Structure-Function Relationships in Human Testis-Determining Factor SRY. AN AROMATIC BUTTRESS UNDERLIES THE SPECIFIC DNA-BENDING SURFACE OF AN HMG BOX* [Cell Biology]

September 25th, 2014 by Racca, J. D., Chen, Y.-S., Maloy, J. D., Wickramasinghe, N., Phillips, N. B., Weiss, M. A.

Human testis determination is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in SRY cause 46, XY gonadal dysgenesis with female somatic phenotype (Swyer′s syndrome) and confer high risk of malignancy (gonadoblastoma). Such mutations cluster in the SRY high-mobility-group (HMG) box, a conserved motif of specific DNA binding and bending. To explore structure-function relationships, we constructed all possible substitutions at a site of clinical mutation (W70L). Our studies thus focused on a core aromatic residue (position 15 of the consensus HMG box) that is invariant among SRY-related HMG-box transcription factors (the SOX family) and conserved as aromatic (Phe or Tyr) among other sequence-specific boxes. In a yeast-1-hybrid system sensitive to specific SRY-DNA binding, the variant domains exhibited reduced (Phe and Tyr) or absent activity (the remaining 17 substitutions). Representative nonpolar variants with partial or absent activity (Tyr, Phe, Leu, and Ala in order of decreasing side-chain volume) were chosen for study in vitro and in mammalian cell culture. The clinical mutation (Leu) was found to markedly impair multiple biochemical and cellular activities as respectively probed through (i) in vitro assays of specific DNA binding, and protein stability; and (ii) cell-culture-based assays of proteosomal degradation, nuclear import, enhancer DNA occupancy, and SRY-dependent transcriptional activation. Surprisingly, however, DNA bending is robust to this or the related Ala substitution that profoundly impairs box stability. Together, our findings demonstrate that the folding, trafficking, and gene-regulatory function of SRY requires an invariant aromatic ″buttress″ beneath its specific DNA-bending surface.
  • Posted in Journal of Biological Chemistry, Publications
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