SOX9 and TCF transcription factors associate to mediate Wnt/β-catenin target gene activation in colorectal cancer

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. 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. Transcriptional regulation by the Wnt/β-catenin pathway is essential for metazoan development and homeostasis (1Clevers H. Loh K.M. Nusse R. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control.Science. 2014; 3461248012Crossref PubMed Scopus (914) Google Scholar), and the transcription factors (TFs) of the T-cell factor/lymphoid enhancer factor family (TCF/LEF family or TCFs) are major effectors of this signaling cascade (2Ramakrishnan A.-B. Cadigan K.M. Wnt target genes and where to find them.F1000Res. 2017; 6: 746Crossref PubMed Scopus (48) Google Scholar). Clusters of TCF-binding sites are characteristic of many Wnt-regulated elements (WREs), the cis-regulatory DNA elements that drive Wnt-responsive transcription (3Archbold H.C. Yang Y.X. Chen L. Cadigan K.M. How do they do Wnt they do?: regulation of transcription by the Wnt/β-catenin pathway.Acta Physiol. 2012; 204: 74-109Crossref PubMed Scopus (106) Google Scholar). WRE activation is triggered by the Wnt-dependent stabilization and accumulation of the protein β-catenin, which is recruited to WREs by TCFs. β-catenin recruits coactivators to induce Wnt target gene transcription (2Ramakrishnan A.-B. Cadigan K.M. Wnt target genes and where to find them.F1000Res. 2017; 6: 746Crossref PubMed Scopus (48) Google Scholar, 4Clevers H. Nusse R. Wnt/β-Catenin signaling and disease.Cell. 2012; 149: 1192-1205Abstract Full Text Full Text PDF PubMed Scopus (4087) Google Scholar). Signaling by Wnt proteins performs many essential developmental functions that do not involve transcriptional regulation through β-catenin (5Acebron S.P. Niehrs C. β-Catenin-Independent roles of Wnt/LRP6 signaling.Trends Cell Biol. 2016; 26: 956-967Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 6Yang Y. Mlodzik M. Wnt-frizzled/planar cell polarity signaling: cellular orientation by facing the wind (Wnt).Annu. Rev. Cell Dev. Biol. 2015; 31: 623-646Crossref PubMed Scopus (236) Google Scholar). For brevity, we use the phrases “Wnt signaling” and “Wnt target gene” in this study to refer to Wnt signaling through β-catenin and genes regulated by the Wnt/β-catenin pathway. While TCFs mediate Wnt target gene activation across cell types, there is great spatiotemporal diversity in the identity of Wnt target genes, with different tissues expressing unique cell type–specific Wnt transcriptional programs. This specificity is thought to arise from interactions between TCFs and other TFs that help determine cell fate and identity. Many non-TCF TFs bind to WREs and affect Wnt target gene expression, but a comprehensive molecular understanding of how these TFs generate target gene specificity is still incomplete (7Nakamura Y. Hoppler S. Genome-wide analysis of canonical Wnt target gene regulation in Xenopus tropicalis challenges β-catenin paradigm.genesis. 2017; 55e22991Crossref PubMed Scopus (13) Google Scholar, 8Söderholm S. Cantù C. The WNT/β-catenin dependent transcription: a tissue-specific business.WIREs Mech. Dis. 2021; 13e1511PubMed Google Scholar). One well-studied example is TFs of the CDX family, which bind to several WREs and recruit TCFs to them (9Lewis A. Freeman-Mills L. de la Calle-Mustienes E. Giráldez-Pérez R.M. Davis H. Jaeger E. et al.A polymorphic enhancer near GREM1 influences bowel cancer risk through differential CDX2 and TCF7L2 binding.Cell Rep. 2014; 8: 983-990Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 10Verzi M.P. Hatzis P. Sulahian R. Philips J. Schuijers J. Shin H. et al.TCF4 and CDX2, major transcription factors for intestinal function, converge on the same cis-regulatory regions.