Abstract
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.
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Acknowledgements
Y.W. discloses support for the research of this work from the National Natural Science Foundation of China (82370254), the New Quality Clinical Specialty Program of High-end Medical Disciplinary Construction in Shanghai Pudong New Area (2025-PWXZ-03), the Science and Technology Research Program of Shanghai (24HC2810100, 23ZR1426000), and the Scientific Research Foundation provided by Pudong Hospital affiliated to Fudan University (YJYJRC202308). X.G. discloses support for the research of this work from the Science and Technology Commission of Shanghai Municipality (24YF2738700) and the Scientific Research Foundation provided by Pudong Hospital affiliated to Fudan University (YJYJRC202402).
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Ge, X., Zhang, Y., Han, Y. et al. RFX5 as a regulator of spatial chromatin contacts and gene expression. Commun Biol (2026). https://doi.org/10.1038/s42003-026-10279-9
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DOI: https://doi.org/10.1038/s42003-026-10279-9
