The cohesin complex has an essential role in maintaining genome organization. However, its role in gene regulation remains largely unresolved. Here we report that the cohesin release factor WAPL creates a pool of free cohesin, in a process known as cohesin turnover, which reloads it to cell-type-specific binding sites. Paradoxically, stabilization of cohesin binding, following WAPL ablation, results in depletion of cohesin from these cell-type-specific regions, loss of gene expression and differentiation. Chromosome conformation capture experiments show that cohesin turnover is important for maintaining promoter–enhancer loops. Binding of cohesin to cell-type-specific sites is dependent on the pioneer transcription factors OCT4 (POU5F1) and SOX2, but not NANOG. We show the importance of cohesin turnover in controlling transcription and propose that a cycle of cohesin loading and off-loading, instead of static cohesin binding, mediates promoter and enhancer interactions critical for gene regulation.
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We thank the NKI Genomics Core Facility for help with sequencing, the NKI Bioimaging Facility for help with microscopy and the NKI Flow Cytometry Facility for help with single-cell sorting of genome-edited cells. We thank M. Kanemaki of the National Institute of Genetics in Japan for his suggestions on AID tagging and sharing antibody to OsTir1. We thank B. Nabet and N. Gray of the Dana Farber Cancer Institute for providing the dTAG-13 molecule. We thank R. Young of the Whitehead Institute for Biomedical Research for providing the OCT4–dTAG mESC line. Work in the de Wit laboratory is supported by ERC StG 637587 (‘HAP-PHEN’) and a Vidi grant from the Netherlands Scientific Organization (NWO, 016.16.316). N.Q.L. is supported by a Veni grant from the Netherlands Scientific Organization (NWO, 016.Veni.181.014). N.Q.L., M.M., T.v.d.B., L.B., H.T., M.M.G.A.S. and E.d.W. are part of Oncode, which is partly financed by the Dutch Cancer Society.
The authors declare no competing interests.
Peer review information Nature Genetics thanks the anonymous reviewers for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Homologous recombination strategy for tagging the endogenous Wapl gene with AID-eGFP. Note that the targeting construct contains a NeoR/KanR resistance gene, but that this has not been used for selecting the first clone. Middle panel shows PCR validation (primers highlighted in left panel) showing homozygous integration of the donor vector (n=1). Right panel shows Sanger sequencing results confirming in-frame tagging of AID tag. b, Left panel shows live cell imaging of WAPL in untreated (0h) and treated (24h) cells (representative images from 3 separate fields). DNA is visualized with Hoechst. Right panel show immunostaining of cohesin subunit RAD21 (representative images from 100 independent cells). DNA is labeled with DAPI. c, WAPL-AID-GFP levels were measured by FACS (gated on all cells besides debris). High temporal resolution shows rapid depletion of WAPL-AID-GFP upon IAA treatment. Median GFP intensity is quantified in bottom plots. Signals are the average of three experiments. Dots indicate mean values. Error bars indicate standard deviation. d, ChIP-seq of WAPL and CTCF before and after IAA treatment. Heatmaps show the distribution of signal for the peaks called in the untreated cells. Average signal is shown above the heatmap. ChIP-seq data is calibrated using spiked-in human HEK-293T cells. Middle two heatmaps show WAPL ChIP-seq signal derived from the human cells.
a, FACS plots showing EdU incorporation to measure the cycle phase of individual cells. All single cells are gated, and G1, S and G2 cells are then separated using the blue, orange and red gates, respectively. b, Quantification of cycle phases based on DAPI/EdU FACS analysis for various times after WAPL depletion (n=3 independent experiments). Height of bar indicates mean value. Error bars show standard deviation. Cyan dots show the value of individual measurements. c, An illustration of cell proliferation assay following WAPL depletion. d, Cell cycles measured following WAPL depletion (n=3 independent experiments). Height of bar indicates mean value. Error bars show standard deviation. Blue dots show the value of individual measurements. e, Western blot showing the protein levels of the pluripotency transcription factors SOX2, OCT4 and NANOG (cropped, n=1). f, Alkaline phosphatase (AP) staining is measured as a marker for pluripotency on the untreated (0 h), treated (96 h) and 48 h treatment followed by 48 h wash-off WAPL-AID and parental cell line (representative images from 2 independent clones). g, An illustration of single cell colony formation experiment following WAPL depletion. h, Single cell colony formation measured following WAPL depletion (n=3 independent experiments). Height of bar indicates mean value. Error bars show standard deviation. Blue dots show the value of individual measurements.