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 15157-15162Crossref PubMed Scopus (68) Google Scholar). Their ability to form complexes with TCFs is essential for the activation of several Wnt target genes (11Ramakrishnan A.-B. Chen L. Burby P.E. Cadigan K.M. Wnt target enhancer regulation by a CDX/TCF transcription factor collective and a novel DNA motif.Nucl. Acids Res. 2021; 49: 8625-8641Crossref PubMed Scopus (4) Google Scholar). Another group of developmentally important TFs that modulate Wnt target gene expression is the SOX family of proteins. While some SOX family members directly bind to TCFs and β-catenin and promote the expression of specific Wnt targets (12Kormish J.D. Sinner D. Zorn A.M. Interactions between SOX factors and Wnt/β-catenin signaling in development and disease.Dev. Dyn. 2010; 239: 56-68PubMed Google Scholar, 13Sinner D. Kordich J.J. Spence J.R. Opoka R. Rankin S. Lin S.-C.J. et al.Sox17 and Sox4 differentially regulate β-catenin/T-cell factor Activity and proliferation of colon carcinoma cells.Mol. Cell. Biol. 2007; 27: 7802-7815Crossref PubMed Scopus (263) Google Scholar, 14Mukherjee S. Chaturvedi P. Rankin S.A. Fish M.B. Wlizla M. Paraiso K.D. et al.Sox17 and β-catenin co-occupy Wnt-responsive enhancers to govern the endoderm gene regulatory network.eLife. 2020; 9e58029Crossref Google Scholar, 15Mukherjee S. Luedeke D.M. McCoy L. Iwafuchi M. Zorn A.M. SOX transcription factors direct TCF-independent WNT/β-catenin responsive transcription to govern cell fate in human pluripotent stem cells.Cell Rep. 2022; 40111247Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar), other SOX proteins are known to repress Wnt/β-catenin signaling, with SOX9 being the most intensively studied. SOX9 has essential functions in the development of cartilage and the skeletal system, and mutations in SOX9 are associated with sex reversal and severe skeletal deformities in humans (16Lefebvre V. Seven - roles and regulation of SOX transcription factors in skeletogenesis.in: Olsen B.R. Current Topics in Developmental Biology. Vertebrate Skeletal Development. 133. Academic Press, Cambridge, MA2019: 171-193Crossref Scopus (58) Google Scholar, 17Lefebvre V. Angelozzi M. Haseeb A. SOX9 in cartilage development and disease.Curr. Opin. Cell Biol. 2019; 61: 39-47Crossref PubMed Scopus (101) Google Scholar). SOX9 is also well known for its role in promoting testis formation during mammalian development (18Sekido R. Lovell-Badge R. Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer.Nature. 2008; 453: 930-934Crossref PubMed Scopus (715) Google Scholar). Genetic studies have shown that testis or ovary commitment in mammalian gonadal development involves a mutual opposition between the Wnt/β-catenin pathway, which promotes ovarian development and inhibits testis fate, and SOX9, which inhibits the Wnt/β-catenin pathway to promote testes growth (19Nicol B. Yao H.H.-C. Gonadal identity in the absence of pro-testis factor SOX9 and pro-ovary factor beta-catenin in Mice1.Biol. Reprod. 2015; 93: 1-12Crossref Scopus (51) Google Scholar). In several different mammalian cell lines, the overexpression of SOX9 causes a reduction in Wnt transcriptional readouts and in β-catenin protein levels (20Akiyama H. Lyons J.P. Mori-Akiyama Y. Yang X. Zhang R. Zhang Z. et al.Interactions between Sox9 and β-catenin control chondrocyte differentiation.Genes Dev. 2004; 18: 1072-1087Crossref PubMed Scopus (643) Google Scholar, 21Sellak H. Wu S. Lincoln T.M. KLF4 and SOX9 transcription factors antagonize β-catenin and inhibit TCF-activity in cancer cells.Biochim. Biophys. Acta (Bba) - Mol. Cell Res. 2012; 1823: 1666-1675Crossref PubMed Scopus (36) Google Scholar, 22Sinha A. Fan V.B. Ramakrishnan A.