, was changed to glutamic acid (G35E).B, EMSAs were performed using in vitro translated wild-type (WT) and mutant (MT) SF-1 (3 μl) and 20 fmol of radiolabeled probes corresponding to SF-1 response elements from murine SCC, murine MIS, rat aromatase, rat LHβ, and murine DAX-1 gene promoters. DNA-protein complexes were resolved on a 0.5× TBE polyacrylamide gel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In general, members of the nuclear receptor superfamily bind to DNA as dimers. Each monomer within these dimer pairs interacts with a half-site sequence, AGNNCA (18Luisi B.F. Xu W.X. Otwinowski Z. Freedman L.P. Yamamoto K.R. Sigler P.B. Nature. 1991; 352: 497-505Crossref PubMed Scopus (1217) Google Scholar, 19Schwabe J.W. Chapman L. Finch J.T. Rhodes D. Cell. 1993; 75: 567-578Abstract Full Text PDF PubMed Scopus (588) Google Scholar, 20Glass C.K. Endocr. Rev. 1994; 15: 391-407PubMed Google Scholar). Based on half-site specificity, nuclear receptors can be divided into two subgroups. The estrogen receptor (ER) subgroup, which also includes the thyroid hormone receptor (TR) and retinoic acid receptor among others, recognizes the core sequence, AGGTCA. The glucocorticoid receptor (GR) subgroup, which includes the androgen receptor and progesterone receptor, has distinct half-site specificity for AGAACA. The two central nucleotides in the half-site sequence therefore largely determine DNA sequence recognition by the two groups of nuclear receptors. Structure-function analyses reveal that nuclear receptors distinguish these two nucleotides via three amino acid residues in the proximal (P)-box of the first zinc finger (21Mader S. Kumar V. de Verneuil H. Chambon P. Nature. 1989; 338: 271-274Crossref PubMed Scopus (358) Google Scholar, 22Danielsen M. Hinck L. Ringold G.M. Cell. 1989; 57: 1131-1138Abstract Full Text PDF PubMed Scopus (257) Google Scholar, 23Umesono K. Evans R.M. Cell. 1989; 57: 1139-1146Abstract Full Text PDF PubMed Scopus (720) Google Scholar). These P-box residues are located within a DNA recognition α-helix that interacts with the major groove of the DNA response element (18Luisi B.F. Xu W.X. Otwinowski Z. Freedman L.P. Yamamoto K.R. Sigler P.B. Nature. 1991; 352: 497-505Crossref PubMed Scopus (1217) Google Scholar).In contrast to these dimeric receptors, a small group of orphan nuclear receptors binds to DNA as monomers. These factors recognize the estrogen response element (ERE)-type half-site, AGGTCA, and include SF-1, FTZ-F1, NGFI-B, RevErbA, and ROR α (24Tsukiyama T. Niwa O. Nucleic Acids Res. 1992; 20: 1477-1482Crossref PubMed Scopus (18) Google Scholar, 25Ueda H. Sun G.C. Murata T. Hirose S. Mol. Cell. Biol. 1992; 12: 5667-5672Crossref PubMed Scopus (171) Google Scholar, 26Wilson T.E. Paulsen R.E. Padgett K.A. Milbrandt J. Science. 1992; 256: 107-110Crossref PubMed Scopus (277) Google Scholar, 27Wilson T.E. Fahrner T.J. Milbrandt J. Mol. Cell. Biol. 1993; 13: 5794-5804Crossref PubMed Scopus (356) Google Scholar, 28Harding H.P. Lazar M.A. Mol. Cell. Biol. 1993; 13: 3113-3121Crossref PubMed Google Scholar, 29Giguere V. Tini M. Flock G. Ong E. Evans R.M. Otulakowski G. Genes Dev. 1994; 8: 538-553Crossref PubMed Scopus (448) Google Scholar). Several of these receptors share a 30-amino acid basic region carboxyl-terminal to the second zinc finger designated the FTZ-F1 box or A-box (25Ueda H. Sun G.C. Murata T. Hirose S. Mol. Cell. Biol. 1992; 12: 5667-5672Crossref PubMed Scopus (171) Google Scholar, 26Wilson T.E. Paulsen R.E. Padgett K.A. Milbrandt J. Science. 1992; 256: 107-110Crossref PubMed Scopus (277) Google Scholar, 27Wilson T.E. Fahrner T.J. Milbrandt J. Mol. Cell. Biol. 1993; 13: 5794-5804Crossref PubMed Scopus (356) Google Scholar). This A-box is proposed to recognize additional nucleotide sequences 5′ to the AGGTCA motif in the minor groove of the response element DNA. For example, SF-1 binds preferentially to the 5′-flanking sequencePyCA AGGTCA (where Py is pyrimidine), whereas NGFI-B recognizes AA AGGTCA.Recently, we reported a SF-1 mutation in a patient with XY sex reversal, primary adrenal insufficiency, dysgenetic testes, and persistent Müllerian structures (30Achermann J.C. Ito M. Ito M. Hindmarsh P.C. Jameson J.L. Nat. Genet. 1999; 22: 125-126Crossref PubMed Scopus (541) Google Scholar). The heterozygous G35E mutation involves the last amino acid in the P-box, an area shown previously to be critical for DNA binding of other nuclear receptors (21Mader S. Kumar V. de Verneuil H. Chambon P. Nature. 1989; 338: 271-274Crossref PubMed Scopus (358) Google Scholar, 22Danielsen M. Hinck L. Ringold G.M. Cell. 1989; 57: 1131-1138Abstract Full Text PDF PubMed Scopus (257) Google Scholar). As expected, this mutant SF-1 protein failed to bind to SF-1 response elements such as those found in the proximal promoter of SCC (Cyp11a), and no transactivation of this gene was seen (30Achermann J.C. Ito M. Ito M. Hindmarsh P.C. Jameson J.L. Nat. Genet. 