Essential roles of CTCF binding sites at TAD boundaries in modulating chromatin interactions and transcriptional regulation at the Ifng locus

Interferon-γ (IFN-γ) is a key cytokine essential for host defense and tumor surveillance, and its expression in T cells is tightly regulated by long-range enhancer-promoter interactions within the <i>Ifng</i> locus. These interactions are organized within topologically associating domains (TADs) and subdomains (intra-TADs), whose boundaries are defined by the architectural protein CTCF. However, the specific contribution of these boundaries to <i>Ifng</i> regulation and chromatin architecture remains incompletely understood. Here, we investigated how chromatin looping within the TAD affects <i>Ifng</i> expression by using chromosome conformation capture (3C) and RNA sequencing in CRISPR-Cas9 genome editing in mice. Deletion of a single CTCF binding site (CBS) at the TAD boundary markedly impaired Th1-mediated immune responses against <i>Cryptococcus neoformans</i> infection and B16 melanoma. This deletion disrupted enhancer-promoter contacts and diminished enhancer-driven activation of <i>Ifng</i>. In contrast, deletion of a CBS within an intra-TAD boundary had no detectable impact on <i>Ifng</i> transcription. Collectively, these findings highlight the essential role of boundary-associated CBS elements in shaping chromatin architecture that enables enhancer-promoter communication and proper transcriptional regulation of the <i>Ifng</i> locus.