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Interplay between cohesin and RNA polymerase II in regulating chromatin interactions and gene transcription

Nature Structural & Molecular Biology (2026)Cite this article

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Abstract

Cohesin is required for chromatin loop formation. However, its precise role in regulating gene transcription remains largely debated. Here we investigated the relationship between cohesin and RNA polymerase II (RNAPII) using single-molecule mapping and live-cell imaging methods in human cells. Cohesin-mediated transcriptional loops were highly correlated with those of RNA polymerase II and followed the direction of gene transcription. Depleting RAD21, a subunit of cohesin, resulted in the loss of long-range (>100 kb) loops between distal (super-)enhancers and promoters of cell-type-specific downregulated genes. By contrast, short-range (<50 kb) loops were insensitive to RAD21 depletion and connected genes that are mostly constitutively expressed. This result explains why only a small fraction of genes are affected by the loss of long-range chromatin interactions in cohesin-depleted cells. Remarkably, RAD21 depletion appeared to upregulate genes that were involved in initiating DNA replication and disrupted DNA replication timing. Our results elucidate the multifaceted roles of cohesin in establishing transcriptional loops, preserving long-range chromatin interactions for cell-specific genes and maintaining timely DNA replication.

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Fig. 1: ChIA-Drop data for mapping multiplex chromatin interactions mediated by CTCF, cohesin and RNAPII.
Fig. 2: Directionality of RNAPII and cohesin loops with respect to TSS and CTCF motif orientations.
Fig. 3: Multiplex transcriptional chromatin interactions involving SEs.
Fig. 4: Effects of RAD21 depletion on chromatin loops with convergent CTCF motifs mediated by CTCF and RNAPII, validated via live-cell imaging.
Fig. 5: Functional roles of RAD21-dependent SE to promoter loop in transcription.
Fig. 6: Distinct roles of RAD21-dependent E-P and RAD21-independent P-P RNAPII loops in gene regulation.
Fig. 7: DNA replication signal patterns in differential genes and the proposed model.

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Data availability

The accession number for the deep-sequencing data reported in this paper is GEO: GSE158897. The following publicly available datasets have also been used in this study: NIPBL ChIP-seq (Gene Expression Omnibus; accession GSM2443453), H3K27ac ChIP-seq (ENCODE data portal88; accession ENCFF340JIF), H3K4me1 ChIP-seq (ENCODE data portal: accession ENCFF831ZHL), RNA-seq (ENCODE data portal: accession ENCLB555AQG), GM12878 Hi-C (4DN data portal89: accession 4DNFI7J8BQ4P, 4DNFI1UEG1HD), list of GM12878 SEs and constituents52, HCT116 Hi-C (4DN data portal: accession 4DNFIP71EWXC, 4DNFIBIV8OUN), HCT116 0 h Repli-seq (4DN data portal: accession 4DNFIR6ZS4LY, 4DNFID2WWTSC, 4DNFIH4B6I1S, 4DNFIFBWQ3QC, 4DNFI6FRVLDB, 4DNFINSRFNDX, 4DNFI3JLMX17, 4DNFIFS513KB, 4DNFIB697UQV, 4DNFIED8FHGM, 4DNFIJ88Z7MW, 4DNFI4FWA2X9, 4DNFIYQQ72X9, 4DNFIPWM5DS1, 4DNFIRBZUG62, 4DNFIQXJN452), HCT116 6 h Repli-seq (4DN data portal: accession 4DNFIXFHUPTI, 4DNFIX6NTFM4, 4DNFISBPS2ZV, 4DNFIMMM331D, 4DNFIPSBAONE, 4DNFIKESZXXD, 4DNFIZT1GRIL, 4DNFIHIHNQSN, 4DNFIQ573AKW, 4DNFI89FPVRX, 4DNFIST28EMP, 4DNFIA9QXIDF, 4DNFIRWK243V, 4DNFI6V9EXOM, 4DNFI1WWTVBY, 4DNFIL37I65A), list of HCT116 SEs (https://asntech.org/dbsuper/download.php), GM12878 ChromHMM states (https://hgsv.washington.edu/cgi-bin/hgFileUi?db=hg18&g=wgEncodeBroadHmm) and HCT116 ChromHMM states (ENCODE data portal: accession ENCFF513PJK). CTCF in situ ChIA-PET data from HFFc6 (4DN data portal: accession 4DNESCQ7ZD21) and MCF10A (ENCODE data portal: accession ENCSR403ZYJ) cells. RNAPII ChIA-PET data, RNA-seq data and ChromHMM states from H1 (4DN data portal: accession 4DNEXF93AC6Q; ENCODE data portal: accession ENCLB555AMA; UCSC Broad ChromHMM), GM12878 (this study; ENCODE data portal: accession ENCLB555AQG; UCSC Broad ChromHMM), K562 (ENCODE data portal: accession ENCSR880DSH, ENCLB555AKN; UCSC Broad ChromHMM), HepG2 (ENCODE data portal: accession ENCSR857MYZ, ENCLB555AQD; UCSC Broad ChromHMM), and MCF7 (ENCODE data portal: accession ENCSR059HDE, ENCLB555AQN, ENCFF506GEX). Gene expression profile in 76 human tissues from the Expression Atlas87 (https://www.ebi.ac.uk/gxa/home). Source data are provided with this paper.

