The DNA-binding specificity of SOX9 and other SOX proteins

Abstract

SOX (SRY-related HMG box) proteins are transcription factors that have critical roles in the regulation of numerous developmental processes. They share at least 50% homology in their HMG domains, which bind the DNA element AACAAT. How different SOX proteins achieve specific regulation of target genes is not known. We determined the DNA-binding specificity of SOX9 using a random oligonucleotide selection assay. The optimal SOX9 binding sequence, AGAACAATGG, contained a core DNA-binding element AACAAT, flanked by 5' AG and 3' GG nucleotides. The specific interaction between SOX9 and AGAACAATGG was confirmed by mobility shift assays, DNA competition and dissociation studies. The 5' AG and 3' GG flanking nucleotides enhance binding by SOX9 HMG domain, but not by the HMG domain of another SOX factor, SRY. For SRY, different 5' and 3' flanking nucleotides are preferred. Our studies support the notion that SOX proteins achieve DNA sequence specificity through subtle preferences for flanking nucleotides and that this is likely to be dictated by signature amino acids in their HMG domains. Furthermore, the related HMG domains of SOX9 and Sox17 have similar optimal binding sites that differ from those of SRY and Sox5, suggesting that SOX factors may co-evolve with their DNA targets to achieve specificity.

Identification and Characterization of an Inner Ear-Expressed Human Melanoma Inhibitory Activity (MIA)-like Gene (MIAL) with a Frequent Polymorphism That Abolishes Translation

“…The binding site for Hmx is also recognized by the homeodomain factor Nkx-2.5 (Amendt et al, 1999). Additional analysis identified three core binding sites (AA-CAAT) for SOX proteins (Mertin et al, 1999).…”

“…In contrast, the MIAL promoter contains no AP-2 or NF-B sites. The MIAL promoter contains three core SOX binding elements, but none with optimal flanking sequence for SOX9 binding (Mertin et al, 1999).…”

Identification of SOX4 target genes using phylogenetic footprinting-based prediction from expression microarrays suggests that overexpression of SOX4 potentiates metastasis in hepatocellular carcinoma

“…Based on the percentage of each nucleotide at each position within the core motif (WWCAAWG) and the three flanking nucleotides, the consensus binding motif for SOX4 is A/T T/A CAA A/t G (Table 2a). Motif specificity was validated by comparing the SOX4 motif with the consensus SOX9 motif (Mertin et al, 1999). In contrast to SOX9, no predominant 5 0 or 3 0 flanking nucleotides were observed for the SOX4 binding motifs in the 31 target genes (Table 2b).…”

Frontal nasal prominence expression driven by Tcfap2a relies on a conserved binding site for STAT proteins

“…Analysis of the sequence in this region revealed an SRY-related HMG box (Sox) binding site (5Ј-ATTGTT-3Ј) (Mertin et al, 1999). Several members of the Sox gene family are required for face and limb development, particularly for skeletogenesis, and so represented important potential candidates for Tcfap2a regulation in these structures (Nakashima and de Crombrugghe, 2003;Richman and Lee, 2003;Sock et al, 2004).…”

“…For these experiments, we generated mutant probes, 95W, 95X, 95Y, and 95Z (Table 1A) and compared their ability to bind Sox6 with either probe 95 or a standard Sox binding site. 95X contained a G to C mutation at position 4 of the Sox binding site within probe 95, a substitution that has previously been shown to interfere with Sox factor-DNA interaction (Mertin et al, 1999). 95W also contains this substitution as well as a second change in the consensus Sox site (Table 1A).…”