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Structure-function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination.

ABSTRACT: The mammalian sex-determining factor SRY comprises a conserved high-mobility group (HMG) box DNA-binding domain and poorly conserved regions outside the HMG box. Mouse Sry is unusual in that it includes a C-terminal polyglutamine (polyQ) tract that is absent in nonrodent SRY proteins, and yet, paradoxically, is essential for male sex determination. To dissect the molecular functions of this domain, we generated a series of Sry mutants, and studied their biochemical properties in cell lines and transgenic mouse embryos. Sry protein lacking the polyQ domain was unstable, due to proteasomal degradation. Replacing this domain with irrelevant sequences stabilized the protein but failed to restore Sry's ability to up-regulate its key target gene SRY-box 9 (Sox9) and its sex-determining function in vivo. These functions were restored only when a VP16 transactivation domain was substituted. We conclude that the polyQ domain has important roles in protein stabilization and transcriptional activation, both of which are essential for male sex determination in mice. Our data disprove the hypothesis that the conserved HMG box domain is the only functional domain of Sry, and highlight an evolutionary paradox whereby mouse Sry has evolved a novel bifunctional module to activate Sox9 directly, whereas SRY proteins in other taxa, including humans, seem to lack this ability, presumably making them dependent on partner proteins(s) to provide this function.

SUBMITTER: Zhao L

PROVIDER: S-EPMC4136622 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Structure-function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination.

Zhao Liang L Ng Ee Ting ET Davidson Tara-Lynne TL Longmuss Enya E Urschitz Johann J Elston Marlee M Moisyadi Stefan S Bowles Josephine J Koopman Peter P

Proceedings of the National Academy of Sciences of the United States of America 20140729 32

The mammalian sex-determining factor SRY comprises a conserved high-mobility group (HMG) box DNA-binding domain and poorly conserved regions outside the HMG box. Mouse Sry is unusual in that it includes a C-terminal polyglutamine (polyQ) tract that is absent in nonrodent SRY proteins, and yet, paradoxically, is essential for male sex determination. To dissect the molecular functions of this domain, we generated a series of Sry mutants, and studied their biochemical properties in cell lines and t ...[more]

PMID: 25074915

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Project description:A critical transcription factor required for mammalian male sex determination is SRY (sex determining region on the Y chromosome). The expression of SRY in precursor Sertoli cells is one of the initial events in testis development. The current study was designed to determine the impact of environmentally induced epigenetic transgenerational inheritance on SRY during gonadal sex determination in the male. The agricultural fungicide vinclozolin and vehicle control (DMSO) exposed gestating females (F0 generation) during gonadal sex determination promoted the transgenerational inheritance of differential DNA methylation in sperm of the F3 generation (great grand-offspring). The fetal gonads in F3 generation males were used to identify potential alterations in SRY binding sites in the developing Sertoli cells. Chromatin immunoprecipitation with an SRY antibody followed by genome-wide promoter tiling array (ChIP-Chip) was used to identify alterations in SRY binding. A total of 81 adjacent oligonucleotide sites and 173 single oligo SRY binding sites were identified to be altered transgenerationally in the Sertoli cell vinclozolin lineage F3 generation males. Observations demonstrate the majority of the previously identified normal SRY binding sites were not altered and the altered SRY binding sites were novel and new additional sites. The chromosomal locations, gene associations and potentially modified cellular pathways were investigated. In summary, environmentally induces epigenetic transgenerational inheritance of germline epimutations appears to alter the cellular differentiation and development of the precursor Sertoli cell SRY binding during gonadal sex determination that influence the developmental origins of adult onset testis disease. The experimental design involved the intraperitoneal exposure of gestating female rats to vinclozolin or a vehicle control (dimethyl sulfoxide, DMSO) transiently from embryonic days E8-E14. Sister littermates were divided into control and vinclozolin treatment groups and mated to similar males to minimize the genetic variation between the control and vinclozolin lineages. Sufficient females were used so no inbreeding (sibling or cousin) occurred in any generation. The F1 generation was bred within the lineage to generate the F2 generation and these F2 generation bred to generate the F3 generation. The only exposure was the F0 generation female. The F3 generation control and vinclozolin lineage embryonic day 13 (E13) embryos were collected and the gonads micro-dissected and then sexed with an SRY PCR protocol. The male gonads were pooled from a minimum of three different litters and the pools used to collect DNA. Three different experiments were performed to collect 3 control and vinclozolin E13 F3 generation testis pools, each with different animals (n=25 gonads/pool). The chromatin DNA (not denatured) from each pool was fragmented and used in an SRY chromatin immunoprecipitation (ChIP) procedure for each pool separately. The control and vinclozolin SRY ChIP DNA were paired for a competitive hybridization on a genome-wide promoter tiling array (ChIP-Chip) assay. The hybridization data obtained was used to identify the SRY binding sites that were different in the F3 generation vinclozolin versus control lineage in E13 testis.

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Structure-function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination.

Publications

Structure-function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination.

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