Code availability

The scripts and downloadable links to the files used in this study are available via GitHub at https://github.com/minjikimlab/cohesin-kim-nsmb-2025.

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Acknowledgements

This study was supported by National Natural Science Foundation of China (32250710678 to Y.R.), the Jackson Laboratory Director’s Innovation Fund (DIF19000-18-02 to Y.R.), 4DN (U54 DK107967 to Y.R.), ENCODE (UM1 HG009409 to Y.R.) consortia, Human Frontier Science Program (RGP0039/2017 to Y.R.), the National Human Genome Research Institute (R01-HG009900 to A.W.C., R01-HG011253 to C.-L.W., R01-GM127531 to C.-L.W., K99-HG011542 to M.K.), and National Science Foundation (CCF-1955712 to O.M., CIF 1956384 to O.M.). We acknowledge Zhihui Li, Xiaoan Ruan, Meizhen Zheng, and Simon Tian for preliminary ChIA-Drop data generation and analyses and thank the Casellas group members for critical feedback on the paper. We also gratefully acknowledge the HCT116-RAD21-mAID-mClover cell line as a gift from Masato Kanemaki (National Institute of Genetics).

Author information

Author notes

  1. These authors contributed equally: Minji Kim, Ping Wang, Patricia A. Clow.

  2. These authors jointly supervised this work: Albert W. Cheng, Yijun Ruan.

Authors and Affiliations

  1. The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA

    Minji Kim, Ping Wang, Patricia A. Clow, Haoxi Chai, Xiyuan Liu, Byoungkoo Lee, Chew Yee Ngan, Jeffrey H. Chuang, Chia-Lin Wei, Albert W. Cheng & Yijun Ruan

  2. Gilbert S. Omenn Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA

    Minji Kim

  3. Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Evanston, IL, USA

    Ping Wang

  4. Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Eli Chien, Jianhao Peng & Olgica Milenkovic

  5. Institute of Reproduction and Development, Shanghai Key Laboratory of Reproduction and Development, Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China