-B. Engelhardt N. Kennell J. Cadigan K.M. Repression of Wnt/β-catenin signaling by SOX9 and Mastermind-like transcriptional coactivator 2.Sci. Adv. 2021; 7eabe0849Crossref Scopus (10) Google Scholar, 23Topol L. Chen W. Song H. Day T.F. Yang Y. Sox9 inhibits Wnt signaling by promoting β-catenin phosphorylation in the nucleus.J. Biol. Chem. 2009; 284: 3323-3333Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Recent work from our group found that SOX9 promoted β-catenin degradation by transcriptionally activating the Notch pathway coactivator MAML2, which associates with β-catenin (22Sinha A. Fan V.B. Ramakrishnan A.-B. Engelhardt N. Kennell J. Cadigan K.M. Repression of Wnt/β-catenin signaling by SOX9 and Mastermind-like transcriptional coactivator 2.Sci. Adv. 2021; 7eabe0849Crossref Scopus (10) Google Scholar). Although primarily characterized as a repressor of Wnt/β-catenin target genes, correlative evidence hints at a more complex relationship between SOX9 and Wnt/β-catenin signaling in the intestinal epithelium and in colorectal cancer (CRC). The villi of the intestinal epithelium consist of short lived terminally differentiated cells that are continuously replenished from a population of intestinal epithelial stem cells. The proliferation of these stem cells and the differentiation of the Paneth cells, which surround and metabolically support them, are both dependent on Wnt/β-catenin signaling (24van Es J.H. Jay P. Gregorieff A. van Gijn M.E. Jonkheer S. Hatzis P. et al.Wnt signalling induces maturation of Paneth cells in intestinal crypts.Nat. Cell Biol. 2005; 7: 381-386Crossref PubMed Scopus (513) Google Scholar, 25van Es J.H. Haegebarth A. Kujala P. Itzkovitz S. Koo B.-K. Boj S.F. et al.A critical role for the Wnt effector Tcf4 in adult intestinal homeostatic self-renewal.Mol. Cell Biol. 2012; 32: 1918-1927Crossref PubMed Scopus (188) Google Scholar). Paneth cells express high levels of SOX9 (26Blache P. van de Wetering M. Duluc I. Domon C. Berta P. Freund J.-N. et al.SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes.J. Cell Biol. 2004; 166: 37-47Crossref PubMed Scopus (380) Google Scholar), and the loss of either SOX9 or Wnt signaling prevents Paneth cell differentiation (24van Es J.H. Jay P. Gregorieff A. van Gijn M.E. Jonkheer S. Hatzis P. et al.Wnt signalling induces maturation of Paneth cells in intestinal crypts.Nat. Cell Biol. 2005; 7: 381-386Crossref PubMed Scopus (513) Google Scholar, 27Bastide P. Darido C. Pannequin J. Kist R. Robine S. Marty-Double C. et al.Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium.J. Cell Biol. 2007; 178: 635-648Crossref PubMed Scopus (378) Google Scholar, 28Mori–Akiyama Y. van den Born M. van Es J.H. Hamilton S.R. Adams H.P. Zhang J. et al.SOX9 is required for the differentiation of Paneth cells in the intestinal epithelium.Gastroenterology. 2007; 133: 539-546Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Additionally, SOX9 overexpression in Wnt-dependent CRC lines induces the expression of many genes characteristic of Paneth cells (29Liang X. Duronio G.N. Yang Y. Bala P. Hebbar P. Spisak S. et al.An enhancer-driven stem cell–like program mediated by SOX9 blocks intestinal differentiation in colorectal cancer.Gastroenterology. 2022; 162: 209-222Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Some of these are also directly activated by Wnt signaling (29Liang X. Duronio G.N. Yang Y. Bala P. Hebbar P. Spisak S. et al.An enhancer-driven stem cell–like program mediated by SOX9 blocks intestinal differentiation in colorectal cancer.Gastroenterology. 2022; 162: 209-222Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 30Wehkamp J. Wang G. Kübler I. Nuding S. Gregorieff A. Schnabel A. et al.The Paneth cell α-defensin deficiency of ileal crohn’s disease is linked to Wnt/Tcf-4.J. Immunol. 