Human Sex Determination at the Edge of Ambiguity: Biophysical Studies of Clinical Mutations in a Master Transcription Factor

Male sex determination in therian mammals is initiated by an architectural transcription factor encoded by the sex-determining region of the Y chromosome. Designated Sry, this factor contains a sequence-specific high-mobility-group (HMG) box, a conserved motif of sharp DNA bending. Sry binds to specific DNA sites within a testis-specific super-enhancer in its principal autosomal target gene Sox9, which in turns regulates the expression of a male-specific gene-regulatory network in the embryonic gonadal ridge. Our overall program of research exploits a growing genetic database of clinical mutations in human SRY leading to XY somatic sex reversal (Swyer syndrome). Such mutations cluster in the HMG box. Here, we focus on a clinical mutation of the conserved “cantilever” residue of human SRY: an Ile that inserts between DNA base pairs to disrupt base stacking but not base pairing at a site of sharp DNA bending. Our studies combine NMR spectroscopy, single-molecule flourescence methods, stopped-flow FRET assays, and biological assays in embryonic gonadal cells to investigate the function of the SRY cantilever. Together, these studies demonstrate that partial DNA intercalation by the cantilever side chain provides a “kinetic anchor” controlling the lifetime of the bent protein-DNA complex. Transcriptional assays in cell culture suggest that a threshold lifetime is required for SRY-directed activation of Sox9 and in turn male development.