Compound effects of point mutations causing campomelic dysplasia/autosomalsex reversal upon SOX9 structure, nuclear transport, DNA binding, and transcriptional activation

Human mutations in the transcription factor SOX9 cause campomelic dysplasia /autosomal sex reversal. Here we identify and characterize two novel hetero zygous mutations, F154L and A158T, that substitute conserved "hydrophobic c ore" amino acids of the high mobility group domain at positions thought to stabilize SOX9 conformation. Circular dichroism studies indicated that. bot h mutations disrupt alpha -helicity within their high mobility group domain , whereas tertiary structure is essentially maintained as judged by fluores cence spectroscopy. In cultured cells, strictly nuclear localization was ob served for wild type SOX9 and the F154L mutant; however, the A158T mutant s howed a 2-fold reduction in nuclear import efficiency. Importin-beta was de monstrated to be the nuclear transport receptor recognized by SOX9, with bo th mutant proteins binding importin-beta with wild type affinity. Whereas D NA bending was unaffected, DNA binding was drastically reduced in both muta nts (to 5% of wild type activity in F154L, 17% in A158T). Despite this larg e effect, transcriptional activation in cultured cells was only reduced to 26% in F154L and 62% in A158T of wild type activity, suggesting that a smal l loss of SOX9 transactivation activity could be sufficient to disrupt prop er regulation of target genes during bone and testis formation. Thus, clini cally relevant mutations of SOX9 affect protein structure leading to compou nd effects of reduced nuclear import and reduced DNA binding, the net effec t being loss of transcriptional activation.