T-cell factor 4 (TCF4), together with β-catenin coactivator, functions as the major transcriptional mediator of the canonical wingless/integrated (Wnt) signaling pathway in the intestinal epithelium. The pathway activity is essential for both intestinal homeostasis and tumorigenesis. To date, several mouse models and cellular systems have been used to analyze TCF4 function. However, some findings were conflicting, especially those that were related to the defects observed in the mouse gastrointestinal tract after Tcf4 gene deletion, or to a potential tumor suppressive role of the gene in intestinal cancer cells or tumors. Here, we present the results obtained using a newly generated conditional Tcf4 allele that allows inactivation of all potential Tcf4 isoforms in the mouse tissue or small intestinal and colon organoids. We also employed the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to disrupt the TCF4 gene in human cells. We showed that in adult mice, epithelial expression of Tcf4 is indispensable for cell proliferation and tumor initiation. However, in human cells, the TCF4 role is redundant with the related T-cell factor 1 (TCF1) and lymphoid enhancer-binding factor 1 (LEF1) transcription factors.

Faculty of Science Charles University Prague Albertov 6 Praha 128 43 Czech Republic

Institute of Molecular Genetics of the CAS v v i Videnska 1083 Prague 142 20 Czech Republic

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Clevers H., Nusse R. Wnt/beta-catenin signaling and disease. Cell. 2012;149:1192–1205. doi: 10.1016/j.cell.2012.05.012. PubMed DOI

Valenta T., Hausmann G., Basler K. The many faces and functions of beta-catenin. EMBO J. 2012;31:2714–2736. doi: 10.1038/emboj.2012.150. PubMed DOI PMC

Polakis P. Wnt signaling in cancer. Cold Spring Harb. Perspect. Biol. 2012;4:a008052. doi: 10.1101/cshperspect.a008052. PubMed DOI PMC

Krausova M., Korinek V. Signal transduction pathways participating in homeostasis and malignant transformation of the intestinal tissue. Neoplasma. 2012;59:708–718. doi: 10.4149/neo_2012_090. PubMed DOI

Van de Wetering M., Sancho E., Verweij C., de Lau W., Oving I., Hurlstone A., van der Horn K., Batlle E., Coudreuse D., Haramis A.P., et al. The beta-catenin/Tcf-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell. 2002;111:241–250. doi: 10.1016/S0092-8674(02)01014-0. PubMed DOI

Barker N., van Es J.H., Kuipers J., Kujala P., van den Born M., Cozijnsen M., Haegebarth A., Korving J., Begthel H., Peters P.J., et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007;449:1003–1007. doi: 10.1038/nature06196. PubMed DOI

Fafilek B., Krausova M., Vojtechova M., Pospichalova V., Tumova L., Sloncova E., Huranova M., Stancikova J., Hlavata A., Svec J., et al. Troy, a tumor necrosis factor receptor family member, interacts with Lgr5 to inhibit Wnt signaling in intestinal stem cells. Gastroenterology. 2013;144:381–391. doi: 10.1053/j.gastro.2012.10.048. PubMed DOI

Clevers H., Loh K.M., Nusse R. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science. 2014;346:1248012. doi: 10.1126/science.1248012. PubMed DOI

Clevers H.C., Bevins C.L. Paneth cells: Maestros of the small intestinal crypts. Annu. Rev. Physiol. 2013;75:289–311. doi: 10.1146/annurev-physiol-030212-183744. PubMed DOI

Siegel R., DeSantis C., Virgo K., Stein K., Mariotto A., Smith T., Cooper D., Gansler T., Lerro C., Fedewa S., et al. Cancer treatment and survivorship statistics, 2012. CA Cancer J. Clin. 2012;62:220–241. doi: 10.3322/caac.21149. PubMed DOI

Korinek V., Barker N., Morin P.J., van Wichen D., de Weger R., Kinzler K.W., Vogelstein B., Clevers H. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC(-/-) colon carcinoma. Science. 1997;275:1784–1787. doi: 10.1126/science.275.5307.1784. PubMed DOI