    Xiaotao Wang

  6. Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, China

    Xiaotao Wang

  7. State Key Laboratory of Eye Health, Eye Hospital, Wenzhou Medical University, Wenzhou, P.R. China

    Xiyuan Liu

  8. Department of Genetics and Genome Sciences, UConn Health, Farmington, CT, USA

    Jeffrey H. Chuang

  9. Hematopoietic Biology and Malignancy, MD Anderson Cancer Center, Houston, TX, USA

    Rafael Casellas

  10. State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, P.R. China

    Albert W. Cheng

  11. School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, USA

    Albert W. Cheng

  12. Life Sciences Institute, Zhejiang University, Hangzhou, P.R. China

    Yijun Ruan

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  1. Minji Kim
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  2. Ping Wang
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  3. Patricia A. Clow
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  4. Eli Chien
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  8. Xiyuan Liu
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  14. Rafael Casellas
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  15. Albert W. Cheng
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Y.R. conceptualized the project. Y.R., M.K. and P.W. designed experiments. P.W., H.C., X.L., C.Y.N. and C.-L.W. generated mapping data. M.K., E.C., X.W., J.P. and B.L. performed computational analyses. O.M., J.H.C., C.-L.W., R.C. and Y.R. assisted data analysis and interpretation. A.W.C., Y.R., P.A.C. and M.K. designed the imaging experiments. P.A.C. and A.W.C. performed the imaging experiments and analyzed the data. Y.R., B.L. and M.K. assisted imaging data analysis and interpretation. Y.R. and M.K. wrote the paper with inputs from P.W., R.C., P.A.C. and A.W.C.

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Correspondence to Yijun Ruan.

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Extended data

Extended Data Fig. 1 Reproducibility and multiplexity of ChIA-Drop data.

(a) A table of ChIP-enriched CTCF, cohesin, and RNA Polymerase II (RNAPII) ChIA-Drop chromatin complexes by the number of fragments per complex (F/C). (b) Stratum-adjusted correlation coefficients (SCC) between all datasets of ChIP-enriched CTCF, RAD21, SMC1A, and RNAPII ChIA-Drop experiments and their replicates. R1, R2, and R3 denote replicates 1, 2, or 3 of a given experiment, respectively. SCC between ChIP-enriched ChIA-Drop and corresponding ChIA-PET data are also computed. (c) Boxplots for quantifications of transcriptional chromatin interactions. Left panel: number of chromatin complexes in CTCF, cohesin, and RNAPII ChIA-Drop data at 1,706 loop loci characterized in Fig. 1d. Right panel: the Jensen-Shannon divergence of pairs of the datasets between RNAPII, cohesin, and CTCF ChIA-Drop (see Methods). p-values are computed from the two-sided Mann-Whitney U test. (d) Left panel: empirical cumulative distribution function (ECDF) of the observed (purple) and expected (grey) number of RNAPII ChIA-Drop complexes with 0, 1, 2, 3, and ≥ 4 promoters (top) and those with 0, 1, 2, 3, and ≥ 4 enhancers (bottom) are plotted. Right panel: the numbers and percentages of RNAPII ChIA-Drop complexes with promoters (top) and enhancers (bottom) that were expected and observed.

Extended Data Fig. 2 Examples showcasing behaviors of RNAPII and cohesin chromatin complexes at CTCF anchoring and cohesin loading sites with and without actively transcribed gene promoters.

(a) The 2D contact maps of RNAPII ChIA-PET, cohesin ChIA-PET, CTCF ChIA-PET, and Hi-C data encompassing all possible interactions for LRCH1 and LPAR1 locus. (b) Similar to Fig. 2b, but at the TSS of PIEZO2 in concordance with CTCF/cohesin anchoring site and TCF4 TSS coinciding with the cohesin loading site. (c) Similar to panel a but for PIEZO2 and TCF4 loci related to Extended Data Fig. 2b. (d) Similar to panel a, but at the TSS of HIVEP1 gene and CTCF-free cohesin loading site without TSS shown in Fig. 2d.

Extended Data Fig. 3 Genome-wide summary statistics of chromatin interactions related to super-enhancers.

(a) Aggregation of 2D pairwise contacts of RNAPII, cohesin, and CTCF ChIA-Drop data at super-enhancer regions and at random regions as negative controls. (b) The 2D contact maps of RNAPII and cohesin ChIA-Drop data at three exemplary regions, each including MYC, MARCKS, XRCC6 genes. (c) A histogram of genomic span of 188 SE-P structures, of which 14 are larger than 2 Mb (left panel). The number of intermediate elements between SE and P are also plotted (right panel). (d) Boxplots of normalized node degrees of SEs, other SE along the path (OSE), enhancers (E), intermediary promoters (IP), and target gene promoters (P) are plotted for the 188 SE-P pairs in RNAPII and cohesin ChIA-Drop data. (e) A scatterplot between node degrees (see Methods) of RNAPII and cohesin ChIA-Drop complexes.

