Fig 2: (See previous page). A Pol II-rich super-enhancer upstream of GUCA2A confers Wnt sensitivity. (A) Nine regions of DNase hypersensitivity were identified from sequencing tracks in Figure 4: CTCF ChIP-seq from human HCT116 colon cancer cells (GSE92879) and DNase-seq from transverse colon (GSE90398; hg19, chr1:42,607,745–42,644,356). ChIP-seq of (B) Pol II and (C) H3K27ac in DLD1(DNTCF) cells with (+) or without (–) 1 µg/mL DOX for 24 hours. (D) Corresponding RNA-seq FPKM quantification illustrating greater transcript expression of GUCA2A than GUCA2B in DLD1(DNTCF) cells. (E, F) Nine regions of DNase hypersensitivity were selected and cloned into luciferase reporters. (E) Diagram of enhancer-luciferase reporters driven by a constitutive (SV40) promoter with no upstream enhancer, upstream TCF sites (TOP), upstream mutant TCF sites (FOP), or upstream GUCA2A DNase-sensitive sites in forward (5'-3') or reverse (3'-5') orientation. (F) Luciferase constructs were expressed in DLD1(DNTCF) cells, and luciferase was quantified with (+) or without (–) 1 µg/mL DOX for 24 hours. (G, H) A luciferase construct driven by the region from +15 to –10,000 relative to the GUCA2A transcription start site was expressed in (G) DLD1(DNTCF) and (H) LS174T (DNTCF) cells. GUCA2A mRNA, GUCA2B mRNA, and luciferase activity were quantified following 1 µg/mL DOX for 0–48 hours. (I) Luciferase constructs containing 5' truncations of the region from +15 to –10,000 relative to the GUCA2A transcription start site were expressed in DLD1(DNTCF) and LS174T (DNTCF) cells and luciferase activity was quantified following 1 µg/mL DOX for 24 hours (DLD1) or 48 hours (LS174T). (B, C) ChIP-seq tracks represent the average of 2 replicate immunoprecipitations. (D) RNA-seq results represent the average of 3 replicates. (G, H) Data points represent the average of 3 wells of cells from a single experiment, with the mean of 4 independent experiments indicated. (F, I) Bars represent the average of 4 independent experiments ± SEM. Significance was determined by (G, H) 1-way, or (F, I) 2-way analysis of variance with matched analysis for independent experiments on log2-transformed results. Data are presented relative to noninduced cells. **P < .01; ***P < .001; ****P < .0001.

Fig 3: Identification of a 2683-bp ß-catenin/TCF-sensitive super-enhancer. (A–E) Luciferase constructs driven by the indicated GUCA2A DNA regions were expressed in DLD1(DNTCF) cells. Luciferase activity was quantified with (+) or without (–) 1 µg/mL DOX for 24 hours, and data are presented relative to noninduced cells. (A-B) Luciferase constructs were driven by the region from +15 to –6000 relative to the GUCA2A transcription start site, and harbored deletions of the indicated DNA positions, revealing a 2683-bp region responsible for ß-catenin-TCF sensitivity. (C) Luciferase constructs were driven by the GUCA2A promoter (+15 to –133) and core enhancer (–1000 to –3683). (D) Luciferase constructs were driven by a constitutive (SV40) promoter and truncations of the GUCA2A upstream region from +15 to –6000. (E) Luciferase constructs were driven by the GUCA2B promoter (+33 to –100) and GUCA2A core enhancer (–1000 to –3683). Bars represent the average of 4 independent experiments ± SEM. Significance was determined by 2-way analysis of variance with matched analysis for independent experiments on log2-transformed results. *P < .05; **P < .01; ***P < .001; ****P < .0001.

Fig 4: Transcriptional coregulation of GUCA2A and GUCA2B within an insulated domain sensitive to Wnt signaling. (A) RNA-seq volcano plots illustrate the fold change of 17,229 genes upon silencing Wnt signaling in the 4 inducible cell lines, with GUCA2A, GUCA2B, HIVEP3, and FOXJ3 indicated. Each gene is represented as a data point, dotted lines denote 2-fold change, and significance is indicated in red (P > .01) or blue (P = .01). (B-C) qRTPCR of (B) HIVEP3 and (C) FOXJ3 in DLD1(DNTCF) cells treated with (+) or without (–) 1 µg/mL DOX for 24 hours. (D–F) GUCA2A mRNA compared with that of (D) HIVEP3, (E) FOXJ3, or (F) GUCA2B, quantified by RNA-seq from human colorectal tumor (n = 380; black) and normal (n = 51; blue) tissue from TCGA (COAD/READ datasets). Significance was determined by 2-tailed Spearman rank correlation (rs), and linear regression (R2) is indicated by the red line. (G, H) Single-cell gene expression data retrieved from The Human Protein Atlas reveals coexpression of (G) GUCA2A and (H) GUCA2B in normal colon epithelial cell clusters (insets show corresponding colon UMAP plots of cell clusters). (B, C) Data points represent the average of 3 wells of cells from a single experiment, with the mean of 3 independent experiments indicated. Significance was determined by Student’s t test with matched analysis for independent experiments on log2-transformed results. Data are presented relative to noninduced cells. *P < .05; ****P < .0001.

Fig 5: Regulatory elements in the GUCA locus are silenced in colorectal cancer. Public datasets reveal regulatory features in the GUCA2A locus. (A) CTCF ChIP-seq from mouse intestinal epithelial cells (GSE98724; mm9, chr4:119,296,885–119,349,526). (B) CTCF ChIP-seq from human HCT116 colon cancer cells (GSE92879; hg19, chr1:42,607,745–42,644,356). (C) DNase-seq from sigmoid (top; GSE90366) and transverse colon (bottom; GSE90398). (D) DNase-seq from normal colon crypts (GSE77737). (E) Formaldehyde-assisted identification of regulatory elements (FAIRE) sequencing from normal colon (GSE94935). (F) Clusters of transcription factor density, representing ChIP-seq of 338 factors in 130 cell types (UCSC Genome Browser, ENCODE Transcription Factor Binding track). (G) H3K27ac ChIP-seq and (H) H3K4me1 ChIP-seq identified poised enhancers in normal colon (top) but not in colon cancer (bottom) (GSE77737). Red lines denote CTCF binding sites. Blue boxes denote gene bodies. Sequencing datasets were obtained from NCBI Gene Expression Omnibus and visualized in the UCSC genome browser.