Fig. 2

Identification of CYR61 enhancers. a From top to bottom: UCSC gene annotation (GRCh38/ hg38) of CYR61; enrichment of H3K27ac in colon adenocarcinoma and normal colon mucosa; enrichment of H3K4me1 in colon adenocarcinoma and normal colon mucosa; enrichment of H3K4me3 in colon adenocarcinoma and normal colon mucosa; GRO-seq of eRNA expression in HCT116 cells; locations of fragments inserted in the pGL3-basic plasmid; positions of primers used in ChIP-qPCR; HindIII digestion sites, and positions of primers used in the 3C assay; DNase I hypersensitivity signals. NC: negative control sequence. b Expression levels of eRNA in different colon cell lines. c RNA polymerase II, H3K27ac, H3K4me1 and H3K4me3 enrichment in NCM460, HCT116 and RKO cells, as assessed by ChIP-qPCR and expressed as fold change over input normalized to the NC. d Relative enhancer luciferase activities, normalized to expression of Renilla luciferase from a co-transfected pRL-SV40 plasmid. e Relative cross-linking frequencies between the constant region (CYR61 promoter) and distal fragments (F1~F11) in the three cell lines, measured by qPCR, normalized to ERCC3 and compared to the control region (fragment F1) to calculate the relative fold change. Significance for all data except a determined by the independent sample t-test, and data are shown as mean ± S.D., n = 3. *P < 0.05, **P < 0.01, ***P < 0.001