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A cohesin traffic pattern genetically linked to gene regulation

Abstract

Cohesin-mediated loop extrusion has been shown to be blocked at specific cis-elements, including CTCF sites, producing patterns of loops and domain boundaries along chromosomes. Here we explore such cis-elements, and their role in gene regulation. We find that transcription termination sites of active genes form cohesin- and RNA polymerase II-dependent domain boundaries that do not accumulate cohesin. At these sites, cohesin is first stalled and then rapidly unloaded. Start sites of transcriptionally active genes form cohesin-bound boundaries, as shown before, but are cohesin-independent. Together with cohesin loading, possibly at enhancers, these sites create a pattern of cohesin traffic that guides enhancer-promoter interactions. Disrupting this traffic pattern, by removing CTCF, renders cells sensitive to knockout of genes involved in transcription initiation, such as the SAGA complexes, and RNA processing such DEAD/H-Box RNA helicases. Without CTCF, these factors are less efficiently recruited to active promoters.

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Fig. 1: Active promoters/TSSs form CTCF-independent chromatin domain boundaries.
Fig. 2: Active TTSs form chromatin boundaries, and a cohesin traffic pattern defines promoter-enhancer interactions.
Fig. 3: Rewiring of enhancer-promoter interactions after CTCF depletion.
Fig. 4: The cohesin traffic pattern is genetically linked to gene regulation factors.
Fig. 5: Altered cohesin traffic pattern following CTCF depletion reduces chromatin binding of DDX55 and TAF5L at active promoters/TSSs.
Fig. 6: DDX55 and TAF5L depletions alter the conformation of active genes independently of CTCF.

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Data availability

The datasets generated in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO SuperSeries accession no. GSE180691. This SuperSeries is composed of the following SubSeries: GSE180922 (Hi-C), GSE180713 (RNA-seq), GSE180690 (ChIP-seq) and GSE180657 (CRISPR screen). The following published datasets were used in this study (Supplementary Table 7): GSE72800, GSE110133, GSE70189, GSE104334, GSE104888, GSE95015; ENCODE, https://www.encodeproject.org/experiments/ENCSR131DVD/; ENCODE, https://www.encodeproject.org/experiments/ENCSR620QNS/; ENCODE, https://www.encodeproject.org/files/ENCFF176NSX/@@download/ENCFF176NSX.bigWig; ENCODE, https://www.encodeproject.org/files/ENCFF364QXM/. Data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

Open2C scripts and notebooks used in this study are publicly available in GitHub: https://github.com/open2c and https://github.com/dekkerlab/ALV-repo.git. No other customized codes were developed for this study.

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Acknowledgements

We thank members of the Dekker and the Mirny laboratories as well as members of the Open Chromosome Collective for creating a collaborative atmosphere and insightful discussions. We thank the Flow Cytometry Core Facility for FACS sorting the cell lines, and the Deep Sequencing Core for the sequencing at the University of Massachusetts Chan Medical School. We thank C. Navarro for help with editing the manuscript. We thank M. Kanemaki (National Institute of Genetics, Mishima, Japan) for sharing the HCT116-RAD21-AID cell line. We thank E. Nora and B. Bruneau (Gladstone Institutes, San Francisco, CA, USA) for sharing plasmids. This work was supported by a grant from the National Human Genome Research Institute (NHGRI) to J.D. (HG003143) and a grant from the National Institute of General Medical Sciences (NIGMS) to A.A.P. (GM133762). J.D. is an investigator of the Howard Hughes Medical Institute. Some of the schematic figures were created with BioRender.com.

Author information

Authors and Affiliations

Authors

Contributions

A.-L.V. and J.D. conceived and designed the study. A.-L.V. engineered cell lines, performed Hi-C, ChIP-seq and all the other experiments. A.-L.V. and S.V.V. analyzed Hi-C, ChIP-seq, RNA-seq and other relevant datasets. A.-L.V., B.M., A.H.Y.T. and J.M. designed the strategy for the CRISPR screens. K.C. generated the lentiviruses for the CRISPR screens. A.-L.V. and B.M. performed the CRISPR screens. A.-L.V., B.M. and M.U. analyzed the CRISPR screen data. E.S.K. and A.A.P. analyzed splicing in the RNA-seq data. A.-L.V. and J.D. wrote the manuscript with input from all the authors.

Corresponding author

Correspondence to Job Dekker.

