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

Abstract	Human mutations in the transcription factor SOX9 cause campomelic dysplasia/autosomal sex reversal. Here we identify and characterize two novel heterozygous mutations, F154L and A158T, that substitute conserved "hydrophobic core" amino acids of the high mobility group domain at positions thought to stabilize SOX9 conformation. Circular dichroism studies indicated that both mutations disrupt alpha-helicity within their high mobility group domain, whereas tertiary structure is essentially maintained as judged by fluorescence spectroscopy. In cultured cells, strictly nuclear localization was observed for wild type SOX9 and the F154L mutant; however, the A158T mutant showed a 2-fold reduction in nuclear import efficiency. Importin-beta was demonstrated to be the nuclear transport receptor recognized by SOX9, with both mutant proteins binding importin-beta with wild type affinity. Whereas DNA bending was unaffected, DNA binding was drastically reduced in both mutants (to 5% of wild type activity in F154L, 17% in A158T). Despite this large effect, transcriptional activation in cultured cells was only reduced to 26% in F154L and 62% in A158T of wild type activity, suggesting that a small loss of SOX9 transactivation activity could be sufficient to disrupt proper regulation of target genes during bone and testis formation. Thus, clinically relevant mutations of SOX9 affect protein structure leading to compound effects of reduced nuclear import and reduced DNA binding, the net effect being loss of transcriptional activation.
Authors	S Preiss, A Argentaro, A Clayton, A John, D A Jans, T Ogata, T Nagai, I Barroso, A J Schafer, V R Harley
Journal	The Journal of biological chemistry (J Biol Chem) Vol. 276 Issue 30 Pg. 27864-72 (Jul 27 2001) ISSN: 0021-9258 [Print] United States
PMID	11323423 (Publication Type: Case Reports, Journal Article, Research Support, Non-U.S. Gov't)
Chemical References	High Mobility Group Proteins Karyopherins Nuclear Proteins SOX9 Transcription Factor SOX9 protein, human Transcription Factors Tryptophan DNA
Topics	Abnormalities, Multiple (genetics) Active Transport, Cell Nucleus (genetics) Adult Amino Acid Sequence Animals Bone and Bones (abnormalities) COS Cells Cell Nucleus (metabolism) Cells, Cultured Circular Dichroism DNA (metabolism) Disorders of Sex Development Electrophoresis, Polyacrylamide Gel Female Genes, Dominant Heterozygote High Mobility Group Proteins (chemistry, genetics, metabolism) Humans Immunohistochemistry Infant, Newborn Karyopherins Karyotyping Kinetics Molecular Sequence Data Mutagenesis, Site-Directed Mutation Nuclear Proteins (metabolism) Phenotype Point Mutation Polymorphism, Single-Stranded Conformational Protein Binding Protein Conformation Protein Denaturation Protein Structure, Tertiary SOX9 Transcription Factor Sequence Analysis, DNA Spectrometry, Fluorescence Structure-Activity Relationship Temperature Transcription Factors (chemistry, genetics, metabolism) Transcriptional Activation Transfection Tryptophan (metabolism)

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