1999; 22: 125-126Crossref PubMed Scopus (541) Google Scholar). However, because SF-1 response elements are variable, we considered the possibility that this P-box mutant might recognize other SF-1 response elements.In this report, we examined mutant SF-1 binding to response elements from a variety of target genes. These studies revealed surprising heterogeneity in the SF-1 binding interactions with various target sequences, thereby allowing detailed analysis of the structural determinants in SF-1 and its recognition sites that mediate receptor binding. We find that interactions between the A-box and the 5′-flanking sequence of the response element stabilize monomeric binding by SF-1, especially in situations where binding affinity is compromised by P-box mutations.DISCUSSIONNuclear receptors regulate gene transcription in diverse biological systems. DNA binding specificity is crucial, therefore, so that different receptors can recognize their specific target genes appropriately. Several studies have shown that the DNA binding specificity of classic ligand-dependent nuclear receptors is determined by the P-box amino acid sequence (for example, GSV for the GR, EGA for the ER, and EGG for the TR (Fig. 4 A) (18Luisi B.F. Xu W.X. Otwinowski Z. Freedman L.P. Yamamoto K.R. Sigler P.B. Nature. 1991; 352: 497-505Crossref PubMed Scopus (1217) Google Scholar, 19Schwabe J.W. Chapman L. Finch J.T. Rhodes D. Cell. 1993; 75: 567-578Abstract Full Text PDF PubMed Scopus (588) Google Scholar, 20Glass C.K. Endocr. Rev. 1994; 15: 391-407PubMed Google Scholar, 21Mader S. Kumar V. de Verneuil H. Chambon P. Nature. 1989; 338: 271-274Crossref PubMed Scopus (358) Google Scholar, 22Danielsen M. Hinck L. Ringold G.M. Cell. 1989; 57: 1131-1138Abstract Full Text PDF PubMed Scopus (257) Google Scholar)). The GS sequence of the GR recognizes the GRE-type half-site, AGAACA, whereas the EG residues of the ER and TR recognize the ERE-type half-site, AGGTCA. Comparatively little is known about the DNA binding specificity of SF-1, an orphan nuclear receptor that has no known ligand and one of the few nuclear receptors that binds DNA as a monomer. SF-1 has a hybrid P-box sequence (ESG) and recognizes a consensus response element consisting of an ERE-type half-site and three preceding nucleotides (PyCA AGGTCA) (35Parker K.L. Schimmer B.P. Endocr. Rev. 1997; 18: 361-377Crossref PubMed Scopus (556) Google Scholar). The recent discovery of a naturally occurring human SF-1 P-box mutation (G35E) (30Achermann J.C. Ito M. Ito M. Hindmarsh P.C. Jameson J.L. Nat. Genet. 1999; 22: 125-126Crossref PubMed Scopus (541) Google Scholar) led us to investigate the structural determinants of DNA binding specificity of SF-1 further.Initial studies showed that the G35E SF-1 mutant did not bind to either of two SF-1 response elements present in the proximal promoter region of the SCC gene (30Achermann J.C. Ito M. Ito M. Hindmarsh P.C. Jameson J.L. Nat. Genet. 1999; 22: 125-126Crossref PubMed Scopus (541) Google Scholar). This lack of binding might be predicted, as a similar glycine to glutamic acid mutation at the third P-box position resulted in loss of TR binding (33Nelson C.C. Hendy S.C. Romaniuk P.J. J. Biol. Chem. 1995; 270: 16988-16994Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 34Nelson C.C. Hendy S.C. Romaniuk P.J. J. Biol. Chem. 1995; 270: 16981-16987Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Also, the patient's phenotype of primary adrenal failure is consistent with impaired transcription of steroidogenic enzymes. However, SF-1 regulates the transcription of a wide array of target genes (35Parker K.L. Schimmer B.P. Endocr. Rev. 1997; 18: 361-377Crossref PubMed Scopus (556) Google Scholar). Many of these have response elements that deviate from the consensus SF-1 sequence such as the murine DAX-1 composite enhancer (Table I; TCG AGGTCA, TCA TGGCCA) (4Yu R.N. Ito M. Jameson J.L. Mol. Endocrinol. 1998; 12: 1010-1022Crossref PubMed Scopus (111) Google Scholar) or the inhibin α gene, where SF-1 regulates gene transcription through its interaction with CREB bound to the cAMP-response element (17Ito M. Park Y. Weck J. Mayo K.E. Jameson J.L. Mol. Endocrinol. 2000; 14: 66-81PubMed Google Scholar). We hypothesized therefore that the G35E mutant might exhibit different DNA binding specificity with respect to other SF-1 response elements.Among the SF-1 response elements studied, the G35E mutant was found to bind to the MIS and aromatase promoters with an affinity approaching that of wild-type SF-1. These promoters share a common SF-1 response element, CCA AGGTCA. Comparison of this sequence with other native SF-1 response elements (Table I) led us to speculate that the first position of the three nucleotides in the 5′-flanking sequence and the fourth and fifth nucleotides of the nuclear receptor half-site itself may influence the DNA binding specificity of the G35E mutant. These nucleotides were replaced with those found in other native promoters to investigate this further. Substitution of the flanking cytosine (CCA) with thymine (TCA) in the aromatase SF-1 response element actually strengthened binding by the mutant SF-1, highlighting the importance of a pyrimidine in this flanking sequence. However, binding was impaired significantly following substitution of this nucleotide with a purine (ACA, GCA) or alteration of the central part of the half-site, supporting the hypothesis that the first nucleotide of the flanking sequence, as well as the composition of the half-site, influence DNA binding by the P-box motif.The role of each of the three P-box amino acid residues in half-site recognition has been studied previously for a variety of nuclear receptors, which allowed us to predict the outcome of mutating these codons in SF-1 (36Zilliacus J. Wright A.P. Carlstedt-Duke J. Gustafsson J.A. Mol. Endocrinol. 