MAMMALIAN TESTIS-DETERMINING FACTOR SRY HAS EVOLVED TO THE EDGE OF AMBIGUITY

by YEN-SHAN CHEN The male sex determination program in eutherian mammals is regulated by Sry, a transcription factor encoded by the Y chromosome. Specific DNA binding is mediated by the high-mobility-group (HMG) box domain. Expression of Sry in the gonadal ridge activates a Sox9-dependent gene-regulatory network leading to testis formation. Mutations in human SRY cause gonadal dysgenesis and a female somatic phenotype. A series of subtle variants define inherited alleles shared by an XY sterile daughter and fertile father. While their specific DNA binding and bending are unaffected, the variant proteins exhibit selective defects in nucleocytoplasmic shuttling (V60L; impairment of Exportin-4-mediated nuclear import, and I90M; impairment of CRM1-regulated nuclear export) or protein stability (F109S and L163X; rapid degradation modulated by proteasomes). Decreased shuttling limits nuclear accumulation of phosphorylated (and hence activated) SRY, in turn reducing occupancy of DNA control sites regulating Sertoli-cell differentiation (the testis-specific Sox9 enhancer). Despite distinct patterns of biochemical and xvi cell-biological perturbations, V60L and I90M each attenuate SRYSox9 activation in transient transfection assays by twofold. The twofold attenuation of SRY-Sox9 regulation was also observed in the hydrophobic core-destabilized mutation, F109S, and the C-terminal truncated SRY, L163X. Accelerated degradation in each case reduces nuclear accumulation of SRY and leads to deficient target enhancer occupancy. The threshold unmasked by these inherited mutations (twofold) is reminiscent of autosomal syndromes of transcription-factor haploinsufficiency, including XY sex reversal associated with mutations in SOX9 (campomelic dysplasia) and SF1 (hypospadias, anorchia). Our results demonstrate that sufficient nuclear accumulation of activated SRY regulated by nucleocytoplasmic shuttling or protein half-life is necessary for robust initiation of testicular development. Although shared by goat and deer Sry, such shuttling is not conserved among rodents wherein impaired nuclear export is compensated by a microsatellite-associated transcriptional activation domain. Human sex reversal due to subtle defects causing the twofold reduced SRY-SOX9 regulation suggests that the transcriptional activity of wild-type SRY lies near the edge of developmental ambiguity. A subset of Sry alleles in superfamily Muroidea (order Rodentia) is remarkable for insertion of DNA microsatellite repeat tracts, most commonly encoding (as in mice) a CAG-repeat-associated glutamine-rich domain (GRD). We xvii provide evidence that this domain functions at a threshold length as a genetic capacitor to facilitate accumulation of variation elsewhere in the protein, including the HMG box. The GRD compensates for otherwise deleterious substitutions in the box and the absence of non-box phosphorylation sites to ensure sufficient occupancy of the DNA target sites. Such compensation enables activation of a male transcriptional program despite perturbations to the HMG box. Whereas human SRY requires nucleocytoplasmic shuttling and coupled phosphorylation, mouse Sry contains a defective nuclear export signal analogous to a variant human SRY associated with inherited sex reversal. We propose that the rodent GRD has (i) fostered accumulation of cryptic intragenic variation and (ii) enabled unmasking of such variation due to DNA replicative slippage. This model highlights genomic contingency as a source of protein novelty at the edge of developmental ambiguity and may underlie emergence of non-Sry-dependent sex determination in the radiation of Muroidea.