The Y-chromosome-encoded transcription factor SRY initiates testis determination in therian mammals by activating SOX9 in embryonic supporting (pre-Sertoli) cells. Racca et al. (2016) showed that the inherited Swyer-syndrome mutation F109S in the SRY DNA-binding domain (HMG box) does not impair DNA binding or bending but destabilizes the domain, leading to enhanced polyubiquitination, accelerated 26S proteasomal degradation, and a twofold reduction in SRY-dependent SOX9 activation — a threshold compatible with the fertile XY father yet sex-reversed XY daughter of the same pedigree. These findings leave several questions open: which cellular E3 ubiquitin ligase(s) recognize the partially unfolded variant HMG box and mark it for degradation; how sequence context, side-chain geometry, and nearby post-translational modifications set ubiquitination susceptibility; whether rodent glutamine-rich domains protect divergent Sry orthologs at the ubiquitination or post-ubiquitination step; how graded SRY molecule number maps onto Sox9 activation and binary gonadal fate in vivo; and whether accelerated ubiquitin-dependent turnover recurs across other inherited Swyer alleles, with autosomal genetic background distinguished from stochastic gene expression in determining penetrance. The problem remains open.

Background

In therian mammals, male sex determination is initiated by SRY (sex-determining region on the Y chromosome), a transcription factor expressed for a narrow developmental window in the embryonic bipotential gonadal ridge. SRY binds — through its conserved DNA-binding motif, the HMG (high-mobility group) box, an L-shaped domain of about 80 amino acids — to specific sites in testis-specific enhancer elements (TES/TESCO and Enh13) upstream of SOX9, sharply bending the DNA and thereby activating SOX9, the master gene that locks embryonic supporting cells into the Sertoli-cell (testis) fate. Loss-of-function mutations in SRY cause 46,XY complete gonadal dysgenesis (Swyer syndrome): affected individuals are chromosomally male but develop as phenotypic females with streak gonads. Most Swyer mutations arise de novo; rare inherited mutations are compatible with male development in some genetic backgrounds (fertile XY fathers) but female development in others (their XY daughters), making them 'experiments of nature' that probe the margin of the switch.

Racca, Chen, Yang, Phillips and Weiss (2016) biochemically and cellularly characterized one such inherited mutation, F109S — a substitution at consensus HMG-box position 54, a conserved aromatic residue — in a pedigree in which the proband (XY female with gonadoblastoma) carries the same allele as her fertile father and uncle. The F109S domain retains native-like specific DNA affinity and DNA bending, but is thermodynamically destabilized and partially unfolded at 37°C. In transfected cell lines (including CH34 cells, derived from the embryonic gonadal ridge, the site and stage of SRY expression), full-length F109S SRY underwent enhanced polyubiquitination — the covalent attachment of ubiquitin chains that signals destruction by the 26S proteasome, the ATP-dependent multi-subunit protease complex — and was degraded faster than wild-type SRY, whereas its ubiquitin-independent degradation by the 20S core proteasome was unchanged in vitro. Proteasome inhibition with MG132 restored enhancer occupancy and SOX9 activation, establishing instability, not loss of DNA-binding function, as the defect. The resulting twofold decrease in SRY-dependent Sox9 activation matched the functional decrement of two other inherited Swyer variants (V60L, impairing nuclear import; I90M, impairing nuclear export), defining a slim 'twofold' transcriptional threshold at the edge of developmental ambiguity. Prior mouse work had shown that the C-terminal glutamine-rich (Q-rich) domain of muroid rodent Sry — encoded by an intragenic microsatellite and acting as a transcriptional activation domain and 'genetic capacitor' (a buffer that tolerates cryptic variation in the HMG box) — also protects the divergent, unstable murine HMG box from proteasomal degradation.

