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

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.