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The authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Daniel Ibrahim and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Carolina Perdigoto, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Characterization of the HAP1-CTCFdegron cell lines.

a, Schematic representing the strategy used to construct the HAP1-CTCFdegron-TIR1 cells. b, Schematic representing CTCF depletion in HAP1-CTCFdegron-TIR1 cells (top). Western blot against CTCF in HAP1-CTCFdegron cells and in HAP1-CTCFdegron-TIR1 cells without/with auxin (NT and IAA). Ponceau is shown for loading control (bottom). c, Flow cytometry for GFP in HAP1-CTCFdegron and HAP1-CTCFdegron-TIR1 cells without/with auxin. d, Flow cytometry for HAP1-CTCFdegron-TIR1 cells without/with auxin stained with Propidium Iodide (PI) to assess the DNA content for cell cycle analysis. e, Hi-C contact frequency as a function of genomic distance, P(s) (top) and its derivative dP/ds (bottom) for HAP1-CTCFdegron and HAP1-CTCFdegron-TIR1 cells without/with auxin. f, Hi-C contact heatmaps at 250kb resolution with the corresponding track of the first Eigenvector (EV1) across chromosome 15 for HAP1-CTCFdegron and HAP1-CTCFdegron-TIR1 cells without/with auxin. g, Genome-wide saddle plots of Hi-C data binned at 100kb resolution for HAP1-CTCFdegron and HAP1-CTCFdegron-TIR1 cells without/with auxin. The compartment strengths are indicated in the corners. h, Hi-C contact heatmaps at 50kb resolution for a 7Mb region on chromosome 10 for HAP1-CTCFdegron and HAP1-CTCFdegron-TIR1 cells without/with auxin. i, Differential interaction heatmaps for HAP1-CTCFdegron and HAP1-CTCFdegron-TIR1 cells. j, Dot pileups for dots characterized in HAP1 cells that have a CTCF peak in either anchor in the Non-Treated sample (4,496 dots) and that have a CTCF peak in both anchors in the auxin sample (1,545 dots) for HAP1-CTCFdegron and HAP1-CTCFdegron-TIR1 cells without/with auxin. The dots were aggregated at the center of a 100kb window at 2kb resolution.

Source data

Extended Data Fig. 2 CTCF sites and active TSSs are chromatin boundaries.

a, Stackups for CTCF-dependent (blue) and promoter/TSS (orange) categories sorted on the change of the first Eigenvector (EV1, 25kb) from left to right flank. CTCF and RAD21 ChIP-seq, calculated insulation and EV1 in HAP1-CTCFdegron-TIR1 cells without/with auxin (NT and IAA) were plotted. b, Stackups for active TSSs, sorted on RAD21 ChIP-seq signal. CTCF and RAD21 ChIP-seq, calculated insulation and RNAseq in the HAP1-CTCFdegron-TIR1 cells without/with auxin were plotted along with the published HAP1 H3K4me3 ChIP-seq signals26. Stackups were flipped according to the orientation of the genes, to have the gene body at the right of the TSSs. c, Dot pileup aggregation plots for remaining CTCF motif orientations represented in Fig. 1e for a 100kb window at 2kb resolution. With orientation (top). CTCF (upstream or downstream)-TSS pairwise interactions are plotted with their quantifications (mean of the 5 central bins at the CTCF site). Nearest analysis (bottom). CTCF (upstream or dowstream)-TSS pairwise interactions are plotted without any CTCF peaks or TSSs in between them with their quantifications (mean of the 5 central bins at the CTCF site). The black arrows represent the CTCF motif and the direction of the arrow, the motif orientation. The double arrows represent the TSSs and the direction of the arrow, the TSSs orientation.

Source data

Extended Data Fig. 3 CTCF, RAD21, WAPL and RNA polII depletion effects on the three types of chromatin boundaries.

a, Oriented interaction pileups for HAP1-CTCFdegron-TIR1, without/with auxin (NT and IAA), aggregated in a 200kb window at 2kb resolution were plotted for the active TTS (without CTCF) with or without R-loops. The black circle on a stick represents the TTS and the gene body is represented by a dash line. b, Stackups for active TTS (without CTCF, with R-loops), sorted on the change of first Eigenvector (EV1, 25kb) signal from left to right flank. RAD21 ChIP-seq levels, calculated insulation and EV1 in HAP1-CTCFdegron-TIR1 cells without/with auxin were plotted along with the published HAP1 H3K4me3 ChIP-seq signals26 and the consensus list of R-loops. c, Stackups for the three categories of insulation: the CTCF peaks (without TSSs, with RAD21) (blue), the active TSSs (orange) and TTSs (green) common between HAP1 and HCT116 cell lines, sorted on the NT RAD21 ChIP-seq signal. The CTCF and RAD21 ChIP-seq signals, the RNA-seq levels and the insulation for the described cell line and condition were plotted along with the published K562 R-loops67 and the HAP126 and HCT116 H3K4me3 ChIP-seq signals. Stackups were flipped according to the orientation of the genes, to have the gene body on the right for the TSSs and on the left of the TTSs. The red dashed rectangle indicates the zoom in Extended Data Fig. 3d. d, Active TSSs common between HAP1 and HCT116 cell lines (without CTCF) were plotted with a different scale to show the remaining RAD21 after RAD21 depletion (red dashed rectangle). e, Average insulation profiles across scaled inactive genes without CTCF at TSSs and TTSs at 5kb resolution for all the Hi-C libraries plotted in Fig. 2b.

