4C-seq to show the effects of insertion of STITCH into MYC+30kb and NEUROG2-65kb positions on the chromatin conformation in human iPSCs and differentiated neural progenitor cells

We reconstituted arrays of CTCF binding sites (L1, L2, L3, L4, R1, R2, and R3) 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 first inserted STITCH into the 30-kb downstream from MYC (STITCH+30kb) in human iPSCs, after one of the two alleles are deleted. We made deletion of each CTCF array, L (delL) and R (delR) in STITCH+30kb. We also inserted STITCH into the 65-kb downstream of NEUROG2 in human iPSCs and integrated a transgene consisting of tetR-KRAB followed by DNA encoding the 2A peptide and the puromycin resistant gene with piggyBac transposition into the genome in the STITCH/NEUROG2 clone (NEUROG2/KRAB). We performed 4C-seq (Circular chromatin conformation capture assay followed by deep-sequencing) from the flanking regions of the STITCH+30kb insertion site as viewpoints to see how STITCH impacts on the chromatin conformation in STITCH+30kb, delL, delR, and the intact clone (Hap). We also performed 4C-seq from the NEUROG2 promoter region in the NEUROG2/KRAB iPSCs and the differentiated neural progenitor cells with and without DOX.