Functional and Structural Studies of Wild Type SOX9 and Mutations Causing Campomelic Dysplasia

Abstract

In humans, mutations in SOX9 result in a skeletal malformation syndrome, campomelic dysplasia (CD). The present study investigated two major classes of 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 mutations 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, the H65Y mutant shows minimal DNA binding, whereas the A19V mutant shows near wild type DNA binding and bends DNA normally. Interestingly, the P70R mutant has altered DNA binding specificity, but also bends DNA normally. The effects of the point mutations were interpreted using a molecular model of the SOX9 HMG domain. We analyzed the effects upon transcription of mutations resembling the truncation and frameshift mutations in CD patients, and found that progressive deletion of the C terminus causes progressive loss of transactivation. Maximal transactivation by SOX9 requires both the C-terminal domain rich in proline, glutamine, and serine and the adjacent domain composed entirely of proline, glutamine, and alanine. Thus, CD arises by mutations that interfere with DNA binding by SOX9 or truncate the C-terminal transactivation domain and thereby impede the ability of SOX9 to activate target genes during organ development.In humans, mutations in SOX9 cause campomelic dysplasia (CD), 1 a skeletal malformation syndrome that is often associated with XY sex reversal (1). Other tissues affected include kidney, heart, and brain, consistent with the expression pattern of Sox9 in developing mouse (2, 3). There are four major classes of mutations causing CD: 1) amino acid substitutions in the HMG domain (Fig. 1A), 2) truncations or frameshifts that alter the C terminus of SOX9 (Fig. 1B), 3) mutations at splice junctions, and 4) chromosomal translocations, of which classes 1 and 2 are investigated here. Most CD patients are heterozygous for wild type and mutant alleles of SOX9. CD appears to result from haploinsufficiency; presumably, a critical dose of SOX9 is required to switch on the appropriate genes during development. The present study reports the identification in a CD patient of a novel amino acid substitution mutation (H65Y) in the HMG domain of SOX9. We report the effects of this and three other point mutations (F12L, A19V, and P70R) on the DNA binding and DNA bending activities of the HMG domain.SOX proteins represent a large class of transcription factors related to SRY, the testis-determining factor, through their HMG domains that bind and bend DNA in a sequence-specific manner. Expression of these proteins in defined cell types at specific stages of development appears to govern cell fate decisions. SOX9 activates expression of type II and type XI collagen in vivo (4 -6), consistent with a role in bone development.SOX proteins fall within a larger group of HMG domain proteins comprising two clas...

Testicular dysgenesis/regression without campomelic dysplasia in patients carrying missense mutations and upstream deletion of SOX9

“…The p.Arg394Gly and p.Arg437Cys substitutions resided within the proline/glutamine/serine (PQS)‐rich domain (also known as SPQ‐rich domain) at the C‐terminus (McDowall et al. 1999) (Fig. 1A).…”

“…2003), high‐mobility group ( HMG : codon 101–184), proline/glutamine/alanine ( PQA : codon 339–379), and proline/glutamine/serine‐rich ( PQS ‐rich: codon 386–509) domains (McDowall et al. 1999). (B) Nucleotide substitutions detected in patients 1 and 2.…”

Isolation of chicken homolog of the FOXL2 gene and comparison of its expression patterns with those of aromatase during ovarian development

“…It leads to the increase in protein sizes and may promote the acquisition of new functions (Mortlock et al, 2000). The high proline, alanine, and glycine content is associated with transcription activation domains in transcription factors (Mitchell and Tjian, 1989;McDowall et al, 1999). On the other hand, alanine repeats or alanine-rich regions may possess repressive activity (Briata et al, 1995;Hanna-Rose and Hansen, 1996;Maurer et al, 2003).…”

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

“…The oligonucleotide used containing the SRY consensus-binding site (underlined) comprised the upper strand sequence GGG TTA ACG TAA ACA ATA AAT CTG GTA GA. Complementary oligonucleotides were annealed and end-filled by using superscript reverse transcriptase in the presence of [␣-32 P]dCTP. EMSAs were performed as described by using either 35 S-labeled in vitrotranslated full-length SRY or recombinant HMG domains (10). For competition experiments, 4 ng of SRY HMG box was incubated with varying amounts of either IMP␣ and͞or IMP␤ before the addition of 20 fmol of SRY DNA consensus probe.…”

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