Abstract
The retinoblastoma protein (RB) is a well-characterized repressor of E2F transcriptional activity that controls genes involved in cell proliferation during the G1 phase. Here, we examine the effect of RB on chromatin organization and uncover a non-canonical role for RB in which it promotes the removal of cohesin from insulators and thereby induces the expression of adjacent genes. We identify that RB colocalizes with cohesin across the human genome. During mitosis, RB facilitates the removal of cohesin from CTCF sites, with this effect persisting into early G1, impacting loop formation and extrusion at topologically associating domain (TAD) boundaries. Chromosome conformation capture assays reveal that RB reduces insulation at TAD boundaries and promotes enhancer-promoter interactions marked by histone 3 lysine 27 acetylation. In this way, RB enhances the transcription of hundreds of genes beyond the E2F transcriptional program, a finding that expands our understanding of this key tumor suppressor and cell cycle regulator.
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Data availability
Source data are provided as a Source Data file. The RB, E2F1, CTCF, c-Jun, H3K4me1, H3K4me3, and H3K27ac ChIP-seq from asynchronous hTERT-RPE1 cells are available at GEO under the Series GSE17603523 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE176035]. The SMC3, acSMC3, H3K27ac, and H3K27me3 ChIP-seq, ATAC-seq, MNase-seq, and RNA-seq datasets from hTERT-RPE1 cells are available at GEO under the SuperSeries GSE246946. Individual accession codes are as follows: ATAC-seq, GSE246942; ChIP-seq, GSE246943; RNA-seq, GSE246944; and MNase-seq, GSE246945. Additionally, STAG1, STAG2, and MED15 ChIP-seq from G1-arrested hTERT-RPE1 cells and SMC3 ChIP-seq from Eg5 inhibitor-treated hTERT-RPE1 cells are available at GEO under the Series GSE315529. Single cell RNA-seq from RBWT and RBKO hTERT-RPE1 cells are available at GEO under the Series GSE315528. The Micro-C dataset is available at the EMBL-EBI ArrayExpress database under the accession code E-MTAB-13566, and the processed.mcool files are available at EMBL-EBI BioStudies database under the accession code S-BSST1221. The H3K27ac HiChIP in G1-arrested hTERT-RPE1 cells is available at GEO under the Series GSE266412. All mass spectrometry data can be accessed through the MassIVE data repository under the accession number MSV000098256 [https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=1c31585ab6ae49a2a3599d8eec197c9d]. Source data are provided with this paper.
Code availability
Source codes utilized for the bioinformatics analyses are publicly available at our GitHub repository79 [https://github.com/hanjunlee21/Lee-et-al-2023].
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Acknowledgements
We thank Dr. Nicholas J. Dyson for his unwavering support and critical reading of the manuscript. We also thank Dr. Miguel N. Rivera and Dr. Christopher J. Ott for their helpful discussions and feedback. This study was funded by National Institutes of Health grant R01CA236538 to Nicholas J. Dyson and Ioannis Sanidas and by Rullo Family Innovation Award to Michael S. Lawrence.
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Conceptualization: H.L., M.S.L., and I.Sanidas. Methodology: H.L., I.-M.G., M.S.L., and I.Sanidas. Investigation: H.L., I.-M.G., C.G.M., B.K., S.A., I.Salinas, R.M., and I.Sanidas. Visualization: H.L. Funding acquisition: M.S.L. and I.Sanidas. Project administration: M.S.-F., W.H., M.S.L., and I.Sanidas. Supervision: M.S.L. and I.Sanidas. Writing – original draft: H.L. Writing – review & editing: H.L., I.-M.G., M.S.L., and I.Sanidas.
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Lee, H., Gkotinakou, IM., McGrath, C.G. et al. RB loss modulates chromatin organization by regulating cohesin-dependent loops and enhancer-promoter interactions. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71655-x
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DOI: https://doi.org/10.1038/s41467-026-71655-x
