VDR/RXR and TCF4/beta-Catenin Cistromes in Colonic Cells of Colorectal Tumor Origin: Impact on c-FOS and c-MYC Gene Expression

Many of the transcriptional and growth regulating activities of 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the intestine and colon are recapitulated in the human colorectal cancer cell LS180. We therefore used this line together with ChIP-seq and gene expression analyses to identify the vitamin D receptor (VDR)/retinoid x receptor (RXR) and TCF7L2(TCF4)/ß-catenin cistromes and the genes that they regulate. VDR and RXR co-localized to predominantly promoter distal, VDRE-containing sites in a largely ligand-dependent manner. These regulatory sites control the expression of both known as well as novel 1,25(OH)2D3 target genes. TCF4 and ß-catenin cistromes partially overlapped, contained TCF/LEF consensus elements, and were only modestly influenced by 1,25(OH)2D3. However, the two heterodimer complexes co-localized at sites near a limited set of genes that included c-FOS and c-MYC; the expression of both genes was modulated by 1,25(OH)2D3. At the c-FOS gene, both VDR/RXR and TCF4/ß-catenin bound to a single distal enhancer located 24 kb upstream of the transcriptional start site. At the c-MYC locus, however, binding was noted at a cluster of sites between -139 and -165 kb and at a site located -335 kb upstream. Examined as isolated enhancer fragments, these regions exhibited basal and 1,25(OH)2D3-inducible activities that were interlinked to both VDR and ß-catenin activation. These data reveal additional complexity in the regulation of target genes by 1,25(OH)2D3 and support a direct action of both VDR and the TCF4/ß-catenin regulatory complex at c-FOS and c-MYC. Overall design: 13 samples are analyzed by ChIP-seq. The Input was used as the normalizing factor for all transcription factor IPs. A minimum of 2 replicates were run per antibody and condition. After analysis, these lanes were combined for greater read depth. RNA was isolated from biological replicate experiments for a total of 6 samples in this study (3x 0hr, 3x 24hr), reverse transcribed and labeled according to Nimblegen Systems, Inc. (Madison, WI) protocols.