Active enhancers strengthen TAD insulation by bRNA mediated CTCF enrichment at the TAD boundaries
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA749037 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15217158_1.fastq.gz | Fastqsanger.gz | |||
| SRR15217158_2.fastq.gz | Fastqsanger.gz | |||
| SRR15217159_1.fastq.gz | Fastqsanger.gz | |||
| SRR15217159_2.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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Project description:The specificity of gene expression during development requires the insulation of regulatory domains to avoid inappropriate enhancer-gene interactions. In vertebrates, this insulator function is mostly attributed to clusters of CTCF sites located at topologically associating domain (TAD) boundaries. However, TAD boundaries allow certain level of physical crosstalk across regulatory domains, which is at odds with the highly specific and precise expression of developmental genes. Here we show that developmental genes and nearby clusters of CTCF sites synergistically foster the robust insulation of regulatory domains. Firstly, we found that the TADs containing developmental genes have distinctive features, including the sequential organization of developmental genes and CTCF clusters near TAD boundaries. Most importantly, by genetically dissecting representative loci in mouse embryonic stem cells, we showed that developmental genes and CTCF sites synergistically strengthened the insulation capacity of nearby boundaries through different mechanisms. Namely, while CTCF sites prevent undesirable enhancer-gene contacts (i.e. physical insulation), developmental genes preferentially contribute to regulatory insulation through non-structural mechanisms involving promoter competition rather than enhancer blocking. Overall, our work provides important insights into the specificity of gene regulation, which in turn might help interpreting the pathological consequences of certain structural variants.
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Project description:The specificity of gene expression during development requires the insulation of regulatory domains to avoid inappropriate enhancer-gene interactions. In vertebrates, this insulator function is mostly attributed to clusters of CTCF sites located at topologically associating domain (TAD) boundaries. However, TAD boundaries allow certain level of physical crosstalk across regulatory domains, which is at odds with the highly specific and precise expression of developmental genes. Here we show that developmental genes and nearby clusters of CTCF sites synergistically foster the robust insulation of regulatory domains. Firstly, we found that the TADs containing developmental genes have distinctive features, including the sequential organization of developmental genes and CTCF clusters near TAD boundaries. Most importantly, by genetically dissecting representative loci in mouse embryonic stem cells, we showed that developmental genes and CTCF sites synergistically strengthened the insulation capacity of nearby boundaries through different mechanisms. Namely, while CTCF sites prevent undesirable enhancer-gene contacts (i.e. physical insulation), developmental genes preferentially contribute to regulatory insulation through non-structural mechanisms involving promoter competition rather than enhancer blocking. Overall, our work provides important insights into the specificity of gene regulation, which in turn might help interpreting the pathological consequences of certain structural variants.
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Project description:CTCF plays an important role in 3D genome organization by adjusting insulation at TAD boundaries, where clustered CBS (CTCF-binding site) elements are often arranged in tandem array with a complex divergent or convergent orientation. Here using cPcdh and HOXD loci as a paradigm, we look into the clustered CTCF TAD boundaries and find that, counterintuitively, outward-oriented CBS elements are crucial for inward enhancer-promoter interactions as well as for gene regulation. Specifically, by combinatorial deletions of a series of putative enhancer elements in vivo or CBS elements in vitro, in conjunction with chromosome conformation capture and RNA-seq analyses, we show that deletions of outward-oriented CBS elements weaken the strength of long-distance intraTAD promoter-enhancer interactions and enhancer activation of target genes. Our data highlight the crucial role of outward-oriented CBS elements within the clustered CTCF TAD boundaries and have interesting implications on the organization principles of clustered CTCF sites within TAD boundaries.
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Project description:CTCF plays an important role in 3D genome organization by adjusting insulation at TAD boundaries, where clustered CBS (CTCF-binding site) elements are often arranged in tandem array with a complex divergent or convergent orientation. Here using cPcdh and HOXD loci as a paradigm, we look into the clustered CTCF TAD boundaries and find that, counterintuitively, outward-oriented CBS elements are crucial for inward enhancer-promoter interactions as well as for gene regulation. Specifically, by combinatorial deletions of a series of putative enhancer elements in vivo or CBS elements in vitro, in conjunction with chromosome conformation capture and RNA-seq analyses, we show that deletions of outward-oriented CBS elements weaken the strength of long-distance intraTAD promoter-enhancer interactions and enhancer activation of target genes. Our data highlight the crucial role of outward-oriented CBS elements within the clustered CTCF TAD boundaries and have interesting implications on the organization principles of clustered CTCF sites within TAD boundaries.
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Project description:CTCF plays an important role in 3D genome organization by adjusting insulation at TAD boundaries, where clustered CBS (CTCF-binding site) elements are often arranged in tandem array with a complex divergent or convergent orientation. Here using cPcdh and HOXD loci as a paradigm, we look into the clustered CTCF TAD boundaries and find that, counterintuitively, outward-oriented CBS elements are crucial for inward enhancer-promoter interactions as well as for gene regulation. Specifically, by combinatorial deletions of a series of putative enhancer elements in vivo or CBS elements in vitro, in conjunction with chromosome conformation capture and RNA-seq analyses, we show that deletions of outward-oriented CBS elements weaken the strength of long-distance intraTAD promoter-enhancer interactions and enhancer activation of target genes. Our data highlight the crucial role of outward-oriented CBS elements within the clustered CTCF TAD boundaries and have interesting implications on the organization principles of clustered CTCF sites within TAD boundaries.
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Project description:CTCF plays an important role in 3D genome organization by adjusting insulation at TAD boundaries, where clustered CBS (CTCF-binding site) elements are often arranged in tandem array with a complex divergent or convergent orientation. Here using cPcdh and HOXD loci as a paradigm, we look into the clustered CTCF TAD boundaries and find that, counterintuitively, outward-oriented CBS elements are crucial for inward enhancer-promoter interactions as well as for gene regulation. Specifically, by combinatorial deletions of a series of putative enhancer elements in vivo or CBS elements in vitro, in conjunction with chromosome conformation capture and RNA-seq analyses, we show that deletions of outward-oriented CBS elements weaken the strength of long-distance intraTAD promoter-enhancer interactions and enhancer activation of target genes. Our data highlight the crucial role of outward-oriented CBS elements within the clustered CTCF TAD boundaries and have interesting implications on the organization principles of clustered CTCF sites within TAD boundaries.
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