Abstract
CTCF (CCCTC-binding factor) is crucial for organizing mammalian genomes into domains and structural loops, yet its role in enhancer-promoter interactions remains unclear. Here, we demonstrate that 3D enhancer architecture undergoes marked reorganization upon CTCF depletion in activated CD4+ T cells. Despite this, active transcription, particularly driven by STAT5-bound super-enhancers, maintains enhancer loops independently of CTCF. Interestingly, robust enhancer-promoter interactions are associated with the release of RNA polymerase II (RNAPII) pausing and require CTCF-dependent 3D genome organization to shape immune-related gene expression patterns in CD4+ T cells. Notably, CTCF depletion reprograms the transcriptional response of CD4+ T cells to JAK inhibitors by rewiring the STAT5 enhancer network rather than altering the upstream JAK/STAT signaling cascade. This study emphasizes the role of 3D enhancer architecture orchestrated by CTCF and active transcription in directing precise cell identity gene expression through RNAPII pause-release in CD4+ T cells.