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

Abstract

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.

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

“…These studies employed the isolated SRY HMG box and were repeated four times (Figure 3D). In accordance with related studies in our companion study (14), the relationship between flexure displacement and electrophoretic mobility (83) in each case implied sharp DNA bending (Supplemental Figure S6A). Whereas DNA bend angles were indistinguishable among the WT, Y72F and Y72W domain-DNA complexes (black, orange and blue boxes in Figure 3E), Y72A led to a marked reduction in inferred bend angle (purple; from to ca.…”

“…1.5 double-helical turns) whose respective 5'-ends were flexibly linked to a fluorescent donor or acceptor (Figure 4A). Whereas sharp DNA bending was maintained in the variant box-DNA complex in accordance with prior PGEbased estimates of DNA bend angle (14), a slight, yet reproducible broadening was observed in the end-to-end distance distribution (Figure 4C, right) in association with reduction in energy-transfer efficiency and slight shift in position of the EEDD peak (Supplemental Table S3). The increased width of the time-resolved FRET-based distance distribution in the WT box-DNA complex relative to the free DNA [itself reflecting primarily the flexibility of the hexanyllinked fluorescent probes (33, 61)] suggests that the native complex exhibits a baseline range of DNA bend angles rather than a single angle.…”

“…Tyr is invariant at this site (box position 72) among mammalian Sry orthologues and broadly conserved among SOX factors (35). Two de novo mutations (Y127H and Y127C in intact human SRY) were found, as expected given the clinical context, to markedly impair SRY-directed transcriptional activation of principal downstream target gene SOX9 in cellular models (14). Respective mechanisms nonetheless differed, in one case via a direct destabilization of the box-DNA interface (His) and in the other case via mutational destabilization of the protein to cellular degradation (Cys).…”

“…In this and our companion article in this thematic collection (14), we have provided evidence that the transcriptional potency of SRY, as assessed in immortalized cellular models of the embryonic pre-Sertoli cell (36,57,75), correlates with the lifetime of the box-DNA complex (k off ) and not its specific affinity (K d ) (90). We imagine that this lifetime in vitro determines in turn the cellular lifetime of one or more SRYdependent and testis-specific enhanceosomes in far-upstream region of the SOX9 gene (Figure 1D) (5,6,76).…”

“…In our companion article (14) we demonstrated that de novo Swyer mutations at 127, box position 72 [arising in paternal spermatogenesis (15,16)] confer marked, yet distinct molecular defects in vitro and in cell culture. Although specific DNA bending was generally retained, as probed by permutation gel electrophoresis (PGE) and steady-state fluorescence resonance energy transfer (FRET), Y127H perturbs box-DNA affinity and hence testisspecific enhanceosome occupancy, whereas Y127C destabilizes the intact TF to proteasomal degradation in cells (14). In each case, gene-regulatory activity was impaired in accordance with DSD phenotypes (36,45).…”