GSE171910
A b-catenin-TCF-sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer
Background & Aims. Sporadic colorectal cancers arise from mutations in APC, producing oncogenic ß-catenin/TCF-dependent transcriptional reprogramming. The tumor suppressor axis regulated by the intestinal epithelial receptor, GUCY2C, is among the earliest pathways silenced in tumorigenesis. Retention of the receptor, but loss of its paracrine ligands, guanylin and uroguanylin, is an evolutionarily conserved feature of colorectal tumors, arising in the earliest dysplastic lesions. Here, we examined a mechanism of GUCY2C ligand transcriptional silencing by ß-catenin/TCF signaling. Methods. We performed RNA-seq analysis of four unique conditional human colon cancer cell models of ß-catenin/TCF signaling to map the core Wnt-transcriptional program. We then performed a comparative analysis of orthogonal approaches, including luciferase reporters, ChIP-seq, CRISPR Cas9 knockout, and CRISPR epigenome editing, which were cross-validated with human tissue ChIP-seq datasets, to identify functional gene enhancers mediating GUCY2C ligand loss. Results. RNA-seq analyses reveal the GUCY2C hormones as two of the most sensitive targets of ß-catenin/TCF signaling, reflecting transcriptional repression. The GUCY2C hormones share an insulated genomic locus containing a novel locus control region upstream of the guanylin promoter that mediates the coordinated silencing of both genes. Targeting this region with CRISPR epigenome editing reconstituted GUCY2C ligand expression, overcoming gene inactivation by mutant ß-catenin/TCF signaling. Conclusions. These studies reveal novel DNA elements regulating co-repression of GUCY2C ligand transcription by ß-catenin/TCF signaling, reflecting a novel pathophysiological step in tumorigenesis. They offer unique genomic strategies that could re-establish hormone expression in the context of canonical oncogenic mutations to reconstitute the GUYC2C axis and oppose transformation. Overall design: Overall Design: Samples are from four human colorectal cancer cell lines expressing stable transgenes enabling inducible silencing of Wnt signaling. RNAseq was performed on all cell lines, with or without the inducing agent, in triplicate (24 samples). RNA Polymerase II- and H3K27ac-ChIPseq was performed on one cell line, with or without the inducing agent, in duplicate (8 experimental samples and 2 input samples).