Abstract
A general problem is posed by analysis of transcriptional thresholds governing cell fate decisions in metazoan development. A model is provided by testis determination in therian mammals. Its key step, Sertoli cell differentiation in the embryonic gonadal ridge, is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in human SRY cause gonadal dysgenesis leading to XY female development (Swyer syndrome). Here, we have characterized an inherited mutation compatible with either male or female somatic phenotypes as observed in an XY father and XY daughter, respectively. The mutation (a crevice-forming substitution at a conserved back surface of the SRY high mobility group box) markedly destabilizes the domain but preserves specific DNA affinity and induced DNA bend angle. On transient transfection of diverse human and rodent cell lines, the variant SRY exhibited accelerated proteasomal degradation (relative to wild type) associated with increased ubiquitination; in vitro susceptibility to ubiquitin-independent ("default") cleavage by the 20S core proteasome was unchanged. The variant's gene regulatory activity (as assessed in a cellular model of the rat embryonic XY gonadal ridge) was reduced by 2-fold relative to wild-type SRY at similar levels of mRNA expression. Chemical proteasome inhibition restored native-like SRY expression and transcriptional activity in association with restored occupancy of a sex-specific enhancer element in principal downstream gene Sox9, demonstrating that the variant SRY exhibits essentially native activity on a per molecule basis. Our findings define a novel mechanism of impaired organogenesis, accelerated ubiquitin-directed proteasomal degradation of a master transcription factor leading to a developmental decision poised at the edge of ambiguity.
Highlights
Regulation of alternative genetic programs is central to the logic of metazoan development [1]
Assignment of Sry as the testis-determining factor was demonstrated in transgenic mice [20] and extended to human embryogenesis through identification of diverse mutations in SRY associated with a distinct disorder of sex development (DSD) [22], designated Swyer syndrome (Fig. 1B)
Clinical Mutation Destabilizes the high mobility group (HMG) Box—Thermal stabilities of the free WT and variant domains were assessed by circular dichroism (CD) (Fig. 2, A and B)
Summary
Regulation of alternative genetic programs is central to the logic of metazoan development [1]. Analogous principles are thought to govern developmental decisions in metazoans [6] wherein organogenesis may be directed by alternative states of gene-regulatory networks [7, 8] Such control systems have been delineated in model organisms, in particular through molecular-genetic analysis of sex determination and sex-specific gene regulation [9, 10]. Assignment of Sry as the testis-determining factor was demonstrated in transgenic mice [20] and extended to human embryogenesis through identification of diverse mutations in SRY associated with a distinct disorder of sex development (DSD) [22], designated Swyer syndrome (Fig. 1B) Such mutations cluster in SRY’s high mobility group (HMG) box, a sequence-specific DNA-bending domain shared by a conserved family of TFs (designated SRY-related HMG box, Sox [23]). Analogous backgrounddependent XY sex reversal has been observed among strains of laboratory mice [31,32,33], highlighting the tenuous function of murine Sry at the threshold of developmental ambiguity [34]
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