Functional and structural studies of wild type SOX9 and mutations causing campomelic dysplasia

In humans, mutations in SOX9 result in a skeletal malformation syndrome, ca mpomelic dysplasia (CD), The present study investigated two major classes o f CD mutations: 1) point mutations in the high mobility group (HMG) domain and 2) truncations and frameshifts that alter the C terminus of the protein . We analyzed the effect of one novel mutation and three other point mutati ons in the HMG domain of SOX9 on the DNA binding and DNA bending properties of the protein. The F12L mutant HMG domain shows negligible DNA binding, t he H65Y mutant shows minimal DNA binding, whereas the A19V mutant shows nea r wild type DNA binding and bends DNA normally. Interestingly, the P70R mut ant has altered DNA binding specificity, but also bends DNA normally. The e ffects of the point mutations were interpreted using a molecular model of t he SOX9 HMG domain. We analyzed the effects upon transcription of mutations resembling the truncation and frameshift mutations in CD patients, and fou nd that progressive deletion of the C terminus causes progressive loss of t ransactivation, Maximal transactivation by SOX9 requires both the C-termina l domain rich in proline, glutamine, and serine and the adjacent domain com posed entirely of proline, glutamine, and alanine, Thus, CD arises by mutat ions that interfere with DNA binding by SOX9 or truncate the C-terminal tra nsactivation domain and thereby impede the ability of SOX9 to activate targ et genes during organ development.