Human Sex Reversal Due to Impaired Nuclear Localization of SRY
2001; Elsevier BV; Volume: 276; Issue: 49 Linguagem: Inglês
10.1074/jbc.c100388200
ISSN1083-351X
AutoresYaqin Li, Wei Zhang, Ging Chan, Agnes Jancso-Radek, Shunhe Liu, Michael A. Weiss,
Tópico(s)Animal Genetics and Reproduction
ResumoSRY, an architectural transcription factor encoded by the sex-determining region of the Y chromosome, initiates testicular differentiation in mammalian embryogenesis. The protein contains a high-mobility group (HMG) box, a DNA-bending motif conserved among a broad class of nuclear proteins. Mutations causing human sex reversal (46, XY pure gonadal dysgenesis) are clustered in this domain. Basic N- and C-terminal regions of the HMG box are each proposed to provide nuclear localization signals. The significance of the C-terminal basic cluster (SRY residues 130–134) is uncertain, however, as its activity in cell culture varies with assay conditions. To test its importance, we have investigated a C-terminal sex-reversal mutation (R133W, position 78 of the HMG box). This de novo mutation impairs nuclear localization but not specific DNA binding or sharp DNA bending. Correlation between these properties and the phenotype of the patient suggests that nuclear localization of SRY is required for testicular differentiation and directed in part by the C-terminal basic cluster. To our knowledge, these results provide the first example of impaired organogenesis due to a nuclear localization signal mutation. SRY, an architectural transcription factor encoded by the sex-determining region of the Y chromosome, initiates testicular differentiation in mammalian embryogenesis. The protein contains a high-mobility group (HMG) box, a DNA-bending motif conserved among a broad class of nuclear proteins. Mutations causing human sex reversal (46, XY pure gonadal dysgenesis) are clustered in this domain. Basic N- and C-terminal regions of the HMG box are each proposed to provide nuclear localization signals. The significance of the C-terminal basic cluster (SRY residues 130–134) is uncertain, however, as its activity in cell culture varies with assay conditions. To test its importance, we have investigated a C-terminal sex-reversal mutation (R133W, position 78 of the HMG box). This de novo mutation impairs nuclear localization but not specific DNA binding or sharp DNA bending. Correlation between these properties and the phenotype of the patient suggests that nuclear localization of SRY is required for testicular differentiation and directed in part by the C-terminal basic cluster. To our knowledge, these results provide the first example of impaired organogenesis due to a nuclear localization signal mutation. high-mobility group gel mobility shift assay lymphoid enhancer factor 1 nuclear localization signal phosphate-buffered saline permutation gel electrophoresis sex-determining region of Y chromosome T-cell factor 1 base pair(s) 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside wild type SRY, the testes-determining factor encoded by the human Y chromosome (1Koopman P. Gubbay J. Vivian N. Goodfellow P. Lovell-Badge R. Nature. 1991; 351: 117-121Crossref PubMed Scopus (1783) Google Scholar), contains a high-mobility group (HMG)1 box (2Gubbay J. Collignon J. Koopman P. Capel B. Economou A. Munsterberg A. Vivian N. Goodfellow P. Lovell-Badge R. Nature. 1990; 346: 245-250Crossref PubMed Scopus (1377) Google Scholar, 3Berta P. Hawkins J.R. Sinclair A.H. Taylor A. Griffiths B.L. Goodfellow P.N. Fellous M. Nature. 1990; 348: 448-450Crossref PubMed Scopus (428) Google Scholar, 4Sinclair A.H. Berta P. Palmer M.S. Hawkins J.R. Griffiths B.L. Smith M.J. Foster J.W. Frischauf A.M. Lovell-Badge R. Goodfellow P.N. Nature. 