Mechanism of Regulatory Target Selection by the SOX High-Mobility-Group Domain Proteins as Revealed by Comparison of SOX1/2/3 and SOX9

Abstract

SOX proteins bind similar DNA motifs through their high-mobility-group (HMG) domains, but their action is highly specific with respect to target genes and cell type. We investigated the mechanism of target selection by comparing SOX1/2/3, which activate ␦-crystallin minimal enhancer DC5, with SOX9, which activates Col2a1 minimal enhancer COL2C2. These enhancers depend on both the SOX binding site and the binding site of a putative partner factor. The DC5 site was equally bound and bent by the HMG domains of SOX1/2 and SOX9. The activation domains of these SOX proteins mapped at the distal portions of the C-terminal domains were not cell specific and were independent of the partner factor. Chimeric proteins produced between SOX1 and SOX9 showed that to activate the DC5 enhancer, the C-terminal domain must be that of SOX1, although the HMG domains were replaceable. The SOX2-VP16 fusion protein, in which the activation domain of SOX2 was replaced by that of VP16, activated the DC5 enhancer still in a partner factor-dependent manner. The results argue that the proximal portion of the C-terminal domain of SOX1/2 specifically interacts with the partner factor, and this interaction determines the specificity of the SOX1/2 action. Essentially the same results were obtained in the converse experiments in which COL2C2 activation by SOX9 was analyzed, except that specificity of SOX9-partner factor interaction also involved the SOX9 HMG domain. The highly selective SOX-partner factor interactions presumably stabilize the DNA binding of the SOX proteins and provide the mechanism for regulatory target selection.An important question of transcriptional regulation is how transcription factors select their correct target genes within the genome. DNA-binding specificity of individual protein factors must be one of the major determinants of target gene selection. A family of closely related transcription factors often have highly similar DNA-binding properties, most often recognizing only ϳ6-bp DNA sequences, with allowance of considerable degeneracy. Nevertheless, these transcription factors activate or repress each specific set of target genes via these binding motifs and exert quite different biological effects even within a protein family. The Sox gene family investigated in this work is a good example of such a family. The problem is the mechanism which enables each SOX protein to select the correct subset of binding sites to achieve its proper in vivo function.Sox genes have been identified on the basis of their homology to the high-mobility-group (HMG) box of the mammalian testis-determining gene Sry (17; for a review, see reference 36). They comprise a large gene family of more than 20 members in mammals and are found in all investigated species of the animal kingdom (10, 49). SOX proteins are grouped into subfamilies based on the amino acid sequence of the HMG domain (49). Within each subfamily, the amino acid sequence of the HMG domain is Ն90% identical and sequence similarity extends outside this domain. Although the ho...

Novel SOX2 partner-factor domain mutation in a four-generation family

“…20,21 In the lens, SOX2 interacts with the lens-specific factor dEF3, both of which bind to the DC5 enhancer and direct d-crystallin lens-specific gene expression. 20 The interaction between SOX2 and dEF3 is dependent on the conserved B group homology region immediately C-terminal to the HMG domain.…”

“…20,21 In the lens, SOX2 interacts with the lens-specific factor dEF3, both of which bind to the DC5 enhancer and direct d-crystallin lens-specific gene expression. 20 The interaction between SOX2 and dEF3 is dependent on the conserved B group homology region immediately C-terminal to the HMG domain. The SOX2 mutation identified in this family, c.368A4G, p.Asp123Gly, leads to an amino acid change from a hydrophilic aspartic acidic residue to a hydrophobic glycine residue in this highly conserved B group partner-factor interaction region (Figure 2c).…”

Quantitative assessment of the knockdown efficiency of morpholino antisense oligonucleotides in zebrafish embryos using a luciferase assay

“…Lysates (seven-embryo equivalent/ lane) were separated by 10% SDS-PAGE and subjected to western transfer, as described by Kamachi et al (1999). To detect the SOX2 protein, anti-SOX2 antibody (AF2018, R&D systems, Minneapolis, MN) at 0.16 lg/ml in Can-Get-Signal immunoreaction enhancer solution (Toyobo, Osaka, Japan) was used.…”

Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers

“…They are recognized as key players in the determination of cell fate (Pevny and Lovell-Badge 1997). Because HMG domains of SOX proteins are similar to each other in their DNA sequence preference (Mertin et al 1999) and in their DNA-bending activity (Kamachi et al 1999), it remains elusive how they are capable of specific target site selection. Assembly with unrelated transcriptional regulator proteins, however, provides a plausible explanation of how they can distinguish their targets as well as act in a cell-specific fashion (Kamachi et al 2000).…”