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Active enhancers strengthen TAD insulation by bRNA mediated CTCF enrichment at the TAD boundaries

By Zubairul Islam, Bharath Saravanan, Kaivalya Walavalkar, Umer Farooq, Anurag Kumar Singh, Jitendra Thakur, Awadhesh Pandit, Radhakrishnan Sabarinathan, Steven Henikoff, Dimple Notani

Posted 14 Jul 2021
bioRxiv DOI: 10.1101/2021.07.13.452118

The genome is partitioned into Topologically Associating 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 clustering 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/stabilise the CTCF even on weaker motifs within these boundaries and supports CTCF binding in clusters, therefore enhancing TAD insulation which favors the intra-TAD enhancer:promoter interactions and robust gene transcription. Functionally, eRNAs within the boundaries are repurposed as more stable bRNAs and their 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. These transcripts trigger CTCF clustering at the boundary resulting in better insulation which favours robust intra-TAD enhancer:promoter interactions to activate the gene transcription.

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