Shimizu Y., Ikeda S., Fujimori M., Kodama S., Nakahara M., Okajima M., Asahara T. Frequent alterations in the Wnt signaling pathway in colorectal cancer with microsatellite instability. Genes Chromosom. Cancer. 2002;33:73–81. doi: 10.1002/gcc.1226. PubMed DOI

Liu W.G., Dong X.Y., Mai M., Seelan R.S., Taniguchi K., Krishnadath K.K., Halling K.C., Cunningham J.M., Qian C.P., Christensen E., et al. Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating beta-catenin/tcf signalling. Nat. Genet. 2000;26:146–147. doi: 10.1038/79859. PubMed DOI

Morin P.J., Sparks A.B., Korinek V., Barker N., Clevers H., Vogelstein B., Kinzler K.W. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science. 1997;275:1787–1790. doi: 10.1126/science.275.5307.1787. PubMed DOI

Krausova M., Korinek V. Wnt signaling in adult intestinal stem cells and cancer. Cell. Signal. 2014;26:570–579. doi: 10.1016/j.cellsig.2013.11.032. PubMed DOI

Cancer Genome Atlas Network Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487:330–337. doi: 10.1038/nature11252. PubMed DOI PMC

Tang W., Dodge M., Gundapaneni D., Michnoff C., Roth M., Lum L. A genome-wide RNAi screen for Wnt/beta-catenin pathway components identifies unexpected roles for TCF transcription factors in cancer. Proc. Natl. Acad. Sci. USA. 2008;105:9697–9702. doi: 10.1073/pnas.0804709105. PubMed DOI PMC

Hrckulak D., Kolar M., Strnad H., Korinek V. TCF/LEF transcription factors: An update from the internet resources. Cancers. 2016;8:70. doi: 10.3390/cancers8070070. PubMed DOI PMC

Korinek V., Barker N., Moerer P., van Donselaar E., Huls G., Peters P.J., Clevers H. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nat. Genet. 1998;19:379–383. doi: 10.1038/1270. PubMed DOI

Van Es J.H., Haegebarth A., Kujala P., Itzkovitz S., Koo B.K., Boj S.F., Korving J., van den Born M., van Oudenaarden A., Robine S., et al. A critical role for the Wnt effector Tcf4 in adult intestinal homeostatic self-renewal. Mol. Cell. Biol. 2012;32:1918–1927. doi: 10.1128/MCB.06288-11. PubMed DOI PMC

Moser A.R., Pitot H.C., Dove W.F. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science. 1990;247:322–324. doi: 10.1126/science.2296722. PubMed DOI

Angus-Hill M.L., Elbert K.M., Hidalgo J., Capecchi M.R. T-cell factor 4 functions as a tumor suppressor whose disruption modulates colon cell proliferation and tumorigenesis. Proc. Natl. Acad. Sci. USA. 2011;108:4914–4919. doi: 10.1073/pnas.1102300108. PubMed DOI PMC

Rodriguez C.I., Buchholz F., Galloway J., Sequerra R., Kasper J., Ayala R., Stewart A.F., Dymecki S.M. High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat. Genet. 2000;25:139–140. doi: 10.1038/75973. PubMed DOI

El Marjou F., Janssen K.P., Chang B.H.J., Li M., Hindie V., Chan L., Louvard D., Chambon P., Metzger D., Robine S. Tissue-specific and inducible cre-mediated recombination in the gut epithelium. Genesis. 2004;39:186–193. doi: 10.1002/gene.20042. PubMed DOI

Tumova L., Pombinho A.R., Vojtechova M., Stancikova J., Gradl D., Krausova M., Sloncova E., Horazna M., Kriz V., Machonova O., et al. Monensin inhibits canonical wnt signaling in human colorectal cancer cells and suppresses tumor growth in multiple intestinal neoplasia mice. Mol. Cancer Ther. 2014;13:812–822. doi: 10.1158/1535-7163.MCT-13-0625. PubMed DOI

Xu Q., Wang Y.S., Dabdoub A., Smallwood P.M., Williams J., Woods C., Kelley M.W., Jiang L., Tasman W., Zhang K., et al. Vascular development in the retina and inner ear: Control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair. Cell. 2004;116:883–895. doi: 10.1016/S0092-8674(04)00216-8. PubMed DOI