Problem Statement

Take the correlative findings of Racca et al. (2016) — enhanced polyubiquitination and accelerated 26S proteasomal degradation of the destabilized F109S SRY variant, rescued by proteasome inhibition to native Sox9 activation, with a twofold functional threshold — to molecular causation and a quantitative model of the human SRY-SOX9 developmental switch. This single objective comprises the following linked questions: (1) Identify the cellular E3 ubiquitin ligase(s) responsible for the enhanced polyubiquitination of destabilized SRY variants in embryonic supporting-lineage cells, and establish the recognition mechanism by which partially unfolded HMG-box surfaces are targeted — including causal tests (loss- and gain-of-function of the ligase, and rescue of variant SRY stability and Sox9 activation). (2) Determine the recognition rules connecting side-chain geometry and aromaticity at consensus box position 54 (e.g., F109L and other non-planar substitutions) to folding, DNA-binding compensation, and ubiquitination susceptibility, together with the contribution of adjacent post-translational modifications — including whether adjacent phospho-acceptor motifs, such as the predicted protein kinase A (PKA) site at residues 31-33, contribute to the same twofold functional threshold. (3) Establish the mechanistic basis of species differences: whether the glutamine-rich domains of rodent Sry orthologs protect divergent, unstable HMG boxes from polyubiquitination or instead hinder 26S degradation after ubiquitination, and quantify how such intragenic features modulate proteasomal targeting across taxa. (4) Construct and validate quantitative experimental and mathematical frameworks (chemical or genetic rheostats, such as regulated expression of degradation-prone or wild-type SRY) that measure how graded changes in SRY molecule number translate into Sox9 activation and binary gonadal-fate outcomes in vivo, across orthologs and genetic backgrounds. (5) Determine, through systematic biochemical and cellular characterization of other inherited Swyer SRY alleles, whether accelerated ubiquitin-dependent turnover is a recurring pathogenic mechanism across distinct clinical variants, and separate the roles of autosomal genetic background versus stochastic gene expression in determining penetrance. Together, these components are constitutive parts of that single objective — a causal, mechanistic account of variant SRY turnover and a quantitative input-output description of the switch — not independent endpoints.

