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In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation [ChIPmentation]

ABSTRACT: In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation [ChIPmentation]

PROVIDER: PRJNA811239 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18174729.fastq.gz	Fastqsanger.gz
	SRR18174730.fastq.gz	Fastqsanger.gz
	SRR18174731.fastq.gz	Fastqsanger.gz
	SRR18174732.fastq.gz	Fastqsanger.gz
	SRR18174733.fastq.gz	Fastqsanger.gz

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Project description: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 association between these transcripts and TAD insulation, enhancer:promoter interactions and transcription of genes remains 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 more specifically on the disease relevant INK4a/ARF TAD. Using a series of CTCF site deletions and bRNA knockdown experiments at this TAD boundary, we show a direct association of CTCF with the bidirectional bRNAs where the loss of bRNA triggers the concomitant loss of: CTCF at the TAD boundary, its insulation, enhancer:promoter interactions and gene transcription within the targeted TAD. We used another series of enhancer deletions and CRISPRi on promoters within INK4a/ARF TAD to better understand the regulation and origins of bRNA itself. We observe that the enhancers interact with boundaries and positively regulate the bRNA transcription at TAD boundaries. In return, the bRNAs recruit/stabilize the CTCF even on weak motifs within these boundaries and supports CTCF binding in clusters, therefore enhancing TAD insulation. This favors the intra-TAD enhancer:promoter interactions and leads to robust gene transcription. Functionally, bRNAs are more stable than eRNAs and their knockdown exactly mimics the CTCF site deletion. Furthermore, transcribing boundaries exhibit high TAD transcription in TCGA tumour datasets. Together, these results show that active enhancers directly mediate better insulation of TADs by activating the transcription at TAD boundaries triggering CTCF clustering at the boundary which results in better insulation and favours robust intra-TAD enhancer:promoter interactions to activate the gene transcription.

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In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation [ChIPmentation]

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