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. 2014 Nov 20;42(22):13615–13632. doi: 10.1093/nar/gku1186

Figure 6.

Figure 6.

Identification of new Wnt target genes. (A) Downregulated genes with an annotated ChIP-seq peak. Note that 733 genes that had an expression change after 2 h of dnTCF1EWT induction (P < 0.02) were assessed for the presence of an annotated peak from the pooled analysis (peak score >67). A significant number of genes contain at least one annotated peak from the 2 h ChIP-seq time point, but not the 0 h (untreated) ChIP-seq sample. There are also many more genes that have a peak associated with the 9 h ChIP-seq than the 0 h ChIP-seq. (B) GSEA was performed on the 42 genes with at least one peak within 30 kb of the TSS as compared to the set of all genes with at least one peak within 30 kb of the TSS at 2 h post-induction and a significant enrichment (P < 0.05) of high scoring peaks was found. (C) Wild-type bound regions from part (A) were cloned into the Thymidine Kinase 100 (TK100) luciferase reporter. Luciferase assays were carried out in COS-1 cells with transient transfection of expression plasmids for β-catenin and full-length TCF1EWT, or TCF1Emut. Mock refers to co-transfection of an empty expression plasmid to control for β-catenin expression. Three biological replicates were included for each luciferase construct tested and luciferase/light unit values were normalized to β-galactosidase levels before comparing to normalized Mock values as described in (42). The TopTK reporter, which contains three multimerized WREs without Helper sites, was activated equally by TCF1EWT/β-catenin and TCF1Emut/β-catenin. The TK100 backbone was not activated by TCF1EWT/β-catenin or TCF1Emut/β-catenin. Helper site-containing ChIP-seq regions with one WRE (GADD45B, AXIN2) conferred activation of the reporter by TCF1EWT but not TCF1Emut. Helper site-containing regions that do not have an identifiable match to a degenerate, shorter WRE (5′-CTTTGW-3′; TGIF, HIST2H4B) also enabled activation by TCF1EWT but not TCF1Emut. Mutation of the three Helper site sequences in the TGIF region (TGIF1mut; TGIF3mut) eliminated regulation (sequences for TOPtk, GADD45B, AXIN2, HIST2H4B and TGIF and its corresponding Helper site mutations are presented in Supplementary Figure S10).