Sanjana N.E., Shalem O., Zhang F. Improved vectors and genome-wide libraries for crispr screening. Nat. Methods. 2014;11:783–784. doi: 10.1038/nmeth.3047. PubMed DOI PMC

Kasparek P., Krausova M., Haneckova R., Kriz V., Zbodakova O., Korinek V., Sedlacek R. Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases. FEBS Lett. 2014;588:3982–3988. doi: 10.1016/j.febslet.2014.09.014. PubMed DOI

Sato T., Vries R.G., Snippert H.J., van de Wetering M., Barker N., Stange D.E., van Es J.H., Abo A., Kujala P., Peters P.J., et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459:262–265. doi: 10.1038/nature07935. PubMed DOI

Sato T., van Es J.H., Snippert H.J., Stange D.E., Vries R.G., van den Born M., Barker N., Shroyer N.F., van de Wetering M., Clevers H. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature. 2011;469:415–418. doi: 10.1038/nature09637. PubMed DOI PMC

Valenta T., Lukas J., Korinek V. Hmg box transcription factor TCF-4’s interaction with CtBP1 controls the expression of the Wnt target Axin2/Conductin in human embryonic kidney cells. Nucleic Acids Res. 2003;31:2369–2380. doi: 10.1093/nar/gkg346. PubMed DOI PMC

Lukas J., Mazna P., Valenta T., Doubravska L., Pospichalova V., Vojtechova M., Fafilek B., Ivanek R., Plachy J., Novak J., et al. Dazap2 modulates transcription driven by the Wnt effector TCF-4. Nucleic Acids Res. 2009;37:3007–3020. doi: 10.1093/nar/gkp179. PubMed DOI PMC

Veeman M.T., Slusarski D.C., Kaykas A., Louie S.H., Moon R.T. Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr. Biol. 2003;13:680–685. doi: 10.1016/S0960-9822(03)00240-9. PubMed DOI

Janeckova L., Fafilek B., Krausova M., Horazna M., Vojtechova M., Alberich-Jorda M., Sloncova E., Galuskova K., Sedlacek R., Anderova M., et al. Wnt signaling inhibition deprives small intestinal stem cells of clonogenic capacity. Genesis. 2016;54:101–114. doi: 10.1002/dvg.22922. PubMed DOI PMC

Gregorieff A., Pinto D., Begthel H., Destree O., Kielman M., Clevers H. Expression pattern of Wnt signaling components in the adult intestine. Gastroenterology. 2005;129:626–638. doi: 10.1016/j.gastro.2005.06.007. PubMed DOI

Buczacki S.J.A., Zecchini H.I., Nicholson A.M., Russell R., Vermeulen L., Kemp R., Winton D.J. Intestinal label-retaining cells are secretory precursors expressing Lgr5. Nature. 2013;495:65–69. doi: 10.1038/nature11965. PubMed DOI

King J.B., von Furstenberg R.J., Smith B.J., McNaughton K.K., Galanko J.A., Henning S.J. Cd24 can be used to isolate Lgr5(+) putative colonic epithelial stem cells in mice. Am. J. Physiol. Gastrointest. Liver Physiol. 2012;303:G443–G452. doi: 10.1152/ajpgi.00087.2012. PubMed DOI PMC

Yui S., Nakamura T., Sato T., Nemoto Y., Mizutani T., Zheng X., Ichinose S., Nagaishi T., Okamoto R., Tsuchiya K., et al. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5(+) stem cell. Nat. Med. 2012;18:618–623. doi: 10.1038/nm.2695. PubMed DOI

Lallemand Y., Luria V., Haffner-Krausz R., Lonai P. Maternally expressed PGK-Cre transgene as a tool for early and uniform activation of the cre site-specific recombinase. Transgen. Res. 1998;7:105–112. doi: 10.1023/A:1008868325009. PubMed DOI

