Fig. 5
ZFP36L2 expression is predominantly driven by the e5 and e11 constituent enhancers. a H3K27ac and DNase-hypersensitivity profiles in E094 cells to identify constituent enhancers e1 through e11 within the super-enhancer region. b Capture-4C experiment assays showed that the super-enhancer region (chr2:43036808-43422165, from e1 to e11) physically interacts with the ZFP36L2 promoter. All of the Capture-C experiments for each cell line were performed with two biological replicates. c Luciferase-reporter assays (n = 3) measuring the activity of e1 through e11 in (upper) SNU-719 cells and (lower) NCI-N87 cells. The pGL3 plasmid without the enhancer region (empty) was used as a negative control. Along the Y-axis, the luciferase signal was first normalized to the Renilla luciferase signal and then normalized to the signal from the empty pGL3 plasmid. P values were derived from t tests. *P ≤ 0.05; ***P ≤ 0.001. d Enhancer activity of duplicated e5 (2 × e5) and e11 (2 × e11) enhancers measured by luciferase-reporter assays (n = 3) in SNU-719 cells and NCI-N87 cells. P values were derived from t tests. *P ≤ 0.05. e Gel pictures of PCR amplification of genomic DNA using primers outside the e5 and e11 enhancer region in (left) SNU-719 and (right) NCI-N87 cells with CRISPR/Cas9-mediated deletion of the e5 and e11 enhancer. sg-Control: empty plasmid; sg-e5del and sg-e11del: pairs of sgRNAs recognizing boundaries of the e5 and e11 enhancer region. f The expression level of ZFP36L2 as measured by quantitative PCR in (left) SNU-719 and (right) NCI-N87 cells with CRISPR/Cas9-mediated deletion of the e5 and e11 enhancer. P values were derived from t tests. **P ≤ 0.01; ***P ≤ 0.001. Error bars represent ±s.d. of three experiments
