Cohesin is positioned in mammalian genomes by transcription, CTCF and Wapl

Abstract

Mammalian genomes are spatially organized by CCCTC-binding factor (CTCF) and cohesin into chromatin loops1,2 and topologically associated domains3,4,5,6, which have important roles in gene regulation1,2,4,5,7 and recombination7,8,9. By binding to specific sequences10, CTCF defines contact points for cohesin-mediated long-range chromosomal cis-interactions1,2,4,5,6,7,11. Cohesin is also present at these sites12,13, but has been proposed to be loaded onto DNA elsewhere14,15 and to extrude chromatin loops until it encounters CTCF bound to DNA16,17,18,19. How cohesin is recruited to CTCF sites, according to this or other models, is unknown. Here we show that the distribution of cohesin in the mouse genome depends on transcription, CTCF and the cohesin release factor Wings apart-like (Wapl). In CTCF-depleted fibroblasts, cohesin cannot be properly recruited to CTCF sites but instead accumulates at transcription start sites of active genes, where the cohesin-loading complex is located14,15. In the absence of both CTCF and Wapl, cohesin accumulates in up to 70 kilobase-long regions at 3′-ends of active genes, in particular if these converge on each other. Changing gene expression modulates the position of these ‘cohesin islands’. These findings indicate that transcription can relocate mammalian cohesin over long distances on DNA, as previously reported for yeast cohesin20,21,22,23, that this translocation contributes to positioning cohesin at CTCF sites, and that active genes can be freed from cohesin either by transcription-mediated translocation or by Wapl-mediated release.

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Figure 1: Cohesin distribution in WT, Ctcf, Smc3 and Wapl KO MEFs.
Figure 2: Cohesin redistribution to transcriptional start sites in Ctcf KO MEFs.
Figure 3: Cohesin accumulation at sites of convergent transcription in Ctcf Wapl DKO MEFs.
Figure 4: Transcription is required for the generation of cohesin islands.

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Acknowledgements

We thank D. Cisneros for help with confocal microscopy, A. Sommer and colleagues at Vienna Biocenter Core Facilities for Illumina sequencing and M. Jaritz for generating the density heat map program. Research in the laboratory of J.-M.P. is supported by Boehringer Ingelheim, the Austrian Science Fund (SFB-F34 and Wittgenstein award Z196-B20) and the Austrian Research Promotion Agency (headquarter grants FFG-834223 and FFG-852936, Laura Bassi Centre for Optimized Structural Studies grant FFG-840283).

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Authors

Contributions

G.A.B. did most experiments; P.v.d.L. performed ChIP–qPCR and the Nipbl and Wapl ChIP-seq experiments; G.A.B. and R.S. performed bioinformatic analyses of ChIP-seq data; R.S. bioinformatically analysed GRO-seq data; E.A. analysed the RNA-seq data; A.T. generated the conditional Wapl mouse; N.G. provided the conditional Ctcf mouse; G.A.B. and J.-M.P. planned the project, designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Jan-Michael Peters.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks G. Natoli and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Characterization of conditional Smc3 KO cells.

a, Schematic representation of the WT, floxed (fl) and deleted (∆) Smc3 alleles (after elimination of the neomycin resistance gene). EcoRV fragments, which were used for allele identification by Southern blot analysis with the indicated exon 8 probe, are shown together with their length (in kilobases). b, Southern blot analysis of tail DNA from WT, Smc3fl/+ and Smc3fl/fl mice. c, Absence of Smc3−/−offspring at birth. The genotype of newborn mice from intercrosses of Smc3+/− mice was determined by PCR genotyping. d, Deletion of the floxed Smc3 allele was detected by PCR genotyping in primary Rosa26CreER/+ Smc3fl/+ MEFs at the indicated days after 4-hydroxytamoxifen (OHT) addition. e, The level of Smc3 protein depletion in primary Rosa26CreER/+ Smc3fl/− MEFs was analysed every second day after OHT addition by immunoblot analysis of whole-cell extracts. Control Rosa26CreER/+ Smc3fl/+ MEFs were additionally analysed together with a dilution series of the day 0 sample. A long and a short exposure of the Smc3 immunoblot are shown. f, Proliferation capacity of WT MEFs and Ctcf, Smc3 and Wapl KO MEFs. The indicated serum-starved cells were stimulated with 10% fetal calf serum, and cell numbers were measured every day using a Casy counter. All three KO cells failed to respond to proliferate, in contrast to WT MEFs. g, The efficiency of protein depletion was analysed by immunoblot analysis of chromatin extracts from starved MEFs at day 10 after OHT treatment (Smc3 and Wapl KO cells) or Adeno-Cre virus infection (Ctcf KO cells). The WT chromatin sample was diluted up to 1:32 to estimate the relative reduction in protein levels (CTCF: >4×; Wapl: >8×; Smc3: >16×). Data are shown for one experiment (d, f), one experiment representative of two experiments (e) and one experiment representative of three experiments (g).