1995; 9: 389-400Crossref PubMed Google Scholar). The first P-box residue (position 31 for SF-1) is a glutamic acid in SF-1, ER, and TR and a glycine in GR. This glutamic acid facilitates binding to response elements containing guanine at the third position of the nuclear receptor half-site (ERE, AGGTCA) and inhibits binding to those containing adenine (GRE, AGAACA) (37Zilliacus J. Wright A.P. Norinder U. Gustafsson J.A. Carlstedt-Duke J. J. Biol. Chem. 1992; 267: 24941-24947Abstract Full Text PDF PubMed Google Scholar). Reduced binding of the SF-1 E31G mutant is consistent with these observations. At the second P-box position (32 for SF-1) SF-1 and GR have a serine, and ER and TR have a glycine. Replacement of this serine with glycine has been shown to increase binding of GR to an ERE-type half-site (37Zilliacus J. Wright A.P. Norinder U. Gustafsson J.A. Carlstedt-Duke J. J. Biol. Chem. 1992; 267: 24941-24947Abstract Full Text PDF PubMed Google Scholar). Consistent with these findings, the S32G SF-1 mutant showed similar DNA binding specificity to wild-type SF-1. Finally, the third P-box residue (position 35 for SF-1) is a glycine in SF-1 and TR, an alanine in ER, and a valine in GR. Like the glutamic acid in the first P-box position, this valine has a dual function of facilitating binding to response elements containing adenine at the fourth position of the nuclear receptor half-site (GRE, AGAACA) and inhibiting binding to those containing thymine (ERE, AGGTCA) (37Zilliacus J. Wright A.P. Norinder U. Gustafsson J.A. Carlstedt-Duke J. J. Biol. Chem. 1992; 267: 24941-24947Abstract Full Text PDF PubMed Google Scholar). Contrary to our prediction, the G35V mutant bound reasonably well to the wild-type response element (half-site; AGGTCA). However, binding of this mutant to the modified half-sites (M4, AGGCTA; M5, AGGCCA) was reduced dramatically presumably due to the mutation introduced into the fourth nucleotide position of the half-site. Taken together, these data demonstrate that the three amino acid residues in the P-box are critical for recognition of the half-site by SF-1, a nuclear receptor that binds to DNA as a monomer. It is also notable that the P-box sequence of SF-1 (ESG) showed the same DNA binding specificity as that of the TR (EGG) because monomeric TR binding has been demonstrated in certain circumstances (38Katz R.W. Koenig R.J. J. Biol. Chem. 1993; 268: 19392-19397Abstract Full Text PDF PubMed Google Scholar).Mutations of the first nucleotide in the 5′-flanking sequence affected binding of several P-box mutants in addition to the G35E mutant protein. This observation provides further evidence that SF-1 binding can be influenced by the 5′-flanking sequence as well as the central part of the nuclear receptor half-site itself. However, it is unclear whether this represents a direct interaction between the P-box motif and the 5′-flanking sequence or whether the flanking sequence has an indirect effect by stabilizing the bound protein-DNA complex. Because it has been suggested that the FTZ-F1/SF-1 A-box can interact directly with this 5′-flanking sequence without interacting with the half-site (25Ueda H. Sun G.C. Murata T. Hirose S. Mol. Cell. Biol. 1992; 12: 5667-5672Crossref PubMed Scopus (171) Google Scholar), we introduced two mutations into the SF-1 A-box based on previous studies of FTZ-F1. The A-box mutations, R92Q (R589Q in FTZ-F1) and K94Q (K591Q in FTZ-F1), did not affect DNA binding on their own. However, introduction of the R92Q mutation in the background of the G35E mutant reduced binding quite dramatically. Taken together, these data support the idea that the A-box of SF-1 can stabilize monomeric binding to half-sites through an interaction with the 5′-flanking sequence in the minor groove of DNA. The additional binding energy provided by this contact may facilitate monomeric binding by receptors, thereby compensating for the stability otherwise provided by receptor dimerization. This interaction may be particularly important when P-box binding is compromised either by mutations in SF-1 or by sequence alterations in its binding site.In this particular patient, activation of the aromatase promoter by the G35E SF-1 mutant probably had little clinical significance because of multiple other deficiencies in steroidogenesis. In addition, activation of the MIS promoter may have had little effect if the dysgenetic testes had reduced capacity to produce MIS. Nevertheless, this study does indicate that a single mutation in SF-1 can exert differential effects on various target genes. This concept may also apply to other transcription factors that regulate multiple downstream target genes with variant response elements. Steroidogenic factor-1 (SF-1)1 (FTZF1) (1Lala D.S. Rice D.A. Parker K.L. Mol. Endocrinol. 1992; 6: 1249-1258Crossref PubMed Scopus (514) Google Scholar, 2Honda S. Morohashi K. Nomura M. Takeya H. Kitajima M. Omura T. J. Biol. Chem. 1993; 268: 7494-7502Abstract Full Text PDF PubMed Google Scholar) is an orphan nuclear receptor that plays an essential role in the development of the adrenal gland, testis, ovary, pituitary gonadotropes, and hypothalamus (3Luo X. Ikeda Y. Parker K.L. Cell. 1994; 77: 481-490Abstract Full Text PDF PubMed Scopus (1357) Google Scholar). SF-1 regulates the transcription of a variety of target genes involved in steroidogenesis and reproduction including DAX-1 (AHC) (4Yu R.N. Ito M. Jameson J.L. Mol. Endocrinol. 1998; 12: 1010-1022Crossref PubMed Scopus (111) Google Scholar), steroidogenic acute regulatory protein (StAR) (5Sugawara T. Holt J.A. Kiriakidou M. Strauss J.F.R. Biochemistry. 1996; 35: 9052-9059Crossref PubMed Scopus (236) Google Scholar), cholesterol side chain cleavage enzyme (SCC, Cyp11a) (6Clemens J.W. Lala D.S. Parker K.L. Richards J.S. Endocrinology. 