Activation of the Wnt/β-catenin pathway regulates gene expression by promoting the formation of a β-catenin–T-cell factor (TCF) complex on target enhancers. In addition to TCFs, other transcription factors interact with the Wnt/β-catenin pathway at different levels to produce tissue-specific patterns of Wnt target gene expression. The transcription factor SOX9 potently represses many Wnt target genes by downregulating β-catenin protein levels. Here, we find using colony formation and cell growth assays that SOX9 surprisingly promotes the proliferation of Wnt-driven colorectal cancer (CRC) cells. In contrast to how it indirectly represses Wnt targets, SOX9 directly co-occupies and activates multiple Wnt-responsive enhancers in CRC cells. Our examination of the binding site grammar of these enhancers shows the presence of TCF and SOX9 binding sites that are necessary for transcriptional activation. In addition, we identify a physical interaction between the DNA-binding domains of TCFs and SOX9 and show that TCF-SOX9 interactions are important for target gene regulation and CRC cell growth. Our work demonstrates a highly context-dependent effect of SOX9 on Wnt targets, with the presence or absence of SOX9-binding sites on Wnt-regulated enhancers determining whether they are directly activated or indirectly repressed by SOX9.

Wnt/β-连环蛋白通路的激活通过促进靶增强子上 β-连环蛋白-T 细胞因子 (TCF) 复合物的形成来调节基因表达。除了 TCF 之外，其他转录因子在不同水平上与 Wnt/β-catenin 通路相互作用，产生 Wnt 靶基因表达的组织特异性模式。转录因子 SOX9 通过下调 β-catenin 蛋白水平来有效抑制许多 Wnt 靶基因。在这里，我们通过集落形成和细胞生长测定发现，SOX9 令人惊讶地促进了 Wnt 驱动的结直肠癌 (CRC) 细胞的增殖。与间接抑制 Wnt 靶标的方式相反，SOX9 直接共占据并激活 CRC 细胞中的多个 Wnt 反应性增强子。我们对这些增强子的结合位点语法的检查表明存在转录激活必需的 TCF 和 SOX9 结合位点。此外，我们还确定了 TCF 和 SOX9 的 DNA 结合域之间的物理相互作用，并表明 TCF-SOX9 相互作用对于靶基因调控和 CRC 细胞生长很重要。我们的工作证明了 SOX9 对 Wnt 靶标的高度依赖性作用，Wnt 调节的增强子上是否存在 SOX9 结合位点决定了它们是否被 SOX9 直接激活或间接抑制。