Extended Data Figure 2 Cohesin relocation in Ctcf KO MEFs.

Binding of Nipbl, CTCF and cohesin (Stag1 and Scc1) at the Nufip2 (a), Gphn (b) and Ublcp1/Rnf145 (c) genes was determined by ChIP-seq analysis in WT MEFs and Ctcf, Smc3 and Wapl KO MEFs. Data are shown for one experiment representative of two independent experiments (ac; CTCF, Stag1 and Scc1 ChIP-seq) per genotype or one experiment (ac; Nipbl ChIP-seq).

Extended Data Figure 3 Analysis of Ctcf KO-specific cohesin sites.

a, Density profiles of CTCF and Stag1 binding at cohesin sites that are commonly found in WT and Ctcf KO cells. The cohesin sites were subdivided on the basis of the detection or absence of a CTCF peak in Ctcf KO cells as determined by the MACS peak-calling program. b, The enrichment of Stag1 binding (n = 2) relative to input is shown for WT (black) and Ctcf KO cells (purple) at two TSSs, one CTCF site and one TTS. Two independent biological experiments were performed and normalized average values with error bars (s.d.) were plotted. c, d, Cohesin (Stag1 and Scc1) ChIP-seq data of replica (rep) experiments are shown as density heat maps of cohesin binding at the different categories of cohesin-binding sites sites (c) and at active and inactive TSSs (d). Binding data are shown for a region extending from −2.5 kb to +2.5 kb relative to the cohesin peak summit. Heat maps were sorted according to the density of Stag1 binding in Ctcf KO cells (rep 2). A density scale from low (grey) to high (yellow) is shown. As indicated, two or three independent ChIP-seq experiments per genotype were performed. e, Categorization of WT-specific, Ctcf KO-specific and commonly found cohesin sites according to their location at active TSSs (H3K4me2+ H3K9ac+) or non-TSS regions with open chromatin (H3K4me2+ H3K9ac+), poised chromatin (H3K4me2+) and no active chromatin marks (rest). Note that one TSS can bind several cohesin sites. Therefore the number of cohesin-bound TSSs and TSS-bound cohesin sites are not necessarily identical.

Extended Data Figure 4 Cohesin redistribution to transcriptional start sites in Ctcf KO MEFs.

a, Binding of cohesin at TSSs of active and inactive genes, which were defined by RNA-seq analysis. Genes with an RPKM >1 were considered as active, whereas genes with an RPKM <1 were classified as inactive. Pie charts indicate the relative binding of cohesin at all annotated TSSs of the RefSeq genome (mm9) in WT and Ctcf KO MEFs. b, Density heat map of cohesin and Nipbl binding at active and inactive TSSs as defined in a. Active and inactive TSSs were sorted according to the read density of Stag1 binding in Ctcf KO cells (replica 3). c, Binding of cohesin at TSSs of active and inactive genes, which were defined by GRO-seq analysis. Genes with a TPM >1 were considered as active, whereas genes with an TPM <1 were classified as inactive. Pie charts indicate the relative binding of cohesin at all annotated TSSs of the RefSeq genome (mm9) in WT and Ctcf KO cells. d, Density heat map of cohesin and Nipbl binding at active and inactive TSSs as defined in c. Active and inactive TSSs were sorted according to the read density of Stag1 binding in Ctcf KO (replica 3). A density scale from low (grey) to high (yellow) is shown (b, d). e, Heat map of cohesin binding at Nipbl peaks in MEFs of the indicated genotypes. The Nipbl peaks were subdivided according to their TSS localization. Peaks were sorted according to the Stag1 binding density in Ctcf KO cells (replica 3). f, Venn diagram indicating the overlap between Nipbl and cohesin peaks in WT or Ctcf KO MEFs. As indicated in b, d and e, two or three independent ChIP-seq experiments per genotype were performed.

