Transcriptional activity of the VTI1A and TCF7L2 promoters.

<p><b> a</b> The pGL4.10-VTI1A-735 and pGL4.10-TCF7L2-1405 plasmids were created by inserting the promoter regions of VTI1A and TCF7L2, respectively, upstream the luciferase gene in the pGL4.10 plasmid. <b>b</b> LS174T cells were transfected with either the pGL4.10-VTI1A-375 or the pGL4.10-TCF7L2-1405 reporter plasmids. Activity is stated in mean values relative to pGL4.10 activity and corrected with β-galactosidase activity. Error bars indicate SD, **** indicates p<0.0001, n = 8.</p

Wnt signaling in colon cancer cell lines.

<p><b> a</b> Wildtype TCF4 contains a β-catenin binding domain, a transcription repression domain, a DNA binding domain, and a nuclear localization signal. With the fusion with VTI1A the resulting fusion protein, VTI1A-TCF4, contains the first three exons of VTI1A and lacks the β-catenin binding domain of TCF4. The rest of the TCF4 domains are still present. <b>b</b> The cells lines LS174T, Caco-2 and NCI-H508 were transfected with the TOPFlash and FOPFlash reporter plasmids, and luciferase activity was measured. Activity is stated in mean values relative to FOPFlash activity for each cell line and corrected with β-galactosidase activity. Error bars indicate SD, **** indicates p<0.0001, n = 4 <b>c</b> The LS174T cell line, showing high Wnt activity, was transfected with the TOPFlash and co-transfected with a plasmid expressing the VTI1A-TCF4 fusion protein. Activity is stated in mean values relative to TOPFlash activity and corrected with β-galactosidase activity. Error bars indicate SD, **** indicates p<0.0001, n = 8.</p

Expression in the colonic crypts.

<p>In normal crypts TCF4 is expressed at the base of the crypts [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200215#pone.0200215.ref008" target="_blank">8</a>] and as cells migrate towards the top of the crypts the expression of TCF4 decreases while expression of CDX2 increases [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200215#pone.0200215.ref017" target="_blank">17</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200215#pone.0200215.ref033" target="_blank">33</a>]. In a proposed model for a crypt with cells carrying the <i>VTI1A</i>-<i>TCF7L2</i> fusion, the VTI1A-TCF4 fusion protein will be expressed throughout the crypt. As β-catenin is not able to bind to VTI1A-TCF4 it may instead bind and activate other members of the TCF/LEF family of transcription factors, e.g. TCF1 and LEF1.</p

Transcriptional activity of CDX2.

<p><b> a</b> LS174T cells were transfected with the pGL4.10-VTI1A plasmid-735 and co-transfected with a CDX2 expression plasmid. Activity is stated in mean values relative to pGL4.10 activity and corrected with β-galactosidase activity. Error bars indicate SD, **** indicates p<0.0001, n = 4. <b>b</b> The LS174T wild type cell line and a LS174T CDX2 knockout cell line was transfected with the pGL4.10-VTI1A plasmid. Activity is stated in mean values relative to pGL4.10 activity and corrected with β-galactosidase activity. Error bars indicate SD, **** indicates p<0.0001, n = 4.</p

CDX2 regulation of <i>VTI1A</i> transcriptional activity.

<p><b>a</b> LS174T CDX2 ChIP-seq data was used to identify a CDX2 peak in the VTI1A promoter region [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200215#pone.0200215.ref030" target="_blank">30</a>], and the JASPAR CORE database identifies two potential binding sites within this region [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200215#pone.0200215.ref031" target="_blank">31</a>]. <b>b</b> qPCR was performed on CDX2 immunoprecipitated DNA from LS174T cells with primers amplifying the 100 bp region seen in fig 4A. Error bars indicates SD, ** indicates p<0.01, n = 4 <b>c</b> Plasmids for deletion analysis with different lengths of the VTI1A promoter inserted in the pGl4.10 plasmid. The two possible CDX2 binding sites are marked. The deletion analysis was carried out in LS174T cells and the luciferase activity is stated in mean values relative to the pGL4.10-VTI1A-735 plasmid and is corrected with β-galactosidase activity. Error bars indicate SD, * indicates p<0.05, *** indicates p<0.001, **** indicates p<0.0001, n = 4.</p