1994; 134: 1499-1508Crossref PubMed Scopus (152) Google Scholar), aromatase (Cyp19) (7Fitzpatrick S.L. Richards J.S. Mol. Endocrinol. 1993; 7: 341-354PubMed Google Scholar,8Lynch J.P. Lala D.S. Peluso J.J. Luo W. Parker K.L. White B.A. Mol. Endocrinol. 1993; 7: 776-786Crossref PubMed Scopus (213) Google Scholar), Müllerian inhibiting substance (M1S, AMH) (9Shen W.H. Moore C.C. Ikeda Y. Parker K.L. Ingraham H.A. Cell. 1994; 77: 651-661Abstract Full Text PDF PubMed Scopus (482) Google Scholar), and luteinizing hormone (LH) β subunit (10Halvorson L.M. Ito M. Jameson J.L. Chin W.W. J. Biol. Chem. 1998; 273: 14712-14720Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). SF-1 belongs to the nuclear receptor superfamily (NR5A1) and shares several well conserved domains with other family members. These regions include an amino-terminal, two zinc finger DNA-binding domain, a putative ligand-binding/dimerization domain, and a carboxyl-terminal AF2 transactivation domain (see Fig. 1 A) (11Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6026) Google Scholar, 12Ito M., Yu, R.N. Jameson J.L. Mol. Endocrinol. 1998; 12: 290-301Crossref PubMed Scopus (123) Google Scholar). SF-1 regulates gene transcription through its interactions with nuclear receptor coactivators such as steroid receptor coactivator-1 and CREB-binding protein (12Ito M., Yu, R.N. Jameson J.L. Mol. Endocrinol. 1998; 12: 290-301Crossref PubMed Scopus (123) Google Scholar, 13Monte D. DeWitte F. Hum D.W. J. Biol. Chem. 1998; 273: 4585-4591Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) as well as by direct interactions with other transcription factors such as DAX-1, Egr-1, SOX9, WT-1, and CREB (10Halvorson L.M. Ito M. Jameson J.L. Chin W.W. J. Biol. Chem. 1998; 273: 14712-14720Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 14Ito M., Yu, R. Jameson J.L. Mol. Cell. Biol. 1997; 17: 1476-1483Crossref PubMed Scopus (390) Google Scholar, 15De Santa Barbara P. Bonneaud N. Boizet B. Desclozeaux M. Moniot B. Sudbeck P. Scherer G. Poulat F. Berta P. Mol. Cell. Biol. 1998; 18: 6653-6665Crossref PubMed Scopus (505) Google Scholar, 16Nachtigal M.W. Hirokawa Y. Enyeart-VanHouten D.L. Flanagan J.N. Hammer G.D. Ingraham H.A. Cell. 1998; 93: 445-454Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 17Ito M. Park Y. Weck J. Mayo K.E. Jameson J.L. Mol. Endocrinol. 2000; 14: 66-81PubMed Google Scholar). In general, members of the nuclear receptor superfamily bind to DNA as dimers. Each monomer within these dimer pairs interacts with a half-site sequence, AGNNCA (18Luisi B.F. Xu W.X. Otwinowski Z. Freedman L.P. Yamamoto K.R. Sigler P.B. Nature. 1991; 352: 497-505Crossref PubMed Scopus (1217) Google Scholar, 19Schwabe J.W. Chapman L. Finch J.T. Rhodes D. Cell. 1993; 75: 567-578Abstract Full Text PDF PubMed Scopus (588) Google Scholar, 20Glass C.K. Endocr. Rev. 1994; 15: 391-407PubMed Google Scholar). Based on half-site specificity, nuclear receptors can be divided into two subgroups. The estrogen receptor (ER) subgroup, which also includes the thyroid hormone receptor (TR) and retinoic acid receptor among others, recognizes the core sequence, AGGTCA. The glucocorticoid receptor (GR) subgroup, which includes the androgen receptor and progesterone receptor, has distinct half-site specificity for AGAACA. The two central nucleotides in the half-site sequence therefore largely determine DNA sequence recognition by the two groups of nuclear receptors. Structure-function analyses reveal that nuclear receptors distinguish these two nucleotides via three amino acid residues in the proximal (P)-box of the first zinc finger (21Mader S. Kumar V. de Verneuil H. Chambon P. Nature. 1989; 338: 271-274Crossref PubMed Scopus (358) Google Scholar, 22Danielsen M. Hinck L. Ringold G.M. Cell. 1989; 57: 1131-1138Abstract Full Text PDF PubMed Scopus (257) Google Scholar, 23Umesono K. Evans R.M. Cell. 1989; 57: 1139-1146Abstract Full Text PDF PubMed Scopus (720) Google Scholar). These P-box residues are located within a DNA recognition α-helix that interacts with the major groove of the DNA response element (18Luisi B.F. Xu W.X. Otwinowski Z. Freedman L.P. Yamamoto K.R. Sigler P.B. Nature. 1991; 352: 497-505Crossref PubMed Scopus (1217) Google Scholar). In contrast to these dimeric receptors, a small group of orphan nuclear receptors binds to DNA as monomers. These factors recognize the estrogen response element (ERE)-type half-site, AGGTCA, and include SF-1, FTZ-F1, NGFI-B, RevErbA, and ROR α (24Tsukiyama T. Niwa O. Nucleic Acids Res. 1992; 20: 1477-1482Crossref PubMed Scopus (18) Google Scholar, 25Ueda H. Sun G.C. Murata T. Hirose S. Mol. Cell. Biol. 1992; 12: 5667-5672Crossref PubMed Scopus (171) Google Scholar, 26Wilson T.E. Paulsen R.E. Padgett K.A. Milbrandt J. Science. 1992; 256: 107-110Crossref PubMed Scopus (277) Google Scholar, 27Wilson T.E. Fahrner T.J. Milbrandt J. Mol. Cell. Biol. 1993; 13: 5794-5804Crossref PubMed Scopus (356) Google Scholar, 28Harding H.P. Lazar M.A. Mol. Cell. Biol. 1993; 13: 3113-3121Crossref PubMed Google Scholar, 29Giguere V. Tini M. Flock G. Ong E. Evans R.M. Otulakowski G. Genes Dev. 1994; 8: 538-553Crossref PubMed Scopus (448) Google Scholar). Several of these receptors share a 30-amino acid basic region carboxyl-terminal to the second zinc finger designated the FTZ-F1 box or A-box (25Ueda H. Sun G.C. Murata T. Hirose S. Mol. Cell. Biol. 1992; 12: 5667-5672Crossref PubMed Scopus (171) Google Scholar, 26Wilson T.E. Paulsen R.E. Padgett K.A. Milbrandt J. Science. 