Extended Data Figure 5 Identification of CTCF- and cohesin-regulated genes.

a, Scatter plot of gene expression differences between WT and Ctcf KO or Smc3 KO MEFs, on the basis of two and four independent RNA-seq experiments, respectively. The normalized expression data of individual genes in the two cell types are plotted as coefficient values. Each symbol represents one gene. Genes with an expression difference greater than twofold, an adjusted P value <0.1 and an RPKM value >1 in WT or KO cells are coloured in blue or red, corresponding to down- or upregulated genes in the indicated KO MEFs, respectively. For evaluation of the RNA-seq data, see Methods. b, Overlap between CTCF- and cohesin-regulated genes, shown as a Venn diagram. ce, Expression of selected regulated genes in WT (black), Ctcf KO (purple) and Smc3 KO (blue) MEFs. The expression of genes, which are commonly regulated by CTCF and cohesin (c), by CTCF alone (d) or by cohesin alone (e), is shown as normalized average expression value (RPKM) with error bars (s.d.) based on ten (WT), two (Ctcf KO) or four (Smc3 KO) independent RNA-seq experiments. f, h, Minimal correlation between CTCF-dependent gene regulation and cohesin (f) or CTCF (h) binding at active promoters. Genes that were down- or upregulated in Ctcf KO MEFs are shown as the percentage of all genes present in the three indicated gene groups that were defined by the presence or absence of cohesin binding at active TSSs in WT and/or Ctcf KO cells. g, i, Little correlation between Smc3-dependent gene regulation and cohesin (g) or CTCF (i) binding at active promoters. Genes that were down- or upregulated in Smc3 KO MEFs are shown as the percentage of all genes that were defined by the presence or absence of cohesin binding at active TSSs in WT cells.

Extended Data Figure 6 Genomic localization of cohesin in Wapl KO cells.

a, The cohesin-binding sites detected in WT and Wapl KO cells were subdivided into WT-specific, Wapl KO-specific and common peaks, as indicated by the Venn diagram. For each subgroup, the most significant DNA-binding motif is shown. The different motifs were detected with the E value indicated in brackets. b, Heat maps of Nipbl and cohesin binding for the different subgroups. Cohesin and Nipbl peaks at cohesin-binding sites (vertical axis) are shown for a region extending from −2.5 kb to +2.5 kb relative to the cohesin peak summit (horizontal axis) and were sorted in each subgroup according to the density of Stag1 binding in Wapl KO cells (replica 3). A density scale from low (grey) to high (yellow) is shown. The number of independent ChIP-seq experiments per genotype are indicated. c, Venn diagram of Wapl and cohesin peaks in WT MEFs. The individual subgroups were further overlapped with Nipbl sites. d, Density profiles of Scc1 binding are shown for Wapl only sites (green) and cohesin/Wapl common sites (blue). Note that cohesin is also enriched (even though at a low level, which was not detected by the peak-calling algorithm we used) at ‘Wapl only’ sites, consistent with our observation that the association of Wapl with chromatin depends on cohesin49 (Extended Data Fig. 1f). e, Examples of the distribution of Nipbl, Wapl and cohesin (Stag1 and Scc1) at two genomic regions, one on chromosome 11 and the other on chromosome 1, as determined by ChIP-seq analysis in WT MEFs.

Extended Data Figure 7 Cohesin islands in Ctcf Wapl DKO MEFs.

Binding of CTCF, Nipbl and cohesin (Stag1 and Scc1) at the convergently transcribed genes Mier1 and Slc35d1 (a) and Mllt1 and Dnajc1 (b) was determined by ChIP-seq analysis in WT MEFs, Ctcf, Smc3 and Wapl KO MEFs and Ctcf Wapl DKO MEFs. c, d, Time-course analysis of the appearance of cohesin islands upon Ctcf and Wapl deletion in Adeno-Cre infected MEFs. c, Scc1 accumulation in the 3′-region of the convergently transcribed Usp47 and Dkk3 genes at the indicated days after Adeno-Cre infection. d, Density profiles of Scc1 accumulation at convergently transcribed genes in response to Adeno-Cre infection. Scc1 binding was centred in the middle of the intervening region between gene pairs with a similarly strong transcription activity (TPM > 5). Data are shown for one experiment representative of two or three independent experiments (a, b; CTCF, Stag1 and Scc1 ChIP-seq) per genotype or one experiment (a, b; Nipbl ChIP-seq) and one experiment (c, d).

