Enhancer activity of distal TFBSs lacking 5hmC in mESCs. (A) Schematic diagram of the experimental setup. (B) Luciferase reporter assay for 5hmC-enriched putative enhancer regions (about 600 bp) including distal TFBSs in mESCs. Control: empty vector, TFBS1: 5hmC-enriched Klf4/Esrrb binding site (Figure 3), TFBS2: 5hmC-enriched Esrrb/Tcfcp2I1 binding site, TFBS3: 5hmC-enriched Tcfcp2I1 binding site, TFBS4: 5hmC-enriched E2f1 binding site, TFBS5: 5hmC-enriched Nanog/Sox2. The normalized luciferase activity of control is set as 1. *p-value < 0.05.