Unknown,Transcriptomics,Genomics,Proteomics

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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

ABSTRACT: 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.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Taro Tsujimura

PROVIDER: E-MTAB-8492 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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

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