biostudies-literatureIslam ZWellcome-IACSIR-SPMDepartment of Science and TechnologyDepartment of Atomic EnergyCouncil of Scientific and Industrial ResearchTIFR-NCBSNational Center for Biological Sciences–Tata Institute of Fundamental ResearchWellcome TrustNCBS-TIFR1-17https://www.ebi.ac.uk/biostudies/studies/S-EPMC9977152biostudies-literatureUnknown33(1)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 <i>INK4a/ARF</i> and <i>MYC</i> 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, enhancer-promoter 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.Genome researchActive enhancers strengthen insulation by RNA-mediated CTCF binding at chromatin domain boundaries.PMC9977152CRG/2019/005714CSIR12-R&amp;D-TFR-5.04-0800IA/1/14/2/501539Walavalkar KSingh AKPandit ASaravanan BHenikoff SNotani DFarooq UIslam ZThakur JRadhakrishnan SfalseActive 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 <i>INK4a/ARF</i> and <i>MYC</i> 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, enhancer-promoter 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.2023-01-01T00:00:00Z2023 Jan2026-05-28T08:08:53.715Z2025-04-04T08:35:44.522ZS-EPMC99771523665005210.1101/gr.276643.122