Source data

Extended Data Fig. 4 Characterization of the HAP1-RPB1-AID cell line.

a, Schematic of the HAP1-RPB1-AID construct b, Western blot against RPB1 in WT HAP1 cells and HAP1-RPB1-AID cells showing RPB1 depletion after 4 hours of auxin treatment (IAA). Ponceau is shown for loading control. Flow cytometry for HAP1-RPB1-AID cells without/with auxin (NT and IAA) stained with Propidium Iodide (PI) to assess the DNA content for cell cycle analysis. c, Hi-C contact frequency as a function of genomic distance, P(s) (top) and its derivative dP/ds (bottom) for HAP1 wild-type cells and HAP1-RPB1-AID cells in absence and presence of auxin. d, Hi-C contact heatmaps at 100kb resolution with the corresponding track of the first Eigenvector (EV1) across a 60Mb region on chromosome 2 for HAP1 and HAP1-RPB1-AID cells without/with auxin. Genome-wide saddle plots of Hi-C data binned at 100kb resolution for HAP1 and HAP1-RPB1-AID cells without/with auxin. The compartment strengths are indicated in the corners. e, Hi-C contact heatmaps at 25kb resolution with the corresponding distribution of EV1 and published RPB1 ChIP-seq signal71 for a 10Mb region on chromosome 14 for HAP1 and HAP1-RPB1-AID cells without/with auxin. The differential interaction heatmap (presence/absence of auxin) is shown on the bottom. Dot pileups for dots found in HAP1 cells that have a CTCF peak in either anchor in the CTCF degron NT sample (4,496 dots) for HAP1-RPB1-AID cells without/with auxin. The dots were aggregated at the center of a 100kb window at 2kb resolution. f, Oriented interaction pileups for HAP1-RPB1-AID, without/with auxin, aggregated in a 200kb window at 2kb resolution were plotted for the active TTS with or without R-loops. The black circle on a stick represents the TTS and the gene body is represented by a dash line on the left of the TTS.

Source data

Extended Data Fig. 5 Genome wide CRISPR screen in context of altered cohesin traffic pattern following CTCF depletion.

a, Western blot against CTCF in HAP1-CTCFdegron and HAP1-CTCFdegron-TIR1 cells without/with auxin (NT and IAA) with the two different auxin concentrations (25μM and 500μM) used in the screen showing the partial CTCF depletion with 25μM IAA and the nearly total CTCF depletion with 500μM IAA. Ponceau is shown for loading control. b, Plot showing the cumulative number of doublings relative to the days cells were passaged during the screen for HAP1-CTCFdegron and HAP1-CTCFdegron-TIR1 for the three auxin concentrations (NT, 25μM and 500μM). c, Fold change distribution of essential and nonessential gene sets at indicated time points for the screens in HAP1-CTCFdegron and HAP1-CTCFdegron-TIR1 cells for the three auxin concentrations (left). Precision-recall curves based on Bayes Factors (BFs) of predefined essential and non-essential gene sets for the screens for HAP1-CTCFdegron and HAP1-CTCFdegron-TIR1 cells for the three auxin concentrations at the indicated time points (right). d, Scatter plots of the log2FC for HAP1-CTCFdegron-TIR1 screens against HAP1-CTCFdegron screens for T6 and T15 time points. Genes with a fold change of ≥ 2 between HAP1-CTCFdegron and HAP1-CTCFdegron-TIR1 screens are highlighted in red. Genes linked to CTCF are indicated (cohesin, TOP2A) along with the two studied gene hits (DDX55 and TAF5L). e, Scatter plots of the log2FC for HAP1-CTCFdegron cells without/with auxin at T6 and T15 time points. Genes with a fold change of ≥ 2 between absence and presence of auxin are highlighted in red and considered as auxin specific genes. Genes linked to CTCF are indicated (cohesin genes, TOP2A) along with the two studied gene hits (DDX55 and TAF5L).

