Generation and mutational analysis of a transgenic mouse model of human<i>SRY</i>

Thomson, Ella; Zhao, Liang; Chen, Yen‐Shan; Longmuss, Enya; Ng, Ee Ting; Sreenivasan, Rajini; Croft, Brittany; Song, Xin; Sinclair, Andrew H.; Weiss, Michael A.; Koopman, Peter; Pelosi, Emanuele

doi:10.1002/humu.24318

Cited by 4 publications

(3 citation statements)

References 56 publications

(83 reference statements)

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“…To gain further insight into structure-activity relationships at box position 72, it would be of future interest to survey the specific enhancer-binding properties (in cells) and specific DNA-binding/bending properties ( in vitro ) of variant SRYs containing a diverse collection of substitutions at this site. The recent development of a CRISPR/Cas9-based mouse model of human SRY promises an opportunity to correlate such data with developmental outcomes in a mammal ( 55 ).…”

Section: Discussionmentioning

confidence: 99%

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Tenuous Transcriptional Threshold of Human Sex Determination. I. SRY and Swyer Syndrome at the Edge of Ambiguity

Chen

Racca²,

Weiss³

2022

Front. Endocrinol.

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Male sex determination in mammals is initiated by SRY, a Y-encoded transcription factor. The protein contains a high-mobility-group (HMG) box mediating sequence-specific DNA bending. Mutations causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box and ordinarily arise de novo. Rare inherited variants lead to male development in one genetic background (the father) but not another (his sterile XY daughter). De novo and inherited mutations occur at an invariant Tyr adjoining the motif’s basic tail (box position 72; Y127 in SRY). In SRY-responsive cell lines CH34 and LNCaP, de novo mutations Y127H and Y127C reduced SRY activity (as assessed by transcriptional activation of principal target gene Sox9) by 5- and 8-fold, respectively. Whereas Y127H impaired testis-specific enhancer assembly, Y127C caused accelerated proteasomal proteolysis; activity was in part rescued by proteasome inhibition. Inherited variant Y127F was better tolerated: its expression was unperturbed, and activity was reduced by only twofold, a threshold similar to other inherited variants. Biochemical studies of wild-type (WT) and variant HMG boxes demonstrated similar specific DNA affinities (within a twofold range), with only subtle differences in sharp DNA bending as probed by permutation gel electrophoresis and fluorescence resonance-energy transfer (FRET); thermodynamic stabilities of the free boxes were essentially identical. Such modest perturbations are within the range of species variation. Whereas our cell-based findings rationalize the de novo genotype-phenotype relationships, a molecular understanding of inherited mutation Y127F remains elusive. Our companion study uncovers cryptic biophysical perturbations suggesting that the para-OH group of Y127 anchors a novel water-mediated DNA clamp.

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Section: Discussionmentioning

confidence: 99%

“…SRY‐mediated transcriptional activation was probed by SOX9/Sox9 mRNA as readouts. Primer sequences for all of the tested genes were applied as described ( 31 , 55 ). An internal control was the specific 5′‐TATAADNA‐binding subunit of TFIID.…”

Section: Methodsmentioning

confidence: 99%

Tenuous Transcriptional Threshold of Human Sex Determination. I. SRY and Swyer Syndrome at the Edge of Ambiguity

Chen

Racca²,

Weiss³

2022

Front. Endocrinol.

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“…After the transient transfection, cellular RNA was extracted and converted to cDNA using the vendor’s protocol (BioRad). SRY-mediated transcriptional activation was probed by SOX9/Sox9 mRNA as functional readouts; primer sequences were as described ( 57 , 77 ). An internal control was provided by the specific 5′‐TATAA DNA‐binding subunit of basal transcription factor TFIID.…”

Section: Methodsmentioning

confidence: 99%

Tenuous transcriptional threshold of human sex determination. II. SRY exploits water-mediated clamp at the edge of ambiguity

et al. 2022

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Y-encoded transcription factor SRY initiates male differentiation in therian mammals. This factor contains a high-mobility-group (HMG) box, which mediates sequence-specific DNA binding with sharp DNA bending. A companion article in this issue described sex-reversal mutations at box position 72 (residue 127 in human SRY), invariant as Tyr among mammalian orthologs. Although not contacting DNA, the aromatic ring seals the domain’s minor wing at a solvent-exposed junction with a basic tail. A seeming paradox was posed by the native-like biochemical properties of inherited Swyer variant Y72F: its near-native gene-regulatory activity is consistent with the father’s male development, but at odds with the daughter’s XY female somatic phenotype. Surprisingly, aromatic rings (Y72, F72 or W72) confer higher transcriptional activity than do basic or polar side chains generally observed at solvated DNA interfaces (Arg, Lys, His or Gln). Whereas biophysical studies (time-resolved fluorescence resonance energy transfer and heteronuclear NMR spectroscopy) uncovered only subtle perturbations, dissociation of the Y72F complex was markedly accelerated relative to wild-type. Studies of protein-DNA solvation by molecular-dynamics (MD) simulations of an homologous high-resolution crystal structure (SOX18) suggest that Y72 para-OH anchors a network of water molecules at the tail-DNA interface, perturbed in the variant in association with nonlocal conformational fluctuations. Loss of the Y72 anchor among SRY variants presumably “unclamps” its basic tail, leading to (a) rapid DNA dissociation despite native affinity and (b) attenuated transcriptional activity at the edge of sexual ambiguity. Conservation of Y72 suggests that this water-mediated clamp operates generally among SRY and metazoan SOX domains.

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Role of nucleobase-specific interactions in the binding and bending of DNA by human male sex determination factor SRY

Racca,

Chen,

Brabender

et al. 2024

Journal of Biological Chemistry

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Generation and mutational analysis of a transgenic mouse model of humanSRY

Cited by 4 publications

References 56 publications

Tenuous Transcriptional Threshold of Human Sex Determination. I. SRY and Swyer Syndrome at the Edge of Ambiguity

Tenuous Transcriptional Threshold of Human Sex Determination. I. SRY and Swyer Syndrome at the Edge of Ambiguity

Tenuous transcriptional threshold of human sex determination. II. SRY exploits water-mediated clamp at the edge of ambiguity

Role of nucleobase-specific interactions in the binding and bending of DNA by human male sex determination factor SRY

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