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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 Cterminal 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, the testes-determining factor encoded by the human Y chromosome (1), contains a high-mobility group (HMG) 1 box (2-4), a conserved motif of DNA bending (Fig. 1, A and C, and Ref. 5). 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. 3 and 6 -8). Clinical mutations cluster in the HMG box 2 and most commonly impair specific DNA binding (7,9,10). SRY is a nuclear protein (11) expressed in the primordial Sertoli cells of the differentiating gonadal ridge (12-14). Although SRY is presumed to function as an architectural transcription factor (9, 15, 16), its downstream genetic pathway is not well characterized (for a review, see Ref. 17).Immunohistochemical studies of murine and human embryos have demonstrated that SRY is a nuclear protein (11,18). Nuclear localization signals (NLSs) in human SRY have been defined in cell culture. Berta and colleagues (11), using microinjection of proteins in adult human fibroblastic cells, identified an NLS in the N-terminal region of the human HMG box 3 ( Fig. 2A; SRY residues 59 -75). This NLS comprises two sets of basic amino acids separated by 12 residues (Fig. 2B), features characteristic of bipartite NLS motifs in diverse proteins (19,20). 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. 2A). 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. 2A). Although these findings appear to exclude a second NLS in SRY, Sü dbeck and Scherer (21) subsequently used a complementary methodology (transient transfection of SRY-␤-galactosidase fusion genes in COS-7 cells; Fig. 2B)...

The testis-determining factor SRY contains an HMG box DNA-bending domain. Human and murine factors (hSRY and mSRY, respectively) exhibit marked sequence divergence and are reported to differ markedly in DNA bending properties. Surprisingly, the combined application of time-resolved fluorescence resonance energy transfer (tr-FRET) and permutation gel electrophoresis demonstrates that the hSRY-DNA complex is more sharply bent than the murine complex and not less bent as previously reported. tr-FRET-based analyses of the distribution of end-to-end distances in the bent DNA-protein complexes further suggest that a broader range of DNA bend angles is populated in the murine ensemble than in the human ensemble. The two domains and their respective DNA complexes nevertheless exhibit similar thermodynamic stabilities. (1)H NMR spectra indicate analogous intercalation of distinct "cantilever" side chains (isoleucine or methionine) with subtle differences in induced DNA structure. Interchange of cantilevers does not affect DNA bending. That transgenic expression of either human or murine Sry in XX mice can confer a male somatic phenotype suggests that SRY-directed transcriptional regulation is robust to enhanced DNA bending and to changes in the precision of DNA bending. We propose that male-specific gene regulation requires DNA bending above a critical threshold set by architectural requirements of enhanceosome assembly.