SRA

The human genome is folded into regulatory units termed 'topologically-associated domains' (TADs), within which the majority of gene-enhancer interactions occur. Genome-wide studies support a global role for the insulator protein CTCF in mediating chromosomal looping and the topological constraint of TAD boundaries3,4. However, the impact of individual insulators on enhancer-gene interactions and transcription remains poorly understood. Here, we investigate epigenome editing strategies for perturbing individual CTCF insulators and evaluating consequent effects on genome topology and transcription. We show that fusions of catalytically-inactive Cas9 (dCas9) to transcriptional repressors (dCas9-KRAB) and DNA methyltransferases (dCas9-DNMT3A, dCas9-DNMT3A3L) can selectively displace CTCF from specific insulators, but only when precisely targeted to the cognate motif. We further demonstrate that stable, mitotically-heritable insulator disruption can be achieved through combinatorial hit-and-run epigenome editing with dCas9-KRAB and dCas9-DNMT3A3L. Finally, we apply these strategies to activate PDGFRA expression in glioblastoma stem cells, thus simulating an insulator loss mechanism implicated in brain tumorigenesis. Our study provides strategies for stably modifying genome organization and gene activity without altering the underlying DNA sequence. Overall design: ChIP-seq, BS-seq, and 4C-seq experiments from HEK293 and GSC6 cells following targeted epigenome editing *** The raw sequencing/alignment data for cell line GSC6 [GSM3937048-GSM3937067] are not provided due to patient confidentiality.