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A b-catenin-TCF-sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer

ABSTRACT: Background & Aims. Sporadic colorectal cancers arise from mutations in APC, producing oncogenic β-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 β-catenin/TCF signaling. Methods. We performed RNA-seq analysis of four unique conditional human colon cancer cell models of β-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 β-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 β-catenin/TCF signaling. Conclusions. These studies reveal novel DNA elements regulating co-repression of GUCY2C ligand transcription by β-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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE171910 | GEO | 2022/01/03

REPOSITORIES: GEO

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Project description:Wnt/β-catenin signaling is a highly conserved molecular pathway that plays a crucial role in stem/progenitor systems and cancer. β-catenin, the main Wnt pathway effector, has two pools within a cell: one for cell-cell adhesion at the membrane and the other for transcriptional functions in the nucleus. However, the mechanism by which β-catenin mediates both roles remain unclear. The tightly controlled, well characterized system of nephrogenesis is an ideal model to decouple the roles of β-catenin at the membrane and in the nucleus. In kidney development, a delicate balance of nephron progenitor cell self-renewal and differentiation is required for the mesenchymal to epithelial transition (MET) in nephrogenesis and is driven by Wnt/β-catenin signaling. Given an ability to isolate and manipulate large numbers of NPCs in tissue culture, we can dissect the dual nature of β-catenin as a transcriptional activator and component of a cell membrane complex in adhesion. We pioneered a method using CRISPR/Cas9 gene editing to rapidly remove β-catenin, Tcf/Lef factors and simultaneous cadherin genes in primary NPCs. We have characterized the effects of modulating β-catenin and integrated RNA-seq results from β-catenin’s removal with mouse ChIP-seq and mouse single cell RNA -seq data. Functional analysis of β-catenin removal provides strong evidence for β-catenin regulation of NPC proliferation, independent of a direct Lef/Tcf associated transcriptional program. Together these data suggest β-catenin mediates aggregation, the first step in MET, through β-catenin mediated cell adhesion complexes while simultaneous transcriptional activation within these structures initiates the nephrogenic program. The studies provide new insight into the direct transcriptional role of Lef/Tcf/β-catenin complexes associated with the initiation of a nephron forming program. Overall, this study enhances an understanding of the molecular mechanisms underlying kidney development and the dual nature of β-catenin stem/progenitor systems at large.

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A b-catenin-TCF-sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer

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