FIG. 1.

β-Catenin and TCF4 bind a region downstream from the c-Myc transcription stop site in HCT116 cells. (A) Diagram of the human c-Myc gene locus. The c-Myc gene is depicted in blue with exons as rectangles, introns as horizontal lines, and the 5′ and 3′ untranslated regions as thin rectangles. An arrow marks the transcription start site. Coordinates of the region of chromosome 8 (chr 8) that contains c-Myc are shown at the top. Below, a red rectangle identifies the characterized 5′ β-catenin/TCF enhancer (21), and the green vertical lines indicate the positions of the β-catenin GSTs identified in the SACO screen (54). Clustered vertical lines below indicate the degree of conservation across mammalian species. This representation was downloaded from the UCSC Genome Browser (http://genome.ucsc.edu/). (B) Real-time PCR analysis of DNA fragments precipitated in a ChIP assay by using an anti-β-catenin antibody (blue bars) or an anti-TCF4 antibody (red bars). Primers were designed to the 5′ promoters of cycD1 and c-Myc to detect specific β-catenin and TCF4 binding and to an internal region of tubulin, an internal region of β-actin, and a region downstream of cycD1 to monitor nonspecific interactions. The primers used to detect binding to the region downstream of c-Myc were designed adjacent to the β-catenin SACO GSTs. As a control, ChIP assays were conducted in TIG fibroblasts which lack appreciable levels of nuclear β-catenin. Data are presented as percent input signal, and error bars indicate standard errors of the means.