Inherited Human Sex Reversal Due to Impaired Nucleocytoplasmic

Inherited human sex reversal due to impaired PNAS PLUS nucleocytoplasmic trafficking of SRY defines a male transcriptional threshold Yen-Shan Chena, Joseph D. Raccaa, Nelson B. Phillipsa, and Michael A. Weissa,b,c,1 Departments of aBiochemistry, bBiomedical Engineering, and cMedicine, Case Western Reserve University, Cleveland, OH 44106 Edited by Patricia K. Donahoe, Massachusetts General Hospital, Boston, MA, and approved August 14, 2013 (received for review January 16, 2013) Human testis determination is initiated by SRY (sex determining studies of Swyer variants (6). Most often arising de novo in region on Y chromosome). Mutations in SRY cause gonadal dys- spermatogenesis and clustering in the HMG box (Fig. 1A), genesis with female somatic phenotype. Two subtle variants (V60L such mutations typically impair specific DNA binding by direct and I90M in the high-mobility group box) define inherited alleles or indirect perturbation of an angular protein–DNA interface shared by an XY sterile daughter and fertile father. Whereas spe- (6, 12). The present study focuses on inherited mutations cific DNA binding and bending are unaffected in a rat embryonic (V60L and I90M; asterisks in Fig. 1A) that by contrast allow pre-Sertoli cell line, the variants exhibited selective defects in near-native DNA binding and bending (13, 14). Such mutations are nucleocytoplasmic shuttling due to impaired nuclear import compatible with alternative developmental outcomes: testicular (V60L; mediated by Exportin-4) or export (I90M; mediated by chro- differentiation leading to virilization (fertile 46,XY father) or na- mosome region maintenance 1). Decreased shuttling limits nuclear scent ovarian differentiation leading to gonadal dysgenesis (sterile 46,XY daughter) (13). In the structure of the wild-type (WT) SRY– accumulation of phosphorylated (activated) SRY, in turn reducing DNA complex (12), V60 and I90 pack within the minor and major occupancy of DNA sites regulating Sertoli-cell differentiation [the fi Sox9 wings (Fig. 1B) of the HMG box (consensus positions 5 and 35, testis-speci c SRY-box 9 ( ) enhancer]. Despite distinct pat- respectively; Fig. 1C). The clinical substitutions each represent a terns of biochemical and cell-biological perturbations, V60L and Sox9 subtle interchange of nonpolar side chains. Known inherited I90M each attenuated expression in transient transfection mutationsinSRYaresummarizedinSI Appendix,TableS1.* assays by twofold. Such attenuation was also observed in studies Inherited mutations in hSRY provide experiments of nature of V60A, a clinical variant associated with ovotestes and hence probing the threshold molecular properties of a developmental ambiguity between divergent cell fates. This shared twofold switch beyond DNA binding and bending. To this end, our threshold is reminiscent of autosomal syndromes of transcrip- studies exploited a model of the central SRY–Sox9 regulatory tion-factor haploinsufficiency, including XY sex reversal associated axis (Fig. 1D) in a rodent fetal pre-Sertoli cell line (14–16). Our with mutations in SOX9. Our results demonstrate that nucleocyto- findings demonstrate that hSRY undergoes nucleocytoplasmic plasmic shuttling of SRY is necessary for robust initiation of testicular development. Although also characteristic of ungulate Significance orthologs, such shuttling is not conserved among rodents wherein impaired nuclear export of the high-mobility group box and import- Mutations in human SRY (sex determining region on Y chro- dependent phosphorylation are compensated by a microsatel- mosome) associated with somatic sex reversal provide a model lite-associated transcriptional activation domain. Human sex re- for the perturbation of a genetic switch in organogenesis. versal due to subtle defects in the nucleocytoplasmic shuttling of Inherited alleles, associated with either testicular or ovarian SRY suggests that its transcriptional activity lies near the edge of differentiation, provide unique probes of threshold biochemical developmental ambiguity. properties, defining mechanistic borders between functional BIOCHEMISTRY and nonfunctional transcription factors. This study exploited organogenesis | sex determination | gonadogenesis | gene-regulatory two such alleles to demonstrate that bidirectional nucleocy- network | protein-DNA interaction toplasmic trafficking (import–export shuttling) enables robust operation of this switch via phosphorylation at a site external to he male phenotype in therian mammals is determined by Sry the DNA-binding motif of the transcription factor. In accordance T(1), a Y-chromosomal gene encoding an architectural tran- with studies of intersexual mice, our results suggest that human scription factor (TF) (2). SRY contains a central high-mobility SRY functions at the edge of ambiguity. group (HMG) box, a conserved motif of specific DNA binding and bending (Fig. 1A) (3, 4). Assignment of Sry as the testis-determining Author contributions: M.A.W. designed research; Y.