SOX2 can associate with chromatin directly by binding to DNA or indirectly via association with other chromatin-bound proteins

It is widely assumed that SOX2 regulates gene expression and facilitates the opening of chromatin by binding directly at SOX motifs. To test this assumption, we created a SOX2 DNA binding mutant to determine whether other regions of SOX2 contribute to gene target specificity. When exogenously expressed in cells, this SOX2 mutant [SOX2(G76P)], like elevated unmodified SOX2, dramatically alters the transcriptome, but it does so by regulating vastly different gene sets and gene networks than SOX2. Consistent with their differential effects on the transcriptome, ChIP-seq analysis demonstrates that SOX2 and SOX2(G76P) associate primarily with different genomic loci, and motif analysis indicates that SOX2 binds primarily at SOX motifs, whereas SOX2(G76P) associates with chromatin at non-SOX motifs, including AP-1 motifs. Additionally, ATAC-seq analysis indicates that SOX2 substantially increases chromatin accessibility, but SOX2(G76P) does not. The findings presented lead to the conclusion that SOX2(G76P) associates with chromatin indirectly by a "piggyback" mechanism through its association with other chromatin-associated proteins, including AP-1 complexes. Remarkably, we also show that SOX2 and SOX2(G76P) each associate with a subset of the same gene loci that contain several different DNA motifs, including AP-1 motifs, but no high confidence SOX motifs. Overall, our findings provide new perspectives on SOX2 and lead to two important conclusions: 1) selection of gene targets by SOX2 is not solely determined by its DNA binding domain, and 2) SOX2 not only associates with chromatin directly by binding to SOX motifs but can also associate with a subset of gene loci indirectly through its association with other chromatin-associated proteins.