References

  1. Racca JD, Chen Y-S, Yang Y, Phillips NB, Weiss MA (2016) Human Sex Determination at the Edge of Ambiguity: Inherited XY Sex Reversal Due to Enhanced Ubiquitination and Proteasomal Degradation of a Master Transcription Factor. Journal of Biological Chemistry 291(42):22173-22195. DOI 10.1074/jbc.M116.741959. https://doi.org/10.1074/jbc.M116.741959
  2. Chen YS, Racca JD, Phillips NB, Weiss MA (2013) Inherited human sex reversal due to impaired nucleocytoplasmic trafficking of SRY defines a male transcriptional threshold. Proceedings of the National Academy of Sciences USA 110(38):E3567-E3576. DOI 10.1073/pnas.1300828110. https://doi.org/10.1073/pnas.1300828110
  3. Chen YS, Racca JD, Sequeira PW, Phillips NB, Weiss MA (2013) Microsatellite-encoded domain in rodent Sry functions as a genetic capacitor to enable the rapid evolution of biological novelty. Proceedings of the National Academy of Sciences USA 110(33):E3061-E3070. DOI 10.1073/pnas.1300860110. https://doi.org/10.1073/pnas.1300860110
  4. Zhao L, Ng ET, Davidson T-L, Longmuss E, Urschitz J, Elston M, Moisyadi S, Bowles J, Koopman P (2014) Structure-function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination. Proceedings of the National Academy of Sciences USA 111(32):11768-11773. DOI 10.1073/pnas.1400666111. https://doi.org/10.1073/pnas.1400666111
  5. Chen YS, Racca JD, Weiss MA (2022) Tenuous Transcriptional Threshold of Human Sex Determination. I. SRY and Swyer Syndrome at the Edge of Ambiguity. Frontiers in Endocrinology 13:945030. DOI 10.3389/fendo.2022.945030. https://doi.org/10.3389/fendo.2022.945030
  6. Racca JD, Chatterjee D, Chen YS, Rai RK, Yang Y, Georgiadis MM, Haas E, Weiss MA (2022) Tenuous transcriptional threshold of human sex determination. II. SRY exploits water-mediated clamp at the edge of ambiguity. Frontiers in Endocrinology 13:1029177. DOI 10.3389/fendo.2022.1029177. https://doi.org/10.3389/fendo.2022.1029177
  7. Racca JD, Chen YS, Brabender AR, Battistin U, Weiss MA, Georgiadis MM (2024) Role of nucleobase-specific interactions in the binding and bending of DNA by human male sex determination factor SRY. Journal of Biological Chemistry 300(9):107683. DOI 10.1016/j.jbc.2024.107683. https://doi.org/10.1016/j.jbc.2024.107683
  8. Thomson E, Zhao L, Chen YS, Longmuss E, Ng ET, Sreenivasan R, Croft B, Song X, Sinclair A, Weiss M, Koopman P, Pelosi E (2022) Generation and mutational analysis of a transgenic mouse model of human SRY. Human Mutation 43(3):362-379. DOI 10.1002/humu.24318. https://doi.org/10.1002/humu.24318
  9. Tsuji-Hosokawa A, Ogawa Y, Tsuchiya I, Terao M, Takada S (2022) Human SRY Expression at the Sex-determining Period is Insufficient to Drive Testis Development in Mice. Endocrinology 163(1):bqab217. DOI 10.1210/endocr/bqab217. https://doi.org/10.1210/endocr/bqab217
  10. Ogata Y, Nishikata M, Kitada K, Mizushima S, Jogahara T, Kuroiwa A (2019) Spiny rat SRY lacks a long Q-rich domain and is not stable in transgenic mice. Developmental Dynamics 248(9):784-794. DOI 10.1002/dvdy.73. https://doi.org/10.1002/dvdy.73
  11. Zhao L, Quinn A, Ng ET, Veyrunes F, Koopman P (2017) Reduced Activity of SRY and its Target Enhancer Sox9-TESCO in a Mouse Species with X*Y Sex Reversal. Scientific Reports 7:41378. DOI 10.1038/srep41378. https://doi.org/10.1038/srep41378
  12. Idris FP, van den Bergen J, Robevska G, Ferreira LGA, Ferreira KR, Kizys MML, Dias da Silva MR, Bruggenwirth HT, van Bever Y, Sinclair AH, Ayers KL (2025) Functional analysis of SRY variants in individuals with 46,XY differences of sex development. Molecular and Cellular Endocrinology 598:112458. DOI 10.1016/j.mce.2025.112458. https://doi.org/10.1016/j.mce.2025.112458
  13. Idris FP, Ferreira LGA, Houston B, Humphrey SJ, Sinclair AH, Ayers KL (2026) Post-translational modification of proteins in the human testis development pathway. Human Reproduction Update (advance article dmag016). DOI 10.1093/humupd/dmag016. https://doi.org/10.1093/humupd/dmag016
  14. Ríos O, Frias S, Rodríguez A, Kofman S, Merchant H, Torres L, Mendoza L (2015) A Boolean network model of human gonadal sex determination. Theoretical Biology and Medical Modelling 12:26. DOI 10.1186/s12976-015-0023-0. https://doi.org/10.1186/s12976-015-0023-0
  15. Vivanco E, Goles E, Montalva-Medel M, Poupin MJ (2024) Dynamical robustness of a Boolean model for the human gonadal sex determination. Computational Biology and Chemistry 113:108225. DOI 10.1016/j.compbiolchem.2024.108225. https://doi.org/10.1016/j.compbiolchem.2024.108225
  16. Houzelstein D, Eozenou C, Lagos CF, Elzaiat M, Bignon-Topalovic J, Gonzalez I, Laville V, Schlick L, Wankanit S, Madon P, Kirtane J, Athalye A, Buonocore F, Bigou S, Conway GS, Bohl D, Achermann JC, Bashamboo A, McElreavey K (2024) A conserved NR5A1-responsive enhancer regulates SRY in testis-determination. Nature Communications 15:2796. DOI 10.1038/s41467-024-47162-2. https://doi.org/10.1038/s41467-024-47162-2