1990; 346: 240-244Crossref PubMed Scopus (2658) Google Scholar), a conserved motif of DNA bending (Fig. 1, A and C, and Ref. 5Ner S.S. Curr. Biol. 1992; 2: 208-210Abstract Full Text PDF PubMed Scopus (101) Google Scholar). Mutations in SRY are associated with 46, XY pure gonadal dysgenesis leading to failure of testicular differentiation and female somatic phenotype (XY sex reversal; Refs. 3Berta P. Hawkins J.R. Sinclair A.H. Taylor A. Griffiths B.L. Goodfellow P.N. Fellous M. Nature. 1990; 348: 448-450Crossref PubMed Scopus (428) Google Scholar and 6Nasrin N. Buggs C. Kong X.F. Carnazza J. Goebl M. Alexander-Bridges M. Nature. 1991; 354: 317-320Crossref PubMed Scopus (155) Google Scholar, 7Harley V.R. Jackson D.I. Hextall P.J. Hawkins J.R. Berkovitz G.D. Sockanathan S. Lovell-Badge R. Goodfellow P.N. Science. 1992; 255: 453-456Crossref PubMed Scopus (379) Google Scholar, 8Hawkins J.R. Taylor A. Berta P. Levilliers J. Van der Auwera B. Goodfellow P.N. Hum. Genet. 1992; 88: 471-474Crossref PubMed Scopus (212) Google Scholar). Clinical mutations cluster in the HMG box 2Of the greater than 30 clinical mutations found in SRY to date only four map outside of the HMG box. One is a familial point mutation N-terminal to the HMG box (S18N) associated with partial gonadal dysgenesis (57Domenice S. Yumie Nishi M. Correia Billerbeck A.E. Latronico A.C. Aparecida Medeiros M. Russell A.J. Vass K. Marino Carvalho F. Costa Frade E.M. Prado Arnhold I.J. Bilharinho Mendonca B. Hum. Genet. 1998; 102: 213-215Crossref PubMed Scopus (61) Google Scholar); the second and third are nonsense mutations at codons 2 and 4 (22Veitia R. Ion A. Barbaux S. Jobling M.A. Souleyreau N. Ennis K. Ostrer H. Tosi M. Meo T. Chibani J. Fellous M. McElreavey K. Hum. Genet. 1997; 99: 648-652Crossref PubMed Scopus (91) Google Scholar, 58Brown S., Yu, C. Lanzano P. Heller D. Thomas L. Warburton D. Kitajewski J. Stadtmauer L. Am. J. Hum. Genet. 1998; 62: 189-192Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), respectively; the fourth causes deletion of the C-terminal 41 residues (59Tajima T. Nakae J. Shinohara N. Fujieda K. Hum. Mol. 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Cell Biol. 1995; 128: 737-748Crossref PubMed Scopus (101) Google Scholar) expressed in the primordial Sertoli cells of the differentiating gonadal ridge (12Koopman P. Munsterberg A. Capel B. Vivian N. Lovell-Badge R. Nature. 1990; 348: 450-452Crossref PubMed Scopus (718) Google Scholar, 13Lovell-Badge R. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 1993; 339: 159-164Crossref PubMed Scopus (33) Google Scholar, 14Hanley N.A. Hagan D.M. Clement-Jones M. Ball S.G. Strachan T. Salas-Cortes L. McElreavey K. Lindsay S. Robson S. Bullen P. Ostrer H. Wilson D.I. Mech. Dev. 2000; 91: 403-407Crossref PubMed Scopus (240) Google Scholar). Although SRY is presumed to function as an architectural transcription factor (9Haqq C.M. King C.Y. Ukiyama E. Falsafi S. Haqq T.N. Donahoe P.K. Weiss M.A. Science. 1994; 266: 1494-1500Crossref PubMed Scopus (230) Google Scholar, 15Dubin R.A. Ostrer H. Mol. Endocrinol. 1994; 8: 1182-1192PubMed Google Scholar, 16Cohen D.R. Sinclair A.H. McGovern J.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4372-4376Crossref PubMed Scopus (66) Google Scholar), its downstream genetic pathway is not well characterized (for a review, see Ref. 17Koopman P. EXS (Basel). 