Active Enhancers Strengthen TAD Insulation by bRNA Mediated CTCF Enrichment at the TAD Boundaries

Abstract

The genome is partitioned into Topologically Associated Domains (TADs). About half of the boundaries of these TADs exhibit transcriptional activity and are correlated with better TAD insulation. However, the role of these transcripts per se in TAD insulation, enhancer:promoter interactions and transcription remain unknown. Here we investigate the functional roles of these bRNAs (boundary-RNA) in boundary insulation and consequent effects on enhancer-promoter interactions and TAD transcription genome-wide and on disease relevant INK4a/ARF TAD. Using series of CTCF sites deletion and bRNA knockdown approaches at this TAD boundary, we show a direct association of CTCF with bidirectional bRNAs where the loss of bRNA triggers the concomitant loss of: CTCF at TAD boundary, its insulation, enhancer:promoter interactions and gene transcription within the targeted TAD. In search of what regulates bRNA expression itself, we used another series of enhancer deletions and CRISPRi on promoters within INK4a/ARF TAD and observed that indeed, enhancers interact with boundaries and positively regulate the bRNA transcription at TAD boundaries. In return, the bRNAs recruit/stabilize the CTCF at these boundaries, therefore enhancing TAD insulation which favors the intra-TAD enhancer:promoter interactions and robust gene transcription. In contrast promoter transcription, their active repression or levels of CTCF do not affect the bRNA expression. Functionally, bRNA knockdown exactly mimics the boundary loss. Furthermore, transcribing boundaries exhibit high TAD transcription in TCGA tumor datasets. Together, these results show that active enhancers directly mediate better insulation of TADs by activating the transcription at TAD boundaries which stabilizes CTCF thus favours robust intra-TAD enhancer:promoter interactions to activate the gene transcription.