The three-dimensional chromatin architecture is critical for gene regulation, yet factors beyond CCCTC-binding factor (CTCF) and cohesin remain poorly characterized. Here, we identify RFX5 as a novel regulator of chromatin organization. Using CRISPR-mediated RFX5 knockout A375 cells, together with RNA-seq, ChIP-seq, ATAC-seq, QHR-4C, and Hi-C, we demonstrate that RFX5 binds to promoters and enhancers, co-localizes with CTCF, RAD21, and H3K27ac, maintains chromatin accessibility, and preserves chromatin loop strength. RFX5 deletion alters the expression of ~2,000 genes, with strong suppression of cancer-associated genes and oncogenic pathways. Loss of RFX5 reduces CTCF/RAD21 occupancy and promoter accessibility at downregulated genes. Notably, RFX5 acts as an insulator to balance chromatin looping: its absence weakens enhancer–promoter contacts at oncogenic loci while enabling inappropriate long-range enhancer interactions at upregulated genes. Hi-C analysis reveals globally diminished loop strength, with only mild effects on TAD insulation and compartmentalization. These findings establish RFX5 as a key architectural factor that links 3D genome structure to transcriptional programs in cancer and immunity. RFX5 acts as a novel architectural regulator that strengthens enhancer–promoter loops and functions as an insulator. Its deletion globally weakens chromatin loop strength, suppresses oncogenic gene expression, and modestly affects TAD insulation.
Ge et al. (Sat,) studied this question.