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. 2021 Aug 6;49(15):8625–8641. doi: 10.1093/nar/gkab657

Figure 6.

Figure 6.

TCF and CDX binding sites and CAG sites are functionally important for CREAX and the c-Myc-335 enhancer. (A) Effect of mutating the 4 TCF sites, 2 CDX sites, or the 5 CAG sites on CREAX-luciferase reporter activity in HEK293T cells. Cells were transfected with the indicated reporters and an empty vector or plasmid expressing β-catenin* and the fold activation by β-catenin* was compared. (B) CDX1 can bind to the CDX sites in CREAX in vitro. A biotin-labelled probe containing CDX sites from CREAX binds to GST-tagged recombinant CDX1 in an EMSA. An unlabelled probe consisting of these CDX sites competes with the biotinylated probe for binding GST-CDX1. (C) Cartoon of the c-Myc-335 enhancer showing TCF binding sites (red), CDX binding sites (green), and CAG sites (teal). (D) Luciferase assay showing that the mutation of TCF, CDX, or CAG sites in the c-Myc-335 enhancer decreases its activity in LS174T cells. Cells were transfected with the indicated reporters and their relative activity was analysed by a luciferase assay. (E) Luciferase assay showing that the mutation of TCF, CDX, or CAG sites in the c-Myc-335 enhancer decreases its β-catenin*-driven activity in HEK293T cells. Data in (A, D, E) are shown as mean ± SD from three replicates (N = 3). P< 0.05; **P< 0.005; ***P< 0.001; ****P < 0.0001; n.s. P > 0.05.