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

Abstract

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-relevantINK4a/ARFandMYCTAD. 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.

Single-molecule imaging reveals a direct role of CTCF’s zinc fingers in SA interaction and cluster-dependent RNA recruitment

“…Permanently paused states could also be caused by polymerase unbinding and CTCF capturing the RNA chain. Transcription could therefore enable CTCF to recruit RNA to CBSs and lead to an increased insulation at domain boundaries 65 . Impaired transcription by CTCF could also regulate Pol II pausing 47 , which has been linked to alternative splicing 35 .…”

Interplay between cohesin kinetics and polymer relaxation modulates chromatin-domain structure and dynamics

“…Theoretical studies have argued that the experimentally observed large chromatin loops and non-random organization are not probable by a purely thermal process and predicted an active loop extrusion process as the potential mechanism [33,[45][46][47][48]. Several studies found supporting evidence for this idea and discovered that dynamic loop formation by cohesins is crucial for chromatin organization in the interphase and CCCTC-binding factor (CTCF) plays an essential role in maintaining TAD boundaries [27,[49][50][51][52][53][54][55][56][57]. These findings led to the emergence of a dynamic picture where, in the interphase, structural maintenance of chromosomes (SMC) complexes like cohesins (and associated proteins) extrude a loop until they encounter a CTCF barrier.…”

Noncoding function of super enhancer derived mRNA in modulating neighboring gene expression and TAD interaction

“…The genome in the nucleus is organized into different layers of higher architecture, ranging from nucleosome to chromatin looping, to topologically associated domains (TADs), and A/B compartments (7,8). Some TAD boundaries overlap with protein-coding genes or non-coding genes (9)(10)(11)(12). The non-coding RNA transcribed from a TAD boundary (bRNA) has been reported to strengthen TAD insulation by recruiting CTCF to the TAD boundary (9).…”

“…Some TAD boundaries overlap with protein-coding genes or non-coding genes (9)(10)(11)(12). The non-coding RNA transcribed from a TAD boundary (bRNA) has been reported to strengthen TAD insulation by recruiting CTCF to the TAD boundary (9). R loops, which are DNA:RNA hybrids, form as natural byproducts of transcription and accumulate around transcription start and termination sites (13).…”