Extended Data Figure 8 Cohesin islands at isolated genes depend on gene transcription.

a, Similar transcriptional activity of individual genes in WT and Ctcf Wapl DKO MEFs. GRO-seq data are shown for three different gene regions in Ctcffl/fl Waplfl/fl MEFs before and after Adeno-Cre infection. b, Density profiles of Scc1 accumulation in WT and Ctcf Wapl DKO cells at all WT cohesin peaks (28,334) or at overlapping canonical peaks (10,390) identified by peak calling in WT and Ctcf Wapl DKO cells. c, The transcriptional activity determines the amount of cohesin accumulation in the 3′-region of isolated genes lacking a neighbouring downstream gene. Density profiles of Scc1 binding are shown for five groups of genes with decreasing GRO-seq signals (TPM values >9, 5–9, 3–5, 1–3 and <1). d, For better visualization of cohesin islands in Wapl KO cells, we restricted our analysis to genes that were >15 kb in length and highly expressed (TPM > 5). The genes were further filtered on the basis of the presence of a cohesin island in Ctcf Wapl DKO cells and the absence of any intragenic CTCF site (assuming that a CTCF site might prevent proper cohesin pushing along the DNA). Density profiles of Scc1 in WT (black), Wapl KO (green) and Ctcf Wapl DKO (turquoise) cells are shown. e, Examples of cohesin island formation in Wapl KO cells are shown for the A230046K03Rik/Appl2 locus. Data are shown for one GRO-seq experiment (a) per treatment and one representative experiment of two or three ChIP-seq experiments (be) per genotype.

Extended Data Figure 9 Transcriptional changes induced by serum stimulation affect positioning of cohesin islands.

a, c, e, g, i, Binding of Scc1 and mRNA profiling upon serum stimulation (20% fetal bovine serum) for 6 h at five genomic regions. The observed differences in cohesin island positioning correlate with increased or decreased mRNA levels of the respective genes as measured by their RPKM value. a, Capn2: 104.88 (0 h) → 248.32 (6 h); c, Dock5: 4.89 → 14.06; e, Abca1: 11.51 → 2.27; g, Pphln1: 5.13 → 8.00 and Prickle1: 14.19 → 7.86; i, Mast4: 7.70 → 16.68). b, d, f, h, j, Visualization of the altered shape of cohesin islands by plotting the cumulative sum of reads per nucleotide starting from −20 kb to +20 kb after the TTSs of Capn2 (b), Dock5 (d), Abca1 (f), Pphln1 (h) and Mast4 (j) genes (see Methods). Data are shown for one experiment.

Extended Data Figure 10 Transcriptional inhibition and disappearance of cohesin islands.

a, c, Loss of cohesin islands in response to transcriptional inhibition by actinomycin D (Act D; 5 μg ml−1) for 2.5 and 5 h followed by Scc1 profiling in Ctcf Wapl DKO cells. b, d, Disappearance and reappearance of cohesin islands in response to inhibition of RNA polymerase II elongation by DRB (100 μM) and its subsequent removal in Ctcf Wapl DKO cells, respectively. The same convergently transcribed gene pairs are shown for the Act D and DRB experiments (a, b, Arid2 and Scaf11; c, d, Ube2k and Pds5a). Note that Nipbl localization in Drosophila is not affected by transcriptional inhibitors50, implying that Nipbl-cohesin interactions may not be sufficient to explain cohesin accumulation at TSSs in DRB-treated cells. Data are shown for one experiment (ad). e, f, Speculative models of how transcription could move one19 (e) or two18 (f) cohesin rings to mediate loop extrusion.

Supplementary information

Supplementary Information

This file contains: Supplementary Figure 1, the source data for western blots, southern blot and DNA agarose gel; Supplementary Table 1, ChIP-seq experiments used in this study; Supplementary Table 2 RNA-seq and GRO-seq experiments used in this study and Supplementary Table 3, a list of Oligonucleotide primers. Supplementary Figures 1a, d, e are uncropped Western blot exposures for (a) Fig. 1a, (d) Extended Data Fig. 1e and (e) Extended Data Fig. 1g. Supplementary Figure 1b shows uncropped Southern blot exposure for Extended Data Fig. 1b and Supplementary Figure 1c shows uncropped agarose gel for Extended Data Fig. 1d. Supplementary Table 1 contains all ChIP-seq experiments used in this study. The number of peaks identified by MACS peak calling is shown for each ChIP-seq experiment, and the common peaks between replica experiments are indicated. In the “merged replica ChIP” column, the ChIP data of the indicated rows were combined into one dataset and analyzed as described in Online Methods. n.a., not applicable. Supplementary Table 2 contains all RNA-seq and GRO-seq data analyzed in the manuscript. (PDF 503 kb)

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Busslinger, G., Stocsits, R., van der Lelij, P. et al. Cohesin is positioned in mammalian genomes by transcription, CTCF and Wapl. Nature 544, 503–507 (2017). https://doi.org/10.1038/nature22063

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