Kennell J.A., O’Leary E.E., Gummow B.A., Hammer G.D., MacDougald O.A. T-cell factor 4N (TCF-4N), a novel isoform of mouse TCF-4, synergizes with beta-catenin to coactivate C/EBPalpha and steroidogenic factor 1 transcription factors. Mol. Cell. Biol. 2003;23:5366–5375. doi: 10.1128/MCB.23.15.5366-5375.2003. PubMed DOI PMC

Vacik T., Stubbs J.L., Lemke G. A novel mechanism for the transcriptional regulation of Wnt signaling in development. Genes Dev. 2011;25:1783–1795. doi: 10.1101/gad.17227011. PubMed DOI PMC

Hoverter N.P., Ting J.-H., Sundaresh S., Baldi P., Waterman M.L. A WNTt/p21 circuit directed by the C-clamp, a sequence-specific DNA binding domain in TCFs. Mol. Cell. Biol. 2012;32:3648–3662. doi: 10.1128/MCB.06769-11. PubMed DOI PMC

Schuijers J., Mokry M., Hatzis P., Cuppen E., Clevers H. Wnt-induced transcriptional activation is exclusively mediated by TCF/LEF. EMBO J. 2014;33:146–156. doi: 10.1002/embj.201385358. PubMed DOI PMC

Feil R., Wagner J., Metzger D., Chambon P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun. 1997;237:752–757. doi: 10.1006/bbrc.1997.7124. PubMed DOI

Soriano P. Generalized lacZ expression with the ROSA26 cre reporter strain. Nat. Genet. 1999;21:70–71. doi: 10.1038/5007. PubMed DOI

Kuraguchi M., Wang X.-P., Bronson R.T., Rothenberg R., Ohene-Baah N.Y., Lund J.J., Kucherlapati M., Maas R.L., Kucherlapati R. Adenomatous polyposis coli (APC) is required for normal development of skin and thymus. PLoS Genet. 2006;2:e146. doi: 10.1371/journal.pgen.0020146. PubMed DOI PMC

Powell A.E., Wang Y., Li Y.N., Poulin E.J., Means A.L., Washington M.K., Higginbotham J.N., Juchheim A., Prasad N., Levy S.E., et al. The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor. Cell. 2012;149:146–158. doi: 10.1016/j.cell.2012.02.042. PubMed DOI PMC

Lustig B., Jerchow B., Sachs M., Weiler S., Pietsch T., Karsten U., van de Wetering M., Clevers H., Schlag P.M., Birchmeier W., et al. Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors. Mol. Cell. Biol. 2002;22:1184–1193. doi: 10.1128/MCB.22.4.1184-1193.2002. PubMed DOI PMC

Hovanes K., Li T.W.H., Munguia J.E., Truong T., Milovanovic T., Marsh J.L., Holcombe R.F., Waterman M.L. Beta-catenin-sensitive isoforms of lymphoid enhancer factor-1 are selectively expressed in colon cancer. Nat. Genet. 2001;28:53–57. doi: 10.1038/ng0501-53. PubMed DOI

Roose J., Huls G., van Beest M., Moerer P., van der Horn K., Goldschmeding R., Logtenberg T., Clevers H. Synergy between tumor suppressor APC and the beta-catenin-Tcf4 target Tcf1. Science. 1999;285:1923–1926. doi: 10.1126/science.285.5435.1923. PubMed DOI

Yan D., Wiesmann M., Rohan M., Chan V., Jefferson A.B., Guo L.D., Sakamoto D., Caothien R.H., Fuller J.H., Reinhard C., et al. Elevated expression of axin2 and hnkd mRNA provides evidence that Wnt/beta-catenin signaling is activated in human colon tumors. Proc. Natl. Acad. Sci. USA. 2001;98:14973–14978. doi: 10.1073/pnas.261574498. PubMed DOI PMC

Weidinger G., Thorpe C.J., Wuennenberg-Stapleton K., Ngai J., Moon R.T. The Sp1-related transcription factors sp5 and sp5-like act downstream of Wnt/beta-catenin signaling in mesoderm and neuroectoderm patterning. Curr. Biol. 2005;15:489–500. doi: 10.1016/j.cub.2005.01.041. PubMed DOI

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