Controlling gene activation by enhancers through a drug-inducible topological insulator

While regulation of gene-enhancer interaction is better understood, its application remains limited. Here, we reconstituted arrays of CTCF binding sites and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. We applied this to dissect MYC regulation in human pluripotent stem cells. Insertion of STITCH between MYC and the enhancer down-regulated MYC and affected its target transcriptome. Progressive mutagenesis of STITCH led to preferential escalation of the gene-enhancer interaction, corroborating the strong insulation ability of STITCH. The STITCH insertion altered epigenetic states around MYC. Time-course analysis by drug induction uncovered deposition and removal of H3K27me3 repressive marks follows and reflects, but does not precede and determine, the expression change. Thus the tool provided important insights in gene regulation, demonstrating its potency.