A β‐catenin‐TCF‐sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer
Authors: Rappaport Jeffrey A. , Entezari Ariana A. , Snook Adam E. , Waldman Scott A. 
Published: May 2022

Background

Colorectal cancer most commonly arises from mutations in the tumor suppressor APC, or its downstream degradation target β‐catenin, initiating an oncogenic program of β‐catenin/TCF‐dependent nuclear transcription. Mechanisms coupling these mutations to tumorigenesis continue to be refined. The tumor suppressor axis regulated by the intestinal receptor guanylyl cyclase C (GUCY2C) and its paracrine ligands, guanylin and uroguanylin, contributes to intestinal homeostasis by regulating the proliferation, migration, and differentiation programs that maintain intestinal epithelial architecture. Furthermore, GUCY2C agonists oppose transformation in multiple mouse models of intestinal tumorigenesis. This signaling axis is among the earliest pathways silenced in tumorigenesis, reflecting an evolutionarily conserved pattern of GUCY2C retention, but ligand loss, in tumors. These observations suggest a pathophysiological model in which transformation orphans the receptor, silencing its signaling and lifting a block on tumorigenesis. Here, we examined the hypothesis that GUCY2C ligand loss arises from transcriptional silencing by β‐catenin/TCF.

Methods

We mapped the β‐catenin/TCF‐transcriptional program by RNA‐seq in four human colon cancer cell models. We then performed a comparative analysis of orthogonal approaches, including luciferase reporters, ChIP‐seq, CRISPR Cas9 knockout, and CRISPR epigenome editing to identify gene enhancers mediating GUCY2C ligand loss.

Results

RNA‐seq revealed a core β‐catenin/TCF‐transcriptional program of 1,289 genes, of which guanylin and uroguanylin were the seventh and twelfth most sensitive transcripts, reflecting transcriptional silencing. ChIP‐seq analyses of colon tissue revealed an evolutionarily conserved genomic locus encompassing the GUCY2C ligand genes, containing multiple regions of DNase hypersensitivity and H3K27ac enrichment, hallmarks of genetic enhancer loci. These markers were observed in normal colon, but not cancer tissue, consistent with enhancer inactivation and transcriptional silencing during transformation. ChIP‐seq in a colon cancer cell line revealed RNA Polymerase II and H3K27ac recruitment to the regions identified in human tissue, reflecting poised transcriptional machinery. Incorporation of the putative enhancer DNA into luciferase reporter constructs revealed a 2,683 bp locus control region (LCR) responsible for coordinated β‐catenin/TCF‐sensitive control of both GUCY2C ligand promoters. Both CRISPR‐Cas9 deletion of the LCR and epigenetic inactivation with a dCas9.KRAB repressor construct abolished β‐catenin/TCF‐sensitivity of GUCY2C ligand expression. Finally, LCR activation with a dCas9.VP64 transcriptional activator construct reconstituted GUCY2C ligand expression, overcoming gene silencing by β‐catenin/TCF.

Conclusions

This study reveals DNA elements regulating co‐repression of GUCY2C ligand transcription by β‐catenin/TCF, reflecting a novel step in tumorigenesis, and offers unique strategies to re‐establish hormone expression to oppose transformation.