2007; 179: 3109-3118Crossref PubMed Scopus (232) Google Scholar, 31Beisner J. Teltschik Z. Ostaff M.J. Tiemessen M.M. Staal F.J.T. Wang G. et al.TCF-1-mediated Wnt signaling regulates Paneth cell innate immune defense effectors HD-5 and -6: Implications for crohn’s disease.Am. J. Physiol. - Gastrointest. Liver Physiol. 2014; 307: G487-G498Crossref PubMed Scopus (27) Google Scholar), suggesting the possibility of SOX9 and Wnt signaling working together to activate gene expression. Understanding the relationship between SOX9 and Wnt signaling in this context requires an understanding of the regulatory logic of target gene WREs regulated by both factors. Aberrant Wnt/β-catenin pathway activation in intestinal stem cells leads to uncontrolled proliferation, and consequently, activating mutations in the Wnt/β-catenin pathway are major drivers of CRC (4Clevers H. Nusse R. Wnt/β-Catenin signaling and disease.Cell. 2012; 149: 1192-1205Abstract Full Text Full Text PDF PubMed Scopus (4087) Google Scholar, 32Gregorieff A. Clevers H. Wnt signaling in the intestinal epithelium: From endoderm to cancer.Genes Dev. 2005; 19: 877-890Crossref PubMed Scopus (538) Google Scholar). Additionally, mutations that increase the activity of WREs that regulate oncogenes such as MYC are also associated with increased cancer risk (33Dave K. Sur I. Yan J. Zhang J. Kaasinen E. Zhong F. et al.Mice deficient of Myc super-enhancer region reveal differential control mechanism between normal and pathological growth.eLife. 2017; 6e23382Crossref PubMed Scopus (39) Google Scholar, 34Konsavage W.M. Yochum G.S. The myc 3′ Wnt-responsive element suppresses colonic tumorigenesis.Mol. Cell. Biol. 2014; 34: 1659-1669Crossref PubMed Scopus (9) Google Scholar, 35Tuupanen S. Turunen M. Lehtonen R. Hallikas O. Vanharanta S. Kivioja T. et al.The common colorectal cancer predisposition SNP rs6983267 at chromosome 8q24 confers potential to enhanced Wnt signaling.Nat. Genet. 2009; 41: 885-890Crossref PubMed Scopus (428) Google Scholar, 36Wright J.B. Brown S.J. Cole M.D. Upregulation of c-MYC in cis through a large chromatin loop linked to a cancer risk-associated single-nucleotide polymorphism in colorectal cancer cells.Mol. Cell. Biol. 2010; 30: 1411-1420Crossref PubMed Scopus (220) Google Scholar). Many Wnt-dependent CRC lines and primary CRC samples also show high levels of SOX9 expression (37Matheu A. Collado M. Wise C. Manterola L. Cekaite L. Tye A.J. et al.Oncogenicity of the developmental transcription factor Sox9.Cancer Res. 2012; 72: 1301-1315Crossref PubMed Scopus (170) Google Scholar, 38Prévostel C. Blache P. The dose-dependent effect of SOX9 and its incidence in colorectal cancer.Eur. J. Cancer. 2017; 86: 150-157Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), and recent reports suggest that SOX9 promotes stemness and survival of some CRC lines (29Liang X. Duronio G.N. Yang Y. Bala P. Hebbar P. Spisak S. et al.An enhancer-driven stem cell–like program mediated by SOX9 blocks intestinal differentiation in colorectal cancer.Gastroenterology. 2022; 162: 209-222Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 39Xu Y. Zhang X. Hu X. Zhou W. Zhang P. Zhang J. et al.The effects of lncRNA MALAT1 on proliferation, invasion and migration in colorectal cancer through regulating SOX9.Mol. Med. 2018; 24: 52Crossref PubMed Scopus (87) Google Scholar, 40Zhou T. Wu L. Ma N. Tang F. Yu Z. Jiang Z. et al.SOX9-activated FARSA-AS1 predetermines cell growth, stemness, and metastasis in colorectal cancer through upregulating FARSA and SOX9.Cell Death Dis. 2020; 11: 1-15Crossref PubMed Scopus (30) Google Scholar). The findings of SOX9 promoting Paneth cell fate and CRC cell survival are at odds with models of SOX9 as a Wnt/β-catenin pathway repressor. In this study, we interrogate the relationship between SOX9 and Wnt signaling in regulating gene expression. In contrast to its previously known role as a repressor of Wnt/β-catenin signaling, we show that SOX9 and Wnt signaling work together