Extended Data Fig. 5: The functional CRE variant rs10871066 regulates PIBF1 and KLF5 expression by interacting with FOXP1 and TCF7L2.
a. The target genes of CRE1 predicted by ABC model among three stages. b. Normalized Hi-C interaction frequencies of small bowel shown as a two-dimensional heatmap denote the potential interaction between rs10871066 and target gene (PIBF1, KLF5) in the region of CRE1 from 3D Genome browser (GRCh37/hg19) from website (http://kobic.kr/3divv1/intro). Genomic tracks represent significant loops. c. The box plots show the expression level (left: PIBF1, middle: KLF5,) and their corresponding chromatin activity, the two-sided P values were calculated by one-way ANOVA (top). The scatter plots represent the correlation between the chromatin activity and gene expression of KLF5 and PIBF1 among colorectal normal (n = 110, left), advanced adenoma (n = 30, middle) and cancer tissues (n = 110, right), The correlation association was calculated by two-sided Pearson correlation. d. eQTL analyses for the association between rs10871066 genotypes and the expression level of PIBF1, KLF5 in CRC samples from TCGA (nAA = 55, nAG = 111, nGG = 159), the two-sided P values were calculated by linear regression analysis. e. The relative luciferase activity of pGL3-promoter vectors containing rs10871066[A] or rs10871066[G] allele in HCT116 cells. n = 9 biologically independent samples. The P values were calculated by unpaired two-sided Student’s t-test. f-g. Footprint profiles of FOXP1 (f) and TCF7L2 (g) at ATAC-seq peaks across all three stages of CRC. h. EMSAs with biotin-labeled probes containing rs10871066 in HCT116 cells. Arrows indicated allele-specific binds that interacted with nuclear protein in the cells. i. Super-shift EMSA using FOXP1 or TCF7L2 antibody in HCT116 cells. ‘I’ represents the allele-specific binding band. ‘II’ represents the super-shifted band. j-k. DNase I footprinting assay. The DNA fragments containing either rs10871066[A] (j) or rs10871066[G] (k) are labeled with 5’-FAM and incubated with different FOXP1 or TCF7L2 concentrations. A black dotted box indicates the region that protein protects from DNase I cleavage. DNA sequencing is shown at the bottom, and the sequence marked in blue represents the protein binding motif. Data were shown as the median, box limits indicate upper and lower quartiles and whiskers indicate the maximum and minimum (c-e).