Extended Data Fig. 4 Cohesin perturbation by RAD21 depletion and its impacts to CTCF and RNAPII.

(a) A schematic of experimental design of using Auxin Inducible Degron (AID) tagged cell line HCT116-RAD21-mAC for auxin (IAA)-inducible degradation of RAD21 to study cohesin’s roles in CTCF- and RNAPII-associated chromatin looping. (b) A western blot result of RAD21-depletion in HCT116 cells with auxin treatment of 0,6,9,24 hours (h). The bands for RAD21-mAC and GAPDH are indicated. (c) An example browser views of ChIP-seq data of RAD21, CTCF, and RNAPII and RNA-seq data in HCT116 cells tagged with Auxin-inducible Degron AID (HCT116-RAD21-mAC) with auxin (IAA) treatment for 0, 6, 9, and 12 hours. Light blue arrows indicate CTCF binding motif and orientations. (d) Boxplots of chromatin loop span in the categories of ‘RAD21-dependent’ (that is, reduced loop strengths) and ‘RAD21-independent’ (unchanged) loops in CTCF and RNAPII ChIA-PET data (see Methods), where n denotes the number of loops in each category and median loop span recorded below. p-values are from the two-sided Mann-Whitney U test. (e) Segmented bar charts for the proportions of CTCF-associated chromatin loops in ‘All’, ‘RAD21-dependent’, and ‘RAD21-independent’ HCT116 loops. CTCF loops with binding motifs in 4 categories: convergent (‘> <’), right tandem (‘> >’), left tandem (‘< <’), divergent (‘< >’). (f) Segmented bar charts for the proportions of RNAPII-associated chromatin loops in ‘All’, ‘RAD21-dependent’, and ‘RAD21-independent’ HCT116 loops. RNAPII loops are first characterized by CTCF binding motifs as convergent (‘> <’), right tandem (‘> >’), left tandem (‘< <’), divergent (‘< >’), and the rest of the CTCF-free loops are further categorized as promoter-promoter (‘P-P’), enhancer-promoter (‘E-P’), and enhancer-enhancer (‘E-E’) loops.

Extended Data Fig. 5 Effects of RAD21 depletion on gene transcription, gene body loops, and super-enhancer to promoter interactions.

(a) Scatter plots of TPM (transcripts per kilobase million) of genes from RNA-seq with various timepoints of auxin treatment. \(\rho\): Pearson’s correlation coefficient. (b) Boxplots of log2(fold-change) of gene expression before and after depleting RAD21 of up-regulated and down-regulated genes from PRO-seq (left) and RNA-seq (right) datasets; up-regulated and down-regulated genes were defined by PRO-seq (Rao et al., 2017). (c) A scatterplot of log2(fold-change) of gene expression between RNA-seq and PRO-seq data for unchanged genes, with colors denoting the density of data points. (d) Scatterplot of RNAPII binding intensity at promoters of unchanged genes between 0 h and 6 h of RAD21-depletion (left panel) and aggregated peaks ±25 kb of TSS (right panel). (e) Gene Ontology terms enriched in down-regulated, up-regulated, and unchanged genes. (f) An example of gene body loops of RNAPII ChIA-PET data before (0 h; h: hours) and after (6 h) depleting RAD21. Top panel: PTEN is an active gene not affected by RAD21 depletion as shown by RNA-seq (this study) and PRO-seq (Rao et al., 2017) recorded by TPM (transcript per kilobase million) and RPKM (reads per kilobase million), respectively. Bottom panel: transcriptional looping over PTEN gene body in RNAPII ChIA-PET fragment views. n denotes the number of chromatin complexes. (g) Boxplots of log10 of number of complexes in the gene-body loops before (0 h) and after (6 h) depleting RAD21, plotted separately for down-regulated genes and unchanged genes. The central line inside the box is the median, the edges of the box are the 25th and 75th percentiles, and whiskers extend to the most extreme data points not considered outliers. All p-values are from the two-sided Mann-Whitney U test. (h) Boxplots of log10 of PET counts in RNAPII ChIA-PET of SE-P and intra-SE interactions within super-enhancer among constituents before (0 h) and after (6 h) RAD21 depletion. (i) Representative Casilio images of SOX9-SE loop anchors (two pairs of probes in circles per nucleus) in control HCT116-RAD21-mAC cells (Auxin 0 h) and cells with 24 hours of auxin treatment for RAD21-degradation (Auxin 24 h). The pair with light blue arrow in each image is further tracked in Fig. 5g. Scale bars, 5 µm. (j) In a large chromatin domain (1.2 Mb) harboring MYC gene and associated cis-regulatory elements super-enhancer (SE), enhancers (E) and promoter (P) demarcated by ChromHMM, tracks of RNAPII ChIA-PET loops/peaks and chromHMM states are shown for 6 cell lines: GM12878, HCT116, HepG2, MCF7, K562, H1.