a, A volcano plot shows differential genes between the untreated parental (PT) and WAPL-AID cells at a false discovery rate (FDR) cutoff of 0.05. b, Transcriptomic changes of PT cells with 96 h IAA treatment following using the same FDR cutoff as (a). c, Transcriptomic changes at different time points following WAPL depletion using the same FDR cutoff as (a, b). d, Heatmap showing the genes that are differentially expressed following IAA treatment in WAPL-AID cells. Genes are clustered according to the timepoint in which they are first observed to be differentially expressed (left panel). e, Gene Set Enrichment Analysis of RNA-seq data following WAPL depletion. Analysis was performed for Gene Ontology databases and the significant (FDR < 0.01) terms with a positive normalized enrichment score (NES) are plotted. Terms that are related to development, differentiation or morphogenesis are highlighted.
Left panel shows signal of the active promoter mark H3K4me3 aligned to the promoters of differentially expressed genes. Right panel shows H3K27ac alignment to enhancers within 10kb up- or downstream of the promoter of a differentially expressed gene.
a, Venn diagram showing the uniquely bound and overlapping RAD21 binding sites identified by ChIP-seq in a second WAPL-AID clone. Peak calling was performed on a subset of 7 M sequencing reads for both of the samples. b, Scatter plot showing the Pearson correlation of RAD21 ChIP-seq data between the two different WAPL-AID clones. Red rectangle highlighting the correlation of the same time points. c, Alignment of RAD21 binding data from a second WAPL-AID clone on the RDCs and RSCs identified in the original WAPL-AID clone. d, Alignment of published ChIP-seq data for five different cohesin subunits generated in V6.5 mESC line (see Supplementary Table 3 for details). e, Example locus showing a region with an RDC and RSC. RAD21 binding is shown for treated and untreated cell for two WAPL-AID clones. Cohesin binding is shown for the five subunits shown in (d). f, Table containing the top 5 (by lowest FDR) categories in a GREAT analysis using the Mouse Genome Informatics (MGI) expression database for RDCs and RSCs.
Extended Data Fig. 6 Molecular features of the Regions of Dynamic Cohesin and the Regions of Stalled Cohesin.
a, Alignments of H3K27ac and MED1 ChIP-seq data on RDCs and RSCs. b, Example region showing the relationship between the factors analyzed in Fig. 1g and the position of a RSC. c, similar alignments as (a) but for SOX2, OCT4 and NANOG ChIP-seq data. d, RAD21 alignment on the transcription start sites (TSS) of the top 1000 most stably expressed housekeeping genes for treated and untreated cells in two different clones.
Extended Data Fig. 7 Global effects of WAPL depletion on genome architecture and nascent transcription.
a, Relative contact probability plot (RCP) showing the distribution of the chance of two loci contacting in the context of the 3D genome as a function of the distance between them on the linear chromosome. b, Example locus showing the formation of extended loops upon WAPL depletion. Heatmaps visualize the contact frequency matrices for the Nfe2l3 locus. Rightmost panel shows the differential contacts between the 24h treated cells and the untreated cells. c, Total number of TADs detected in control (0 h) and WAPL depleted (24 h) cells. d, TAD size from control (0 h) and WAPL depleted (24 h) depleted cells (Box plot elements: center line, median; box, inter quartile range/IQR; whiskers, 1.5 times the IQR from the median value; dots, instances lower or higher than the IQR from the median value). e, Distribution of intra- and inter-TAD interactions. f, Global effects of WAPL depletion on nascent transcription at early time points (6 and 24 h). g, Nascent transcription changes following WAPL depletion are plotted using the same gene clusters described in Extended Data Fig. 3d.
