Active enhancers strengthen insulation by RNA-mediated CTCF binding at chromatin domain boundaries

Vertebrate genomes are partitioned into chromatin domains or topologically associating domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at domain boundaries correlates with better insulation; however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF and MYC TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancerpromoter interactions, and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters, we show that active TAD enhancers, but not promoters, induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs.

Automated summary

Vertebrate genomes are divided into chromatin domains called topologically associating domains (TADs), which are typically bound by pairs of CTCF binding sites. Transcription at domain boundaries is correlated with better insulation, and this study shows that boundary-associated RNAs aid in the recruitment and clustering of CTCF at TAD borders. These boundary-associated RNAs enhance TAD insulation, enhancer-promoter interactions, and gene expression within the TAD.