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The essential role of CTCF binding site at TAD boundaries in modulating Ifng locus chromatin interactions and transcriptional regulation [ChIP-seq]

ABSTRACT: Interferon gamma (IFN-g) is a vital cytokine for host defense and tumor surveillance. Its gene expression in T cells is precisely controlled by long-range enhancer-promoter interactions within the Ifng locus. These interactions depend on topologically associating domains (TADs) and intra-TADs, whose boundaries are maintained by the architectural protein CTCF. Despite their importance, the specific contributions of these boundaries to Ifng regulation and their interplay in domain formation are poorly understood. Here, using CRISPR-Cas9 genome editing in mice combined with chromosome conformation capture (3C) and RNA-seq, we explored how chromatin loop dynamics within the TAD modulate Ifng expression. We found that deleting a single CTCF binding site (CBS) at the TAD boundary impaired Th1-associated immune response to Cryptococcus neoformans infection and B16 melanoma cells. This deletion significantly weakened enhancer-promoter interactions and enhancer-driven Ifng activation. Interestingly, disrupting a CBS at an intra-TAD boundary had no impact on Ifng transcription. This study highlights the essential role of CBS elements at TAD boundaries in shaping the chromatin architecture to enable enhancer-promoter communication and subsequent gene regulation.

ORGANISM(S): Mus musculus

PROVIDER: GSE300739 | GEO | 2025/08/20

REPOSITORIES: GEO

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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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The essential role of CTCF binding site at TAD boundaries in modulating Ifng locus chromatin interactions and transcriptional regulation [ChIP-seq]

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