Targeted intragenic demethylation initiates chromatin rewiring for gene activation

Aberrant Transcription Start Site (TSS) surrounding region DNA methylation is a hallmark of gene silencing in cancer. Currently approved demethylating agents lack specificity and exhibit high toxicity. Herein we show, using the p16 gene locus as an example, that targeted demethylation of the exon 1-intron 1 region initiates an epigenetic wave of local chromatin remodeling and distal long-range interactions, culminating in gene-locus specific activation. Through development of CRISPR-DiR (DNMT1-interacting RNA), an innovative technology wherein ad-hoc edited guides block methyltransferase activity in a locus-specific fashion, we demonstrate that demethylation is coupled to epigenetic and topological changes. These results suggest the existence of a specialized “demethylation firing center (DFC)” which can be switched on by an adaptable and selective RNA-mediated approach for locus-specific transcriptional activation. Overall design: Wild type SNU-398 and Decitabine (DAC 2.5uM) treated 3 days SNU-398 cells DNA methylation were profiled through WGBS. The long range chromatine interactions in SNU-398 CRISPR-DiR Day 13 Control (GN2) sample and D1+D3 targeted (G19, G36, G110, G111) sample were profiled by 4C-seq.