1992; 256: 107-110Crossref PubMed Scopus (277) Google Scholar, 27Wilson T.E. Fahrner T.J. Milbrandt J. Mol. Cell. Biol. 1993; 13: 5794-5804Crossref PubMed Scopus (356) Google Scholar). This A-box is proposed to recognize additional nucleotide sequences 5′ to the AGGTCA motif in the minor groove of the response element DNA. For example, SF-1 binds preferentially to the 5′-flanking sequencePyCA AGGTCA (where Py is pyrimidine), whereas NGFI-B recognizes AA AGGTCA. Recently, we reported a SF-1 mutation in a patient with XY sex reversal, primary adrenal insufficiency, dysgenetic testes, and persistent Müllerian structures (30Achermann J.C. Ito M. Ito M. Hindmarsh P.C. Jameson J.L. Nat. Genet. 1999; 22: 125-126Crossref PubMed Scopus (541) Google Scholar). The heterozygous G35E mutation involves the last amino acid in the P-box, an area shown previously to be critical for DNA binding of other nuclear receptors (21Mader S. Kumar V. de Verneuil H. Chambon P. Nature. 1989; 338: 271-274Crossref PubMed Scopus (358) Google Scholar, 22Danielsen M. Hinck L. Ringold G.M. Cell. 1989; 57: 1131-1138Abstract Full Text PDF PubMed Scopus (257) Google Scholar). As expected, this mutant SF-1 protein failed to bind to SF-1 response elements such as those found in the proximal promoter of SCC (Cyp11a), and no transactivation of this gene was seen (30Achermann J.C. Ito M. Ito M. Hindmarsh P.C. Jameson J.L. Nat. Genet. 1999; 22: 125-126Crossref PubMed Scopus (541) Google Scholar). However, because SF-1 response elements are variable, we considered the possibility that this P-box mutant might recognize other SF-1 response elements. In this report, we examined mutant SF-1 binding to response elements from a variety of target genes. These studies revealed surprising heterogeneity in the SF-1 binding interactions with various target sequences, thereby allowing detailed analysis of the structural determinants in SF-1 and its recognition sites that mediate receptor binding. We find that interactions between the A-box and the 5′-flanking sequence of the response element stabilize monomeric binding by SF-1, especially in situations where binding affinity is compromised by P-box mutations. DISCUSSIONNuclear receptors regulate gene transcription in diverse biological systems. DNA binding specificity is crucial, therefore, so that different receptors can recognize their specific target genes appropriately. Several studies have shown that the DNA binding specificity of classic ligand-dependent nuclear receptors is determined by the P-box amino acid sequence (for example, GSV for the GR, EGA for the ER, and EGG for the TR (Fig. 4 A) (18Luisi B.F. Xu W.X. Otwinowski Z. Freedman L.P. Yamamoto K.R. Sigler P.B. Nature. 1991; 352: 497-505Crossref PubMed Scopus (1217) Google Scholar, 19Schwabe J.W. Chapman L. Finch J.T. Rhodes D. Cell. 1993; 75: 567-578Abstract Full Text PDF PubMed Scopus (588) Google Scholar, 20Glass C.K. Endocr. Rev. 1994; 15: 391-407PubMed Google Scholar, 21Mader S. Kumar V. de Verneuil H. Chambon P. Nature. 1989; 338: 271-274Crossref PubMed Scopus (358) Google Scholar, 22Danielsen M. Hinck L. Ringold G.M. Cell. 1989; 57: 1131-1138Abstract Full Text PDF PubMed Scopus (257) Google Scholar)). The GS sequence of the GR recognizes the GRE-type half-site, AGAACA, whereas the EG residues of the ER and TR recognize the ERE-type half-site, AGGTCA. Comparatively little is known about the DNA binding specificity of SF-1, an orphan nuclear receptor that has no known ligand and one of the few nuclear receptors that binds DNA as a monomer. SF-1 has a hybrid P-box sequence (ESG) and recognizes a consensus response element consisting of an ERE-type half-site and three preceding nucleotides (PyCA AGGTCA) (35Parker K.L. Schimmer B.P. Endocr. Rev. 1997; 18: 361-377Crossref PubMed Scopus (556) Google Scholar). The recent discovery of a naturally occurring human SF-1 P-box mutation (G35E) (30Achermann J.C. Ito M. Ito M. Hindmarsh P.C. Jameson J.L. Nat. Genet. 1999; 22: 125-126Crossref PubMed Scopus (541) Google Scholar) led us to investigate the structural determinants of DNA binding specificity of SF-1 further.Initial studies showed that the G35E SF-1 mutant did not bind to either of two SF-1 response elements present in the proximal promoter region of the SCC gene (30Achermann J.C. Ito M. Ito M. Hindmarsh P.C. Jameson J.L. Nat. Genet. 1999; 22: 125-126Crossref PubMed Scopus (541) Google Scholar). This lack of binding might be predicted, as a similar glycine to glutamic acid mutation at the third P-box position resulted in loss of TR binding (33Nelson C.C. Hendy S.C. Romaniuk P.J. J. Biol. Chem. 1995; 270: 16988-16994Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 34Nelson C.C. Hendy S.C. Romaniuk P.J. J. Biol. Chem. 1995; 270: 16981-16987Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Also, the patient's phenotype of primary adrenal failure is consistent with impaired transcription of steroidogenic enzymes. However, SF-1 regulates the transcription of a wide array of target genes (35Parker K.L. Schimmer B.P. Endocr. Rev. 1997; 18: 361-377Crossref PubMed Scopus (556) Google Scholar). Many of these have response elements that deviate from the consensus SF-1 sequence such as the murine DAX-1 composite enhancer (Table I; TCG AGGTCA, TCA TGGCCA) (4Yu R.N. Ito M. Jameson J.L. Mol. Endocrinol. 1998; 12: 1010-1022Crossref PubMed Scopus (111) Google Scholar) or the inhibin α gene, where SF-1 regulates gene transcription through its interaction with CREB bound to the cAMP-response element (17Ito M. Park Y. Weck J. Mayo K.E. Jameson J.L. Mol. Endocrinol. 