to promote the growth and survival of CRC cells. Among the target genes activated by Wnt/β-catenin signaling and SOX9 is the oncogene MYC. In CRC cells, SOX9 is associated with many WREs, including several cancer risk-associated enhancers of MYC. Our characterization of the cis-regulatory grammar that allows SOX9 to activate Wnt target genes reveals the presence of SOX9-binding sites in the c-myc-335 WRE, which regulate enhancer activity along with TCF sites. A similar regulatory logic is also seen in the promoters of Defa5 and Defa6, two markers of Paneth cells, that are synergistically upregulated under high Wnt, high SOX9 conditions. With reporter mutagenesis and synthetic reporters, we show that the combination of TCF- and SOX-binding sites is necessary and sufficient for synergistic activation by Wnt and SOX9. Mechanistically, we show that SOX9 directly binds to TCFs. Through a novel SOX9 separation-of-function mutant that is defective in TCF binding, we show that the activation of WREs and CRC cell growth both require not just the activities of TCFs and SOX9 but also the activity of a TCF-SOX9 complex. Our work demonstrates that in addition to its role as a repressor of the Wnt/β-catenin pathway, SOX9 works together with the Wnt/β-catenin pathway to activate a subset of Wnt target genes. To explore the roles of SOX9 and Wnt/β-catenin signaling in CRC, we tested the consequences of depleting SOX9 and/or β-catenin in LS174T CRC cells. These cells contain an activating mutation in β-catenin (S45F) and are dependent on Wnt signaling for their growth and survival (41van de Wetering M. Oving I. Muncan V. Fong M.T.P. Brantjes H. Leenen D. van et al.Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector.EMBO Rep. 2003; 4: 609-615Crossref PubMed Scopus (463) Google Scholar, 42Li V.S.W. Ng S.S. Boersema P.J. Low T.Y. Karthaus W.R. Gerlach J.P. et al.Wnt signaling through inhibition of β-catenin degradation in an intact Axin1 complex.Cell. 2012; 149: 1245-1256Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar). We used LS174T cells containing a doxycycline (DOX)-inducible β-catenin shRNA expression cassette (41van de Wetering M. Oving I. Muncan V. Fong M.T.P. Brantjes H. Leenen D. van et al.Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector.EMBO Rep. 2003; 4: 609-615Crossref PubMed Scopus (463) Google Scholar) (referred to as LS174T-pTER-β-cat cells) and transduced them with constructs expressing either nontargeting (Scrambled) or SOX9-targeting shRNAs (Table S1). Western blots showed reduced SOX9 expression in the two SOX9 shRNA lines (Fig. 1A, lanes 2,3). This reduction of SOX9 protein levels was not accompanied by an upregulation of β-catenin protein, as would be expected if SOX9 was promoting β-catenin turnover (Fig. 1A, lanes 2,3). This suggested that SOX9 does not repress β-catenin levels in these cells. SOX9 protein levels were not affected by β-catenin depletion, suggesting that SOX9 is not a target of Wnt signaling in these cells (Fig. 1A, lanes 1,4). To understand whether SOX9 and Wnt signaling worked together to promote the survival of LS174T-pTER-β-cat cells, we tested for a SOX9 requirement in a colony formation assay with and without DOX-induced β-catenin depletion. Knocking down SOX9 alone resulted in fewer colonies, and the combined depletion of β-catenin and SOX9 reduced the clonogenicity of these cells even further (Fig. 1, B and C). We then tested the impact of Wnt signaling and SOX9 on the growth rate of these cells. We treated cells with or without DOX for 48 h and measured metabolic activity as a proxy for cell number using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The depletion of β-catenin or SOX9 led to slower growth, and combined depletion of β-catenin and SOX9 reduced growth 4- to 5-fold (Fig. 1D). To ensure that our findings