Human Sex Reversal Due to Impaired Nuclear Localization of SRY

Abstract

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)...

Human SRY inhibits β-catenin-mediated transcription

“…Interestingly, R133W and R76P, which have similar nuclear import defects, 52% and 50% of wild-type activity respectively (Harley et al, 2003, Sim et al, 2005, displayed differing abilities to inhibit TOPFLASH activity induced by β-catenin. R133W, which retains almost wild-type DNA binding and bending activities (Harley et al, 2003, Li et al, 2001, was less potent in inhibiting β-catenin-induced TOPFLASH activity than R76P which retains only 33% of DNA binding activity (Harley et al, 2003). We conclude that SRY does not seem to require DNA-binding properties to inhibit β-catenin signaling.…”

“…Error bars indicate standard errors of the means for triplicate samples from two independent experiments. (Mitchell and Harley, 2002); 2 (Assumpcao et al, 2002); 3 (Pontiggia et al, 1994); 4 (Harley et al, 2003); 5 (Li et al, 2001)). (C).…”

Defective importin β recognition and nuclear import of the sex-determining factor SRY are associated with XY sex-reversing mutations

“…The oligonucleotide used containing the SRY consensus-binding site (underlined) comprised the upper strand sequence GGG TTA ACG TAA ACA ATA AAT CTG GTA GA. Complementary oligonucleotides were annealed and end-filled by using superscript reverse transcriptase in the presence of [␣-32 P]dCTP. EMSAs were performed as described by using either 35 S-labeled in vitrotranslated full-length SRY or recombinant HMG domains (10). For competition experiments, 4 ng of SRY HMG box was incubated with varying amounts of either IMP␣ and͞or IMP␤ before the addition of 20 fmol of SRY DNA consensus probe.…”

Nuclear localization signals and human disease

“…2A). However, the mutations identified in the Swyer syndrome females result in decreased nuclear localization of SRY leading to lower activation of testes-specific genes required for proper XY male testes formation (81,82).…”

“…Clearly, if the import receptor does not recognize the NLS-cargo, then that cargo will remain in the cytoplasm, which could be deleterious if the nuclear role is critical for proper cell function. A recent example where loss of nuclear localization of a key developmental protein has been linked to disease is in Swyer syndrome where developmental defects include male-to-female sex reversal (81,82).…”