2000; 14: 66-81PubMed Google Scholar). We hypothesized therefore that the G35E mutant might exhibit different DNA binding specificity with respect to other SF-1 response elements.Among the SF-1 response elements studied, the G35E mutant was found to bind to the MIS and aromatase promoters with an affinity approaching that of wild-type SF-1. These promoters share a common SF-1 response element, CCA AGGTCA. Comparison of this sequence with other native SF-1 response elements (Table I) led us to speculate that the first position of the three nucleotides in the 5′-flanking sequence and the fourth and fifth nucleotides of the nuclear receptor half-site itself may influence the DNA binding specificity of the G35E mutant. These nucleotides were replaced with those found in other native promoters to investigate this further. Substitution of the flanking cytosine (CCA) with thymine (TCA) in the aromatase SF-1 response element actually strengthened binding by the mutant SF-1, highlighting the importance of a pyrimidine in this flanking sequence. However, binding was impaired significantly following substitution of this nucleotide with a purine (ACA, GCA) or alteration of the central part of the half-site, supporting the hypothesis that the first nucleotide of the flanking sequence, as well as the composition of the half-site, influence DNA binding by the P-box motif.The role of each of the three P-box amino acid residues in half-site recognition has been studied previously for a variety of nuclear receptors, which allowed us to predict the outcome of mutating these codons in SF-1 (36Zilliacus J. Wright A.P. Carlstedt-Duke J. Gustafsson J.A. Mol. Endocrinol. 1995; 9: 389-400Crossref PubMed Google Scholar). The first P-box residue (position 31 for SF-1) is a glutamic acid in SF-1, ER, and TR and a glycine in GR. This glutamic acid facilitates binding to response elements containing guanine at the third position of the nuclear receptor half-site (ERE, AGGTCA) and inhibits binding to those containing adenine (GRE, AGAACA) (37Zilliacus J. Wright A.P. Norinder U. Gustafsson J.A. Carlstedt-Duke J. J. Biol. Chem. 1992; 267: 24941-24947Abstract Full Text PDF PubMed Google Scholar). Reduced binding of the SF-1 E31G mutant is consistent with these observations. At the second P-box position (32 for SF-1) SF-1 and GR have a serine, and ER and TR have a glycine. Replacement of this serine with glycine has been shown to increase binding of GR to an ERE-type half-site (37Zilliacus J. Wright A.P. Norinder U. Gustafsson J.A. Carlstedt-Duke J. J. Biol. Chem. 1992; 267: 24941-24947Abstract Full Text PDF PubMed Google Scholar). Consistent with these findings, the S32G SF-1 mutant showed similar DNA binding specificity to wild-type SF-1. Finally, the third P-box residue (position 35 for SF-1) is a glycine in SF-1 and TR, an alanine in ER, and a valine in GR. Like the glutamic acid in the first P-box position, this valine has a dual function of facilitating binding to response elements containing adenine at the fourth position of the nuclear receptor half-site (GRE, AGAACA) and inhibiting binding to those containing thymine (ERE, AGGTCA) (37Zilliacus J. Wright A.P. Norinder U. Gustafsson J.A. Carlstedt-Duke J. J. Biol. Chem. 1992; 267: 24941-24947Abstract Full Text PDF PubMed Google Scholar). Contrary to our prediction, the G35V mutant bound reasonably well to the wild-type response element (half-site; AGGTCA). However, binding of this mutant to the modified half-sites (M4, AGGCTA; M5, AGGCCA) was reduced dramatically presumably due to the mutation introduced into the fourth nucleotide position of the half-site. Taken together, these data demonstrate that the three amino acid residues in the P-box are critical for recognition of the half-site by SF-1, a nuclear receptor that binds to DNA as a monomer. It is also notable that the P-box sequence of SF-1 (ESG) showed the same DNA binding specificity as that of the TR (EGG) because monomeric TR binding has been demonstrated in certain circumstances (38Katz R.W. Koenig R.J. J. Biol. Chem. 1993; 268: 19392-19397Abstract Full Text PDF PubMed Google Scholar).Mutations of the first nucleotide in the 5′-flanking sequence affected binding of several P-box mutants in addition to the G35E mutant protein. This observation provides further evidence that SF-1 binding can be influenced by the 5′-flanking sequence as well as the central part of the nuclear receptor half-site itself. However, it is unclear whether this represents a direct interaction between the P-box motif and the 5′-flanking sequence or whether the flanking sequence has an indirect effect by stabilizing the bound protein-DNA complex. Because it has been suggested that the FTZ-F1/SF-1 A-box can interact directly with this 5′-flanking sequence without interacting with the half-site (25Ueda H. Sun G.C. Murata T. Hirose S. Mol. Cell. Biol. 1992; 12: 5667-5672Crossref PubMed Scopus (171) Google Scholar), we introduced two mutations into the SF-1 A-box based on previous studies of FTZ-F1. The A-box mutations, R92Q (R589Q in FTZ-F1) and K94Q (K591Q in FTZ-F1), did not affect DNA binding on their own. However, introduction of the R92Q mutation in the background of the G35E mutant reduced binding quite dramatically. Taken together, these data support the idea that the A-box of SF-1 can stabilize monomeric binding to half-sites through an interaction with the 5′-flanking sequence in the minor groove