Extended Data Fig. 8 Depletion of WAPL and RAD21 reorganizes the 3D genome and affects gene expression.
a, Effects of WAPL depletion on gene expression similar to Fig. 2d but using RNA-seq. The yellow stars indicate the observed over expected enrichment of the RNA-seq data (n=2 independent experiments), and the violin plots show distribution of enrichment based on 10,000 random circular permutations (red lines: median values, orange lines: interquartile range, *: P<0.001, **: P<0.0001). b, High-resolution 4C-seq data for the Klf4 locus (viewpoint: Klf4 enhancer, overlapping with the distal RDC of the Klf4 gene). The 4C-seq data is visualized in the same way as Fig. 2e. The bottom two rows show nascent transcription of the Klf4 gene and neighboring region in the untreated (0 h) and treated (24 h) ES cells. c, Expression of the Klf4 gene was quantified by RT-qPCR (n=3 independent experiments) after 24 h of WAPL depletion and 24 h depletion followed by 24/48 h wash-off (two-sided student’s t-test without multiple testing correction, bar height: mean values, error bars: standard deviations, red dots: individual data points). d, High-resolution 4C-seq analysis of the Klf4 locus in the RAD21 depletion experiment (viewpoint: the same as (b)). The 4C-seq data is visualized in the same way as Fig. 2e. The bottom two rows show expression of the Klf4 gene and neighboring region in the untreated (0 h) and treated (24 h) ES cells. e, Global effects of RAD21 depletion on gene expression at early time points (6 and 24 h).
a–c, heatmaps showing the binding of pluripotency transcription factor SOX2 (a), OCT4 (b) and NANOG (c) remain unchanged following WAPL depletion. d, A published OCT4-FKBP-mCherry cell lines was used. The FKBP degron can be degraded with the dTAG-13 molecule. e, A western blot shows protein levels of OCT4 after 6 and 24 hours of dTAG-13 treatment (cropped). f, g, ChIP-seq heatmaps showing MED1 binding at OCT4 binding sites and NANOG binding sites in the OCT4-FKBP (f) and NANOG-FKBP (g) lines, respectively. h, i, ChIP-seq heatmaps showing RAD21 binding and chromatin accessibility at CTCF binding sites in OCT4-FKBP (h), SOX2-FKBP (i, left) and NANOG-FKBP (i right). For OCT4-FKBP CTCF sites from V6.5 mESCs were used for alignment and for Nanog-FKBP, which are derived from E14 mESCs CTCF sites identified in WAPL-AID cells were used.
a, Staining for Nestin (Green) and GFAP (red) in NPCs derived from the WAPL-AID cell line (representative images from 2 experiments and 3 separate fields per experiment). b, Staining for alkaline phosphatase in NPCs derived from the WAPL-AID cell line (n=1). c, Comparison of the differential genes between ESC and NPC identified in a published study and in the WAPL-AID line. d, Venn diagram shows overlap of RAD21 binding sites measured ChIP-seq for untreated and 24h treated WAPL-AID NPCs. Only sites that showed consistent unique or shared binding across two replicates were included. Peak calling was performed on a subset of 7 M sequencing reads for all the samples. e, MA-plot showing the log2 fold-change as a function of the number of reads in a RAD21 peak. Highlighted dots indicate significantly changed RAD21 levels based on DESeq2 with an FDR <0.01 and a fold-change of at least 2 up or down. Red is increase in RAD21, blue is decrease. f, Annotation of the motifs identified in Fig. 5d.
Unprocessed western blots for Fig. 1.
Unprocessed western blots for Fig. 3.
Unprocessed western blots for Fig. 4.
Unprocessed western blots for Extended Data Fig. 2.
Unprocessed western blots for Extended Data Fig. 9.
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Liu, N.Q., Maresca, M., van den Brand, T. et al. WAPL maintains a cohesin loading cycle to preserve cell-type-specific distal gene regulation. Nat Genet 53, 100–109 (2021). https://doi.org/10.1038/s41588-020-00744-4
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