BPS2026 – Kinetic regulation of a developmental decision by protein-DNA intercalation

Protein-directed DNA bending provides a general structural mechanism of eukaryotic gene regulation. Our studies focus on the contribution of an intercalative side chain to (1) the biophysical properties of a bent protein-DNA complex and (2) correlation with developmental outcomes in a cell-fate decision and organogenesis. A model is provided by the sequence-specific high-mobility-group (HMG) box as exemplified by the human male-determining factor SRY and homologous SOX domains in Drosophila melanogaster . A “cantilever” side chain (conserved among metazoans as Met, Ile, Leu, or Phe) inserts between consecutive AT base pairs to disrupt DNA base stacking while preserving base pairing. Stopped-flow FRET studies and NMR titrations demonstrate that the identity of the cantilever side chain modulates protein-DNA on/off rates without change in equilibrium dissociation constants; degree of DNA bending is essential the same. Comparative co-crystal structures of specific WT and variant SRY-DNA complexes reveal analogous modes of intercalation with only minor changes in the structure of the non-canonical bent DNA site. Remarkably, clinical mutation of the cantilever in human SRY causes XY sex reversal, whereas its genetic variation among rodents is associated with female-predominant communities (XX females, XY females, and XY males). CRISPR-Cas9-based modification of the cantilever residue in an homologous SOX gene in fruit flies demonstrates that the kinetic properties of the specific HMG box-DNA complex can regulate developmental gene regulation. To our knowledge, this is the first demonstration of kinetic control of organogenesis by an architectural transcription factor.