Inherited human sex reversal due to impaired nucleocytoplasmic trafficking of SRY defines a male transcriptional threshold

Abstract

Human testis determination is initiated by SRY (sex determining region on Y chromosome). Mutations in SRY cause gonadal dysgenesis with female somatic phenotype. Two subtle variants (V60L and I90M in the high-mobility group box) define inherited alleles shared by an XY sterile daughter and fertile father. Whereas specific DNA binding and bending are unaffected in a rat embryonic pre-Sertoli cell line, the variants exhibited selective defects in nucleocytoplasmic shuttling due to impaired nuclear import (V60L; mediated by Exportin-4) or export (I90M; mediated by chromosome region maintenance 1). Decreased shuttling limits nuclear accumulation of phosphorylated (activated) SRY, in turn reducing occupancy of DNA sites regulating Sertoli-cell differentiation [the testis-specific SRY-box 9 (Sox9) enhancer]. Despite distinct patterns of biochemical and cell-biological perturbations, V60L and I90M each attenuated Sox9 expression in transient transfection assays by twofold. Such attenuation was also observed in studies of V60A, a clinical variant associated with ovotestes and hence ambiguity between divergent cell fates. This shared twofold threshold is reminiscent of autosomal syndromes of transcription-factor haploinsufficiency, including XY sex reversal associated with mutations in SOX9. Our results demonstrate that nucleocytoplasmic shuttling of SRY is necessary for robust initiation of testicular development. Although also characteristic of ungulate orthologs, such shuttling is not conserved among rodents wherein impaired nuclear export of the high-mobility group box and importdependent phosphorylation are compensated by a microsatellite-associated transcriptional activation domain. Human sex reversal due to subtle defects in the nucleocytoplasmic shuttling of SRY suggests that its transcriptional activity lies near the edge of developmental ambiguity.organogenesis | sex determination | gonadogenesis | gene-regulatory network | protein-DNA interaction

Structure-Function Relationships in Human Testis-determining Factor SRY

“…Following the initiator Met, the cloning site encoded a hemagglutinin (HA) tag in triplicate. In selected constructs, an element encoding a heterologous nuclear localization signal (NLS; sequence PRRRKV as derived from the large T antigen of simian virus 40 (SV40)) was inserted after the HA-related codons (38). Mutations in SRY were introduced using QuikChange TM (Stratagene).…”

“…Mammalian Cell Culture-CH34 cells (kindly provided by Dr. P. K. Donahoe, Massachusetts General Hospital, Boston) (38,42) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% heat-inactivated fetal bovine serum at 37°C under 5% CO 2 . For proteasome-inhibitor studies, transfected cells were maintained for 24 h in serum-free conditions and then treated with the proteasome inhibitor MG132 for 6 h followed by 18 h of incubation in 5% serum-containing medium.…”

“…This study focused on the in vitro properties of the variant SRY domains (the HMG box) in relation to the following: (a) its wild-type (WT) structure (25,28) and (b) the biological activities of the intact protein (38). The apparent lack of organized structure within the non-box segments of SRY (12), their general lack of conservation (11), and the similar DNA-binding and DNA-bending properties of intact protein and the isolated domain (39,40) support the logic of such a modular approach.…”

“…Because box position 15 is part of its hydrophobic core, priority was given to nonpolar and aromatic substitutions with particular attention to DSD-associated mutation W70L. These studies culminated in analysis of transcriptional regulatory activities in an embryonic rat pre-Sertoli cell line (42), previously characterized as a model of the bipotential gonadal ridge at a stage of development just prior to its morphological differentiation (38). This model enables dissection of distinct cellular processes (including TF expression, proteasome-mediated turnover, and nuclear import) underlying the operation of a sex-specific GRN.…”

“…Although Exp4 was originally designated as an "exportin" in relation to its Ran-dependent role in the nuclear export of eukaryotic translational initiation factor 5A (94), Demmers and co-workers (47) have shown that Exp4 mediates nuclear import of SRY via interactions with the N-terminal segment of the HMG box (NLS residues 59 -77 in intact human SRY); an homologous interaction contributes to nuclear import of SOX9 (69). Nuclear exports of SRY and SOX9 are instead mediated by an interaction of CRM1 with a classical nuclear export signal (38,95 defects that together block SRY-directed transcriptional activation of Sox9. Although the ability of the SRY variants to direct sharp DNA bending in vitro was in each case retained, altered core packing led to mild (W70F and W70Y), moderate (W70A), or severe (W70L) perturbations to the lifetime of the bent protein⅐DNA complex.…”