-S.C., J.D.R., and N.B.P. performed factor is supported by transgenic mouse models (5) and human research; Y.-S.C., J.D.R., N.B.P., and M.A.W. analyzed data; and Y.-S.C. and M.A.W. wrote mutations leading to gonadal dysgenesis with female somatic the paper. phenotype (Swyer syndrome) (6). Expressed in the pre-Sertoli cells The authors declare no conflict of interest. of the differentiating gonadal ridge, Sry activates Sox9, an auto- This article is a PNAS Direct Submission. somal gene broadly conserved among vertebrate sex-determining 1 pathways (7). Direct binding of Sry to specific DNA sites within the To whom correspondence should be addressed. E-mail: [email protected]. testis-specific core enhancer of Sox9 (TESCO) activates a male- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1300828110/-/DCSupplemental. specific gene-regulatory network in the fetal gonadal ridge (7). Sustained Sox9 expression orchestrates programs of cell–cell *Seven inherited Swyer alleles of SRY have been reported (SI Appendix, Table S1). Anal- ysis of specific DNA binding of one variant (V60L) by a gel mobility-shift assay provided communication, migration, and differentiation leading to gonado- apparent evidence for a severe impairment (>50-fold as estimated by absence of genesis (8), regression of the female primordia (following Sertoli- a shifted band) (53). Although this finding would imply a high transcriptional threshold cell secretion of anti-Müllerian Hormone/Müllerian Inhibiting (as so-marked a perturbation would seem required to render gonadogenesis ambigu- Substance; AMH/MIS) (9), and somatic virilization (through fetal ous), these data are not in accordance with equilibrium-based measurement of the near- Leydig-cell secretion of testosterone) (10). Mutations in this native properties of the variant HMG box (Table 1 and Ref. 14) and so presumably reflected accelerated dissociation of the variant complex during electrophoresis. Tran- pathway are associated with disorders of sexual development (11). scriptional activities of four other inherited variants were probed by reporter-gene Structure–function relationships in human SRY (hSRY) have cotransfection assays on overexpression in Chinese hamster ovarian cells but yielded been investigated through comparative biochemical and biophysical a range of results (13), precluding assessment of an SRY transcriptional threshold. www.pnas.org/cgi/doi/10.1073/pnas.1300828110 PNAS | Published online September 3, 2013 | E3567–E3576 Downloaded by guest on October 1, 2021 tains an active NES (IxxxLxxxxxML), microsatellite-associated rodent variants contain the variant sequence IxxxLxxxxxSL (22). Such variation is associated with clade-specific insertion of a DNA microsatellite in the divergent evolution of the Y chromosome in superfamily Muroidea (order Rodentia) (23). Mammalian TFs regulating tissue- and stage-specific gene expression are ordinarily present at low abundance (1-100 nM representing 102 to 104 molecules per nucleus) (24). Studies in cell culture, however, often use strong viral promoters (such as that derived from the cytomegalic virus) (25), which typically confer high-level TF expression (1-10 μM representing 105 to 106 molecules per nucleus). Because such overexpression can alter quantitative patterns of gene expression and qualitative biological responses (26), the contribution of NCS-linked phosphorylation to the transcriptional activity of hSRY was estimated at progressively lower levels of protein expression within the physiological range. In each case, the limiting extent of attenuation was twofold, a quantitative threshold reminiscent of disorders of sexual development-associated syndromes of autosomal transcription-factor haploinsufficiency (11). An analogous Sry-related threshold in XY mice has been inferred based on studies of intersexual phenotypes due to the strain-specific phenomenon of Y chromosome/autosome incompatibility (27–29). This seeming paradox of mice and men highlights the tenuous beginning of the male program (11). Defining a critical boundary between testicular self-organization and gonadal dysgenesis, inherited human sex reversal associated with subtle perturbation of hSRY thus violates the principle of Waddington canalization (30). Whereas the latter emphasizes the importance of robustness in de- Fig. 1. Structure and function of human SRY. (A) Domain organization and velopmental pathways (31), we speculate that SRY has evolved to Swyer mutations. Residues 56–141 comprise HMG

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