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 ␣-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-␤ was demonstrated to be the nuclear transport receptor recognized by SOX9, with both mutant proteins binding importin-␤ 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. Campomelic dysplasia/autosomal sex reversal (CD/SRA1)1 is a severe skeletal malformation syndrome associated with XY male-to-female sex reversal caused by mutations in the SOX9 gene (1, 2). CD/SRA1 is an autosomal dominant disorder characterized by congenital bowing of the long bones and other skeletal malformations (narrow ilia, hypoplastic ischiopubic rami, micrognathia, cleft palate, and retroglossia), absence of olfactory bulbs and tracts, heart and renal malformations, hypoplastic lungs, narrow thoracic cage, defective tracheobronchial cartilages, small scapulae, and delayed bone age and psychomotor disorders (3, 4). In 75% of cases there is 46 XY testicular dysgenesis (5). The phenotypic changes seen in CD/ SRA1 patients correlate with sites of expression of SOX9 and suggest SOX9 is essential for the normal development of many organs. CD/SRA1 individuals are heterozygous resulting in haploinsufficiency of SOX9; presumably a critical dose of SOX9 is required to switch on appropriate genes during development. The present study reports the identification in two CD patients with novel amino acid substitution mutations, A154L and A158T in the HMG domain of SOX9, the latter in an XY female.To understand the functional consequences of these mutations, we attempted to correlate in vitro studies addressing protein structure, DNA binding, DNA bending, and importin recognition with in vivo studies of nuclear transport and transcriptional activation in cultured cells.

Defective importin β recognition and nuclear import of the sex-determining factor SRY are associated with XY sex-reversing mutations

“…Similar to the cNLS, the HMG domain was recognized poorly by IMP␣ (K d ϭ 112 nM), although IMP␤ recognition was slightly increased in the presence of IMP␣ (K d ϭ 2.4 nM). Thus, SRY joins a less common but growing list of arginine-rich NLScontaining proteins recognized by IMP␤ rather than IMP␣ (14,22,29,(31)(32)(33) that include the Rev (RqaRRnRRRRwR) from HIV type 1 (32, 34), the Rex protein of human T cell leukemia virus type 1 (mpKtRRRpRRsqRKRppt) (33), and the AP-1 family transcription factor CREB (26).…”

“…Immunocytochemistry with a mouse anti-FLAG antibody was carried out essentially as described (22). Cells were imaged by confocal laser scanning microscopy (CLSM, Bio-Rad MRC-500), and image analysis was performed by using NIH IMAGE 1.60 public-domain software (24).…”

“…Wild-type and mutant SRY HMG domains were expressed in Escherichia coli strain BL21 (DE3) (10) and purified by FPLC as described (22).…”

Human SRY inhibits β-catenin-mediated transcription

“…A similar strategy was used to generate N+HMG, C+HMG and HMG deletion constructs of SRY. HA-tagged human SOX9 expression plasmid was previously described (Preiss et al, 2001). V5-tagged mouse SOX17, V5-tagged mouse SOX17ΔC and GST-β-catenin plasmids were a kind gift of Dr. Aaron Zorn (Zorn et al, 1999).…”

Suppression of growth and tumorigenicity in the prostate tumor cell line M12 by overexpression of the transcription factor SOX9

“…SOX9 belongs to a family of transcription factors named for their DNA-binding domain (SRY-related HMG box); it activates transcription and causes DNA bending (Marshall and Harley, 2000;Koopman et al, 2001). Mutations or deletions in humans result in campomelic dysplasia syndrome manifest as chondro-dysplastic dwarfism, chondrocyte sarcomas, and sex reversal or intersex if the subject is XY (Kanai and Koopman, 1999;Olney et al, 1999;Preiss et al, 2001). SOX9 is a downstream effector of SRY, which in turn is dependent on the activity of androgens and the androgen receptor (AR) (Kent et al, 1996;Bowles and Koopman, 2001).…”