were not specific to LS174T cells, we also tested the role of SOX9 in promoting the survival of DLD-1 cells. DLD-1 cells also have elevated levels of Wnt signaling, but unlike LS174T cells, express a WT β-catenin protein and a truncated form of the destruction complex component APC (43Kishida S. Yamamoto H. Ikeda S. Kishida M. Sakamoto I. Koyama S. et al.Axin, a negative regulator of the Wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of β-catenin∗.J. Biol. Chem. 1998; 273: 10823-10826Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). In DLD-1 cells, SOX9 depletion caused a 3-fold reduction in colony formation (Fig. 1, E and F). Just like in LS174T cells, SOX9 depletion did not cause an upregulation of β-catenin protein levels, and the inhibition of Wnt signaling through a dominant negative TCF (dnTCF) construct (44van de Wetering M. Sancho E. Verweij C. de Lau W. Oving I. Hurlstone A. et al.The β-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.Cell. 2002; 111: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1748) Google Scholar) did not reduce SOX9 protein levels (Fig. S1A). We then examined the importance of Wnt signaling and SOX9 across a panel of CRC lines using data from the Cancer Cell Line Encyclopedia (CCLE) (45Barretina J. Caponigro G. Stransky N. Venkatesan K. Margolin A.A. Kim S. et al.The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.Nature. 2012; 483: 603-607Crossref PubMed Scopus (4972) Google Scholar, 46Ghandi M. Huang F.W. Jané-Valbuena J. Kryukov G.V. Lo C.C. McDonald E.R. et al.Next-generation characterization of the cancer cell line Encyclopedia.Nature. 2019; 569: 503-508Crossref PubMed Scopus (1279) Google Scholar). First, we examined gene expression data and correlated it with dependency scores calculated from CRISPR KO experiments using the Chronos algorithm (47Dempster J.M. Boyle I. Vazquez F. Root D.E. Boehm J.S. Hahn W.C. et al.Chronos: a cell population dynamics model of CRISPR experiments that improves inference of gene fitness effects.Genome Biol. 2021; 22: 343Crossref PubMed Scopus (43) Google Scholar). As expected in CRC, 49/53 lines examined showed high β-catenin (CTNNB1) expression and dependence based on our cutoffs (Fig. S1B). A similar analysis for SOX9 revealed that 38/53 lines showed high SOX9 expression and SOX9-dependent growth (Fig. S1C). Dependence on SOX9 was highly correlated with β-catenin-dependence, with 40/56 lines being highly dependent on both β-catenin and SOX9 (Fig. S1D). As a negative control, we looked at SOX17, which represses Wnt targets and inhibits the proliferation of CRC cells (13Sinner D. Kordich J.J. Spence J.R. Opoka R. Rankin S. Lin S.-C.J. et al.Sox17 and Sox4 differentially regulate β-catenin/T-cell factor Activity and proliferation of colon carcinoma cells.Mol. Cell. Biol. 2007; 27: 7802-7815Crossref PubMed Scopus (263) Google Scholar). Just 2/56 lines showed a dependence on both genes (Fig. S1E). Across all cancers in the CCLE database (n = 1070), β-catenin was the seventh-most correlated gene with SOX9 in terms of essentiality (Fig. S1F). In conjunction with our experimental data, these analyses strongly indicated that Wnt signaling and SOX9 working together was a general feature of CRC. Since both Wnt signaling and SOX9 are major regulators of transcription, we wondered whether SOX9 could cooperate with the Wnt/β-catenin pathway to activate a common transcriptional program in CRC. One of the best-characterized Wnt targets in the context of CRC is the MYC oncogene (48Van der Flier L.G. Sabates–Bellver J. Oving I. Haegebarth A. De Palo M. Anti M. et al.The intestinal Wnt/TCF signature.Gastroenterology. 2007; 132: 628-632Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar), which is regulated by several WREs associated with CRC risk (33Dave K. Sur I. Yan J. Zhang J. Kaasinen E. Zhong F. et al.Mice deficient of Myc super-enhancer region reveal differential control mechanism between normal and pathological growth.eLife. 