2001; 91: 25-56PubMed Google Scholar). Immunohistochemical studies of murine and human embryos have demonstrated that SRY is a nuclear protein (11Poulat F. Girard F. Chevron M.P. Goze C. Rebillard X. Calas B. Lamb N. Berta P. J. Cell Biol. 1995; 128: 737-748Crossref PubMed Scopus (101) Google Scholar, 18Salas-Cortes L. Jaubert F. Barbaux S. Nessmann C. Bono M.R. Fellous M. McElreavey K. Rosemblatt M. Int. J. Dev. Biol. 1999; 43: 135-140PubMed Google Scholar). Nuclear localization signals (NLSs) in human SRY have been defined in cell culture. Berta and colleagues (11Poulat F. Girard F. Chevron M.P. Goze C. Rebillard X. Calas B. Lamb N. Berta P. J. Cell Biol. 1995; 128: 737-748Crossref PubMed Scopus (101) Google Scholar), using microinjection of proteins in adult human fibroblastic cells, identified an NLS in the N-terminal region of the human HMG box 3Although residues in the N-terminal NLS of SRY contribute to the minor groove DNA-binding surface of the domain, NLS activity does not require DNA binding: a sex-reversal mutation elsewhere in the HMG box (Y127C; Fig. 1 B) impairs specific DNA binding but not nuclear localization (11Poulat F. Girard F. Chevron M.P. Goze C. Rebillard X. Calas B. Lamb N. Berta P. J. Cell Biol. 1995; 128: 737-748Crossref PubMed Scopus (101) Google Scholar). In the structure of an SRY·DNA complex (43Werner M.H. Bianchi M.E. Gronenborn A.M. Clore G.M. Biochemistry. 1995; 34: 11998-12004Crossref PubMed Scopus (51) Google Scholar) Tyr-127 (residue 72) contacts the DNA backbone.(Fig. 2 A; SRY residues 59–75). This NLS comprises two sets of basic amino acids separated by 12 residues (Fig. 2 B), features characteristic of bipartite NLS motifs in diverse proteins (19Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1290) Google Scholar, 20Boulikas T. Crit. Rev. Eukaryot. Gene Expr. 1993; 3: 193-227PubMed Google Scholar). An isolated N-terminal SRY peptide (residues 58–78) was shown to be sufficient to direct nuclear translocation of coupled rabbit IgG (protein SRY21 in Fig. 2 A). By contrast the remainder of the HMG box (residues 74–137) was unable to direct nuclear translocation of coupled rabbit IgG (protein SRY64 in Fig.2 A). Although these findings appear to exclude a second NLS in SRY, Südbeck and Scherer (21Sudbeck P. Scherer G. J. Biol. Chem. 1997; 272: 27848-27852Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) subsequently used a complementary methodology (transient transfection of SRY-β-galactosidase fusion genes in COS-7 cells; Fig. 2 B) to identify a basic cluster NLS (residues 130–134;underlined in Fig.1 B, top sequence) in the C-terminal tail of the HMG box (highlighted in red in Fig. 1, A–C). Whereas the microinjection assay suggested that the N-terminal NLS is sufficient to direct complete nuclear localization, in the transient transfection assay both N- and C-terminal NLS motifs are required (21Sudbeck P. Scherer G. J. Biol. Chem. 1997; 272: 27848-27852Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). These differences may be due to assay procedures and/or cell lines. 4It is possible that in a fibroblastic cell line the C-terminal NLS functions too weakly to be detected given the low peptide-IgG coupling ratio used in the microinjection assay (11Poulat F. Girard F. Chevron M.P. Goze C. Rebillard X. Calas B. Lamb N. Berta P. J. Cell Biol. 1995; 128: 737-748Crossref PubMed Scopus (101) Google Scholar). The function of the NLSs of SRY has not been established in vivo as to date no clinical mutations have been shown to impair nuclear localization. In this communication we provide evidence for the physiological importance of the putative C-terminal NLS. Experimental design exploits a de novo sex-reversal mutation in the C-terminal basic cluster (R133W; Fig. 1 B and Ref. 22Veitia R. Ion A. Barbaux S. Jobling M.A. Souleyreau N. Ennis K. Ostrer H. Tosi M. Meo T. Chibani J. Fellous M. McElreavey K. Hum. Genet. 