title: 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.affiliation: Department of Genetics, University of Melbourne, Howard Florey Institute, University of Melbourne, Parkville 3052, Australia.
authors.forename: S
authors.initials: S
authors.lastname: Preiss
authors.forename: A
authors.initials: A
authors.lastname: Argentaro
authors.forename: A
authors.initials: A
authors.lastname: Clayton
authors.forename: A
authors.initials: A
authors.lastname: John
authors.forename: D A
authors.initials: DA
authors.lastname: Jans
authors.forename: T
authors.initials: T
authors.lastname: Ogata
authors.forename: T
authors.initials: T
authors.lastname: Nagai
authors.forename: I
authors.initials: I
authors.lastname: Barroso
authors.forename: A J
authors.initials: AJ
authors.lastname: Schafer
authors.forename: V R
authors.initials: VR
authors.lastname: Harley
mesh_terms: D000015:Abnormalities, Multiple / Q000235:genetics*; D021581:Active Transport, Cell Nucleus / Q000235:genetics*; D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D001842:Bone and Bones / Q000002:abnormalities*; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D012734:Disorders of Sex Development; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005799:Genes, Dominant; D006579:Heterozygote; D006609:High Mobility Group Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007231:Infant, Newborn; D028884:Karyopherins; D007621:Karyotyping; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D010641:Phenotype; D017354:Point Mutation; D018807:Polymorphism, Single-Stranded Conformational; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D055758:SOX9 Transcription Factor; D017422:Sequence Analysis, DNA; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014364:Tryptophan / Q000378:metabolism
chemical_list: D006609:High Mobility Group Proteins; D028884:Karyopherins; D009687:Nuclear Proteins; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D014157:Transcription Factors; D014364:Tryptophan; D004247:DNA