of DNA. The additional binding energy provided by this contact may facilitate monomeric binding by receptors, thereby compensating for the stability otherwise provided by receptor dimerization. This interaction may be particularly important when P-box binding is compromised either by mutations in SF-1 or by sequence alterations in its binding site.In this particular patient, activation of the aromatase promoter by the G35E SF-1 mutant probably had little clinical significance because of multiple other deficiencies in steroidogenesis. In addition, activation of the MIS promoter may have had little effect if the dysgenetic testes had reduced capacity to produce MIS. Nevertheless, this study does indicate that a single mutation in SF-1 can exert differential effects on various target genes. This concept may also apply to other transcription factors that regulate multiple downstream target genes with variant response elements. Nuclear receptors regulate gene transcription in diverse biological systems. DNA binding specificity is crucial, therefore, so that different receptors can recognize their specific target genes appropriately. Several studies have shown that the DNA binding specificity of classic ligand-dependent nuclear receptors is determined by the P-box amino acid sequence (for example, GSV for the GR, EGA for the ER, and EGG for the TR (Fig. 4 A) (18Luisi B.F. Xu W.X. Otwinowski Z. Freedman L.P. Yamamoto K.R. Sigler P.B. Nature. 1991; 352: 497-505Crossref PubMed Scopus (1217) Google Scholar, 19Schwabe J.W. Chapman L. Finch J.T. Rhodes D. Cell. 1993; 75: 567-578Abstract Full Text PDF PubMed Scopus (588) Google Scholar, 20Glass C.K. Endocr. Rev. 1994; 15: 391-407PubMed Google Scholar, 21Mader S. Kumar V. de Verneuil H. Chambon P. Nature. 1989; 338: 271-274Crossref PubMed Scopus (358) Google Scholar, 22Danielsen M. Hinck L. Ringold G.M. Cell. 1989; 57: 1131-1138Abstract Full Text PDF PubMed Scopus (257) Google Scholar)). The GS sequence of the GR recognizes the GRE-type half-site, AGAACA, whereas the EG residues of the ER and TR recognize the ERE-type half-site, AGGTCA. Comparatively little is known about the DNA binding specificity of SF-1, an orphan nuclear receptor that has no known ligand and one of the few nuclear receptors that binds DNA as a monomer. SF-1 has a hybrid P-box sequence (ESG) and recognizes a consensus response element consisting of an ERE-type half-site and three preceding nucleotides (PyCA AGGTCA) (35Parker K.L. Schimmer B.P. Endocr. Rev. 1997; 18: 361-377Crossref PubMed Scopus (556) Google Scholar). The recent discovery of a naturally occurring human SF-1 P-box mutation (G35E) (30Achermann J.C. Ito M. Ito M. Hindmarsh P.C. Jameson J.L. Nat. Genet. 1999; 22: 125-126Crossref PubMed Scopus (541) Google Scholar) led us to investigate the structural determinants of DNA binding specificity of SF-1 further. Initial studies showed that the G35E SF-1 mutant did not bind to either of two SF-1 response elements present in the proximal promoter region of the SCC gene (30Achermann J.C. Ito M. Ito M. Hindmarsh P.C. Jameson J.L. Nat. Genet. 1999; 22: 125-126Crossref PubMed Scopus (541) Google Scholar). This lack of binding might be predicted, as a similar glycine to glutamic acid mutation at the third P-box position resulted in loss of TR binding (33Nelson C.C. Hendy S.C. Romaniuk P.J. J. Biol. Chem. 1995; 270: 16988-16994Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 34Nelson C.C. Hendy S.C. Romaniuk P.J. J. Biol. Chem. 1995; 270: 16981-16987Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Also, the patient's phenotype of primary adrenal failure is consistent with impaired transcription of steroidogenic enzymes. However, SF-1 regulates the transcription of a wide array of target genes (35Parker K.L. Schimmer B.P. Endocr. Rev. 1997; 18: 361-377Crossref PubMed Scopus (556) Google Scholar). Many of these have response elements that deviate from the consensus SF-1 sequence such as the murine DAX-1 composite enhancer (Table I; TCG AGGTCA, TCA TGGCCA) (4Yu R.N. Ito M. Jameson J.L. Mol. Endocrinol. 1998; 12: 1010-1022Crossref PubMed Scopus (111) Google Scholar) or the inhibin α gene, where SF-1 regulates gene transcription through its interaction with CREB bound to the cAMP-response element (17Ito M. Park Y. Weck J. Mayo K.E. Jameson J.L. Mol. Endocrinol. 2000; 14: 66-81PubMed Google Scholar). We hypothesized therefore that the G35E mutant might exhibit different DNA binding specificity with respect to other SF-1 response elements. Among the SF-1 response elements studied, the G35E mutant was found to bind to the MIS and aromatase promoters with an affinity approaching that of wild-type SF-1. These promoters share a common SF-1 response element, CCA AGGTCA. Comparison of this sequence with other native SF-1 response elements (Table I) led us to speculate that the first position of the three nucleotides in the 5′-flanking sequence and the fourth and fifth nucleotides of the nuclear receptor half-site itself may influence the DNA binding specificity of the G35E mutant. These nucleotides were replaced with those found in other native promoters to investigate this further. Substitution of the flanking cytosine (CCA) with thymine (TCA) in the aromatase SF-1 response element actually strengthened binding by the mutant SF-1, highlighting the importance of a pyrimidine in this flanking sequence. However, binding was impaired significantly following substitution of this nucleotide with a purine (ACA, GCA) or alteration of the central part of the half-site, supporting the hypothesis that the first nucleotide of the flanking sequence, as well as the composition of the half-site, influence DNA binding by the P-box motif. The role of each of the three P-box amino acid residues in half-site recognition has been studied previously for a variety of nuclear receptors, which allowed us to predict the outcome of mutating these codons in SF-1 (36Zilliacus J. Wright A.P. Carlstedt-Duke J. Gustafsson J.A. Mol. Endocrinol. 