1997; 99: 648-652Crossref PubMed Scopus (91) Google Scholar) to distinguish between DNA binding and nuclear localization. The site of mutation is disordered in the NMR structure of a specific SRY·DNA complex (Fig. 1 C and Ref. 10Werner M.H. Huth J.R. Gronenborn A.M. Clore G.M. Cell. 1995; 81: 705-714Abstract Full Text PDF PubMed Scopus (435) Google Scholar). Although the variant SRY domain exhibits essentially native DNA recognition, the mutation impairs nuclear translocation in a rat embryogenic gonadal ridge cell line. Control substitution R133A also impairs NLS function, suggesting that mislocalization is due to loss of the native side chain rather than specific interference by tryptophan. Correlation between impaired nuclear localization and phenotype (XY sex reversal) strongly suggests that the C-terminal basic cluster contributes to the nuclear localization of SRY in vivo and that such localization is required for testicular differentiation. To our knowledge, this is the first example of impaired organogenesis associated with mutation of an NLS. Native and variant SRY HMG box domains (84 amino acid; residues 57–140) were expressed in Escherichia coli as thrombin-cleavable fusion proteins and purified as described previously (23King C.Y. Weiss M.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11990-11994Crossref PubMed Scopus (122) Google Scholar). The final SRY fragment (86 residues) contains two additional N-terminal residues (Gly-Ser) derived from the thrombin site. Purity was >98% as assessed by SDS-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography. Predicted molecular masses of SRY fragments were verified by mass spectrometry. Spectra were obtained using an Aviv spectropolarimeter in 50 mm KCl and 10 mm potassium phosphate (pH 7.4) as described previously (24Haqq C.M. King C.Y. Donahoe P.K. Weiss M.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1097-1101Crossref PubMed Scopus (141) Google Scholar). The sequence specificity of the SRY HMG box has previously been described (6Nasrin N. Buggs C. Kong X.F. Carnazza J. Goebl M. Alexander-Bridges M. Nature. 1991; 354: 317-320Crossref PubMed Scopus (155) Google Scholar, 7Harley V.R. Jackson D.I. Hextall P.J. Hawkins J.R. Berkovitz G.D. Sockanathan S. Lovell-Badge R. Goodfellow P.N. Science. 1992; 255: 453-456Crossref PubMed Scopus (379) Google Scholar, 9Haqq C.M. King C.Y. Ukiyama E. Falsafi S. Haqq T.N. Donahoe P.K. Weiss M.A. Science. 1994; 266: 1494-1500Crossref PubMed Scopus (230) Google Scholar, 10Werner M.H. Huth J.R. Gronenborn A.M. Clore G.M. Cell. 1995; 81: 705-714Abstract Full Text PDF PubMed Scopus (435) Google Scholar, 23King C.Y. Weiss M.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11990-11994Crossref PubMed Scopus (122) Google Scholar). For use in the gel mobility shift assay (GMSA), a 15-bp DNA probe (23King C.Y. Weiss M.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11990-11994Crossref PubMed Scopus (122) Google Scholar) was prepared containing sequence 5′-GTGATTGTTCAG-3′ and complement (core target site in bold). The probe was labeled with 33P and annealed. Each reaction contained 6.25–50 nm protein (see caption to Fig.2 C) and 95% of COS-7 cells (108 of 111 cells counted) and ∼80% of CH34 cells (124 of 154 cells counted) (Fig. 3,column 4). Similar results were obtained by the immunohistochemical assay. Like the SRY-D3 negative control, R133W and R133A constructions (Fig. 3, columns 2 and 3, respectively) gave rise to pancellular distribution in more than 80% of cells analyzed by either assay. β-Galactosidase staining in COS-7 cells was pancellular in 83 of 101 cells counted (R133W) and 108 of 122 (R133A); corresponding values in CH34 cells were 57 of 69 (R133W) and 114 of 124 (R133A). Similarly, immunoreactive staining in COS-7 cells was pancellular in 39 of 42 cells counted (R133W) and 21 of 22 (R133A); corresponding values in CH34 cells were 19 of 21 (R133W) and 19 of 22 (R133A). Any differences in outcome between cell lines (Fig. 3,A versus B) or among methods of visualization (Fig. 3, rows a and band a′ and b′) are not of statistical significance. Impairment of nuclear localization due to mutations R133W or R133A is in each case similar to that due to deletion of the basic cluster. Their partial nuclear import presumably reflects continued activity of the N-terminal NLS. SRY was originally identified by genetic analysis of patients with intersex abnormalities (4Sinclair A.H. Berta P. Palmer M.S. Hawkins J.R. Griffiths B.L. Smith M.J. Foster J.W. Frischauf A.M. Lovell-Badge R. Goodfellow P.N. Nature. 