2017; 6e23382Crossref PubMed Scopus (39) Google Scholar). In DLD-1 cells, MYC transcripts were downregulated by the combination of SOX9 knockdown and Wnt inhibition by dnTCF (Fig. 1G). After finding that MYC transcript levels were upregulated by Wnt signaling and SOX9, we wanted to study the regulatory interactions that allowed SOX9 to activate this Wnt target. One of the best-characterized WREs regulating MYC is the c-myc-335 enhancer containing a SNP (rs6983267) that increases CRC risk in humans and is regulated by TCF activity (11Ramakrishnan A.-B. Chen L. Burby P.E. Cadigan K.M. Wnt target enhancer regulation by a CDX/TCF transcription factor collective and a novel DNA motif.Nucl. Acids Res. 2021; 49: 8625-8641Crossref PubMed Scopus (4) Google Scholar, 49Tomlinson I. Webb E. Carvajal-Carmona L. Broderick P. Kemp Z. Spain S. et al.A genome-wide association scan of tag SNPs identifies a susceptibility variant for colorectal cancer at 8q24.21.Nat. Genet. 2007; 39: 984-988Crossref PubMed Scopus (705) Google Scholar, 50Zanke B.W. Greenwood C.M. Rangrej J. Kustra R. Tenesa A. Farrington S.M. et al.Genome-wide association scan identifies a colorectal cancer susceptibility locus on chromosome 8q24.Nat. Genet. 2007; 39: 989-994Crossref PubMed Scopus (617) Google Scholar). Examination of a previously published SOX9 chromatin immunoprecipitation sequencing (ChIP-seq) dataset (51Shi Z. Chiang C.-I. Labhart P. Zhao Y. Yang J. Mistretta T.-A. et al.Context-specific role of SOX9 in NF-Y mediated gene regulation in colorectal cancer cells.Nucl. Acids Res. 2015; 43: 6257-6269Crossref PubMed Scopus (47) Google Scholar) showed that c-myc-335 was bound by SOX9 in HT-29 CRC cells (Fig. 2A). We verified this result in LS174T cells by performing ChIP using antibodies against β-catenin and SOX9. To detect ChIP enrichment, we compared quantitative PCR (qPCR) signals from primers located inside the enhancer to those from two primer sets flanking the enhancer (Fig. 2B) and found that β-catenin and SOX9 were both enriched at the enhancer (Fig. 2C). Previous work from our group had generated a luciferase reporter driven by c-myc-335, which showed Wnt-dependent activity (11Ramakrishnan A.-B. Chen L. Burby P.E. Cadigan K.M. Wnt target enhancer regulation by a CDX/TCF transcription factor collective and a novel DNA motif.Nucl. Acids Res. 2021; 49: 8625-8641Crossref PubMed Scopus (4) Google Scholar). In LS174T cells, we found that RNAi-mediated SOX9 depletion reduced c-myc-335 reporter activity (Fig. 2D). Wnt/β-catenin pathway inhibition with dnTCF repressed, while SOX9 overexpression activated the c-myc-335 reporter (Fig. 2E). In combination with the ChIP data, these results were consistent with a model of SOX9 directly binding and activating this enhancer in conjunction with TCFs and β-catenin. To understand how SOX9 was recruited to this enhancer, we searched its sequence for potential SOX9-binding sites. Our previous work on the cis-regulatory logic of c-myc-335 had identified four TCF-binding sites, two sites bound by CDX proteins, and five repeated motifs, we named CAG sites, all of which contributed to enhancer activity (11Ramakrishnan A.-B. Chen L. Burby P.E. Cadigan K.M. Wnt target enhancer regulation by a CDX/TCF transcription factor collective and a novel DNA motif.Nucl. Acids Res. 2021; 49: 8625-8641Crossref PubMed Scopus (4) Google Scholar). Using SOX9-binding site data from the JASPAR database, we scanned the c-myc-335 sequence for putative clusters of SOX9-binding sites. This search identified a pair of putative SOX-binding sites located adjacently in an inverted repeat orientation (Figs. 2F and S1A). The ability of SOX9 to dimerize and bind DNA is an importan