Extended Data Fig. 6 Examples and genome-wide statistics of RAD21-dependent E-P and RAD21-independent P-P RNAPII loops.

(a) A 300 kb region including a down-regulated CDKN2B gene, where RNAPII ChIA-PET, RNA-seq, and ChomHMM chromatin states are shown for HCT116 and 5 other cell lines: H1, GM12878, K562, HepG2, and MCF7. 2D contact maps of RNAPII ChIA-PET in HCT116 cells before (0 h) and after (6 h) RAD21 depletion are also shown. (b) A 170 kb region encompasses RAD21-independent RNAPII loops connecting promoters (P) of active genes. Annotations are consistent with those in panel a. (c) An empirical cumulative distribution function (CDF) of the normalized Shannon entropy (see Methods) quantified over 6 cell lines gene expression (top panel) and chromatin interaction strengths (bottom panel) involved in RAD21-dependent enhancer-promoter (E-P) and RAD21-independent promoter-promoter (P-P) loops. (d) The Spearman’s correlation coefficient between the genomic profiles between all pairs of 6 cell lines, clustered via hierarchical clustering (see Methods). The left panel characterizes loop strengths of RAD21-dependent enhancer-promoter (E-P) loops and the right panel includes genes involved in these loops. (e) A similar plot as panel d for the RAD21-independent promoter-promoter (P-P) interactions and associated genes therein.

Extended Data Fig. 7 A list of up-regulated genes associated with DNA replication, and examples of replication signals encompassing up-regulated and down-regulated genes.

(a) A list of 14 genes identified to be associated with DNA replication in Gene Ontology of up-regulated genes, along with their fold-change of TPM (6 h/0 h) and descriptions of functions. (b) An example of an up-regulated gene RRM2 along with RNA-seq signal (top panel) and 16-stage Repli-seq (Emerson et al., 2022) signal from early P02 to late P17 stages in HCT116 cells before (0 h; middle panel) and after (6 h; bottom panel) RAD21 depletion. Blue bars are replication initiation zones identified using 0 h data in Emerson et al. with specific labels early or late/mid replication. (c) Similar to panel b, but for a down-regulated FRAS1 gene.

Supplementary information

Supplementary Information

Supplementary Methods.

Reporting Summary

Supplementary Video 1

Time-lapse video of BCL6 loop of pair N13 auxin 0 h. Scale bar: 1 mm. Related to Fig. 4.

Supplementary Video 2

Time-lapse video of BCL6 loop of pair N24 RAD21 degraded auxin 24 h. Scale bar: 1 mm. Related to Fig. 4.

Supplementary Video 3

Time-lapse video of SOX9 loop of auxin 0 h. Scale bar: 1 mm. Related to Fig. 5.

Supplementary Video 4

Time-lapse video of SOX9 loop of RAD21 degraded auxin 24 h. Scale bar: 1 mm. Related to Fig. 5.

Supplementary Data 1

Source data

Source Data Fig. 1

Unprocessed western blots.

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Kim, M., Wang, P., Clow, P.A. et al. Interplay between cohesin and RNA polymerase II in regulating chromatin interactions and gene transcription. Nat Struct Mol Biol (2026). https://doi.org/10.1038/s41594-025-01708-0

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