1990; 346: 240-244Crossref PubMed Scopus (2658) Google Scholar). In the absence of a prioribiochemical information, the mechanism of action of SRY was hypothesized on the basis of homology between its HMG box and a conserved family of architectural transcription factors. This hypothesis has been corroborated in part by a striking correlation between sites of sex-reversal mutations (3Berta P. Hawkins J.R. Sinclair A.H. Taylor A. Griffiths B.L. Goodfellow P.N. Fellous M. Nature. 1990; 348: 448-450Crossref PubMed Scopus (428) Google Scholar, 6Nasrin N. Buggs C. Kong X.F. Carnazza J. Goebl M. Alexander-Bridges M. Nature. 1991; 354: 317-320Crossref PubMed Scopus (155) Google Scholar, 7Harley V.R. Jackson D.I. Hextall P.J. Hawkins J.R. Berkovitz G.D. Sockanathan S. Lovell-Badge R. Goodfellow P.N. Science. 1992; 255: 453-456Crossref PubMed Scopus (379) Google Scholar, 8Hawkins J.R. Taylor A. Berta P. Levilliers J. Van der Auwera B. Goodfellow P.N. Hum. Genet. 1992; 88: 471-474Crossref PubMed Scopus (212) Google Scholar) and sites in the HMG box required for folding or DNA binding (7Harley V.R. Jackson D.I. Hextall P.J. Hawkins J.R. Berkovitz G.D. Sockanathan S. Lovell-Badge R. Goodfellow P.N. Science. 1992; 255: 453-456Crossref PubMed Scopus (379) Google Scholar, 9Haqq C.M. King C.Y. Ukiyama E. Falsafi S. Haqq T.N. Donahoe P.K. Weiss M.A. Science. 1994; 266: 1494-1500Crossref PubMed Scopus (230) Google Scholar, 30Giese K. Amsterdam A. Grosschedl R. Genes Dev. 1991; 5: 2567-2578Crossref PubMed Scopus (222) Google Scholar, 31Lorenz M. Hillisch A. Payet D. Buttinelli M. Travers A. Diekmann S. Biochemistry. 1999; 38: 12150-12158Crossref PubMed Scopus (68) Google Scholar). Almost all clinical mutations in SRY cluster in the HMG box, and almost allde novo mutations impair DNA binding (9Haqq C.M. King C.Y. Ukiyama E. Falsafi S. Haqq T.N. Donahoe P.K. Weiss M.A. Science. 1994; 266: 1494-1500Crossref PubMed Scopus (230) Google Scholar). The present study highlights a mutation in the C-terminal tail of the HMG box (R133W; Ref. 22Veitia R. Ion A. Barbaux S. Jobling M.A. Souleyreau N. Ennis K. Ostrer H. Tosi M. Meo T. Chibani J. Fellous M. McElreavey K. Hum. Genet. 1997; 99: 648-652Crossref PubMed Scopus (91) Google Scholar) that impairs nuclear localization but not DNA binding. The mutation alters an invariant arginine in a basic cluster previously proposed as an accessory NLS (21Sudbeck P. Scherer G. J. Biol. Chem. 1997; 272: 27848-27852Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). That an alanine substitution likewise impairs nuclear localization suggests that the defect is due to loss of the basic side chain rather than specific interference by the bulky Trp-133 side chain. Because alterations in the C-terminal basic cluster leave intact the N-terminal NLS, effects of C-terminal substitutions are incomplete, leading to a broad pattern of cytoplasmic and nuclear localization. Genetic corroboration of a putative NLS in a transcription factor can be confounded by the location of such signals within DNA-binding domains (32Vandromme M. Cavadore J.C. Bonnieu A. Froeschle A. Lamb N. Fernandez A. Proc. Natl. Acad. Sci. 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