1995; 9: 389-400Crossref PubMed Google Scholar). The first P-box residue (position 31 for SF-1) is a glutamic acid in SF-1, ER, and TR and a glycine in GR. This glutamic acid facilitates binding to response elements containing guanine at the third position of the nuclear receptor half-site (ERE, AGGTCA) and inhibits binding to those containing adenine (GRE, AGAACA) (37Zilliacus J. Wright A.P. Norinder U. Gustafsson J.A. Carlstedt-Duke J. J. Biol. Chem. 1992; 267: 24941-24947Abstract Full Text PDF PubMed Google Scholar). Reduced binding of the SF-1 E31G mutant is consistent with these observations. At the second P-box position (32 for SF-1) SF-1 and GR have a serine, and ER and TR have a glycine. Replacement of this serine with glycine has been shown to increase binding of GR to an ERE-type half-site (37Zilliacus J. Wright A.P. Norinder U. Gustafsson J.A. Carlstedt-Duke J. J. Biol. Chem. 1992; 267: 24941-24947Abstract Full Text PDF PubMed Google Scholar). Consistent with these findings, the S32G SF-1 mutant showed similar DNA binding specificity to wild-type SF-1. Finally, the third P-box residue (position 35 for SF-1) is a glycine in SF-1 and TR, an alanine in ER, and a valine in GR. Like the glutamic acid in the first P-box position, this valine has a dual function of facilitating binding to response elements containing adenine at the fourth position of the nuclear receptor half-site (GRE, AGAACA) and inhibiting binding to those containing thymine (ERE, AGGTCA) (37Zilliacus J. Wright A.P. Norinder U. Gustafsson J.A. Carlstedt-Duke J. J. Biol. Chem. 1992; 267: 24941-24947Abstract Full Text PDF PubMed Google Scholar). Contrary to our prediction, the G35V mutant bound reasonably well to the wild-type response element (half-site; AGGTCA). However, binding of this mutant to the modified half-sites (M4, AGGCTA; M5, AGGCCA) was reduced dramatically presumably due to the mutation introduced into the fourth nucleotide position of the half-site. Taken together, these data demonstrate that the three amino acid residues in the P-box are critical for recognition of the half-site by SF-1, a nuclear receptor that binds to DNA as a monomer. It is also notable that the P-box sequence of SF-1 (ESG) showed the same DNA binding specificity as that of the TR (EGG) because monomeric TR binding has been demonstrated in certain circumstances (38Katz R.W. Koenig R.J. J. Biol. Chem. 1993; 268: 19392-19397Abstract Full Text PDF PubMed Google Scholar). Mutations of the first nucleotide in the 5′-flanking sequence affected binding of several P-box mutants in addition to the G35E mutant protein. This observation provides further evidence that SF-1 binding can be influenced by the 5′-flanking sequence as well as the central part of the nuclear receptor half-site itself. However, it is unclear whether this represents a direct interaction between the P-box motif and the 5′-flanking sequence or whether the flanking sequence has an indirect effect by stabilizing the bound protein-DNA complex. Because it has been suggested that the FTZ-F1/SF-1 A-box can interact directly with this 5′-flanking sequence without interacting with the half-site (25Ueda H. Sun G.C. Murata T. Hirose S. Mol. Cell. Biol. 1992; 12: 5667-5672Crossref PubMed Scopus (171) Google Scholar), we introduced two mutations into the SF-1 A-box based on previous studies of FTZ-F1. The A-box mutations, R92Q (R589Q in FTZ-F1) and K94Q (K591Q in FTZ-F1), did not affect DNA binding on their own. However, introduction of the R92Q mutation in the background of the G35E mutant reduced binding quite dramatically. Taken together, these data support the idea that the A-box of SF-1 can stabilize monomeric binding to half-sites through an interaction with the 5′-flanking sequence in the minor groove of DNA. The additional binding energy provided by this contact may facilitate monomeric binding by receptors, thereby compensating for the stability otherwise provided by receptor dimerization. This interaction may be particularly important when P-box binding is compromised either by mutations in SF-1 or by sequence alterations in its binding site. In this particular patient, activation of the aromatase promoter by the G35E SF-1 mutant probably had little clinical significance because of multiple other deficiencies in steroidogenesis. In addition, activation of the MIS promoter may have had little effect if the dysgenetic testes had reduced capacity to produce MIS. Nevertheless, this study does indicate that a single mutation in SF-1 can exert differential effects on various target genes. This concept may also apply to other transcription factors that regulate multiple downstream target genes with variant response elements. We thank T. Kotlar and L. Sabacan for assistance with DNA sequencing." @default.
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• W2076249557 title "A Naturally Occurring Steroidogenic Factor-1 Mutation Exhibits Differential Binding and Activation of Target Genes" @default.
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