Source data

Extended Data Fig. 6 DDX55 and TAF5L chromatin binding.

a, Western blot co-IP replicates against DDX55 and TAF5L in HAP1-CTCFdegron-TIR1 without/with auxin (NT and IAA), treated with either turbonuclease (DNA - and RNA -) or RNAseA (RNA -) for CTCF, cohesin (RAD21 and SMC1A), DDX55 and TAF5L. b, Western blot co-IP replicates against DDX55 and TAF5L in HCT116-RAD21-AID cells without/with auxin, treated with either turbonuclease or RNAseA for CTCF, DDX55 and TAF5L. c, Western blot co-IP (two replicates) against TAF6L in HAP1-CTCFdegron-TIR1 cells without/with auxin, treated with either turbonuclease or RNAseA for CTCF, cohesin (RAD21 and SMC1A) and TAF6L. d, Representative western blots against CTCF, RAD21, DDX55 and β-ACTIN (loading control) showing the CTCF depletion efficiency in the HAP1-CTCFdegron-TIR1 co-IP (left) and the RAD21 depletion efficiency in the HCT116-RAD21-AID cells co-IP (right). e, Efficient DNA digestion by turbonuclease (TURBO) during DDX55, TAF5L and TAF6L co-IPs. DNA digestion was assessed by qPCR using primers specific to ACTB and POLR2A gene locations. Data are presented as mean values ± SD, n = 8 biologically independent co-IP experiments. f, Efficient RNA digestion by turbonuclease (TURBO) and RNaseA (RNASEA) during DDX55, TAF5L and TAF6L co-IPs. RNA digestion was assessed by qPCR using primers specific to ACTB and POLR2A genes. Data are presented as mean values ± SD, n = 8 biologically independent co-IP experiments. g, Stackups for CTCF (without TSSs, with RAD21), sorted on the Non-Treated (NT) DDX55 ChIP-seq signal. CTCF ChIP-seq, RAD21 ChIP-seq, calculated insulation, DDX55 ChIP-seq, TAF5L ChIP-seq and RNA seq signals in HAP1-CTCFdegron-TIR1 cells without/with auxin were plotted along with published HAP1 H3K4me3 ChIP-seq26. For TTSs, stackups were flipped according to the orientation of the genes, to have the gene body on the left of the TTS. h, Stackup quantification for CTCF (without TSSs, with RAD21) for DDX55 and TAF5L ChIP-seq for two replicates. The distribution of ratios between auxin-treated and non-treated signals is shown. A fold change < 1 represents less binding of DDX55 or TAF5L at CTCF (without TSSs, with RAD21) after CTCF depletion.

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Extended Data Fig. 7 Overview of the different genome-wide data generated in this study, ChIP-seq replicate 1.

a, A genomic region on chr1 with many CTCF and RAD21 peaks. b, A genomic region on chr1 with a DDX55 peak at an active promoter which is reduced after CTCF depletion (red arrow). c, A genomic region in chr1 with a TAF5L peak at an active promoter which is reduced after CTCF depletion (red arrow). Genes on the forward strand are represented in red (plus) and genes on the reverse strand are represented in blue (minus). Hi-C contact heatmaps are binned at 2kb resolution.

Extended Data Fig. 8 Overview of the different genome-wide data generated in this study, ChIP-seq replicate 2.

a, A genomic region on chr1 with many CTCF and RAD21 peaks. b, A genomic region on chr1 with a DDX55 peak at an active promoter which is reduced after CTCF depletion (red arrow). c, A genomic region in chr1 with a TAF5L peak at an active promoter which is reduced after CTCF depletion (red arrow). Genes on the forward strand are represented in red (plus) and genes on the reverse strand are represented in blue (minus). Hi-C contact heatmaps are binned at 2kb resolution.

Extended Data Fig. 9 Characterization of DDX55 and TAF5L depletions in HAP1-CTCFdegron-TIR1 cells.

a, Western blots against CTCF, DDX55, TAF5L and β-ACTIN (loading control) showing DDX55 and TAF5L depletions, by siRNA and mutations in DDX55 and TAF5L genes, compared to siRNA controls and mutations at the AAVS1 non-coding sequence (CTRL clone). b, RNA-seq expression (TPM) for HAP1-CTCFdegron-TIR1 cells without/with auxin (NT and IAA) treated with siRNA (siCTRL, siDDX55 and siTAF5L) and CTRL, DDX55 and TAF5L clones for key genes (DDX55, TAF5L, CTCF, RAD21, SMC1A, SMC3, WAPAL, NIPBL, STAG1 and STAG2). c, Flow cytometry for HAP1-CTCFdegron-TIR1 cells without/with auxin (NT and IAA) with siRNA (siCTRL (control), siDDX55 and siTAF5L) (top) and CTRL (control), DDX55 and TAF5L clones (bottom) stained with Propidium Iodide (PI) to assess the DNA content for cell cycle analysis.

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Extended Data Fig. 10 Effect of DDX55 and TAF5L depletions on Hi-C and RNA-seq.

a, Hi-C contact frequency as a function of genomic distance P(s) and its derivative dP/ds for HAP1-CTCFdegron-TIR1 cells without/with auxin (NT and IAA) treated with siRNA (siCTRL, siDDX55 and siTAF5L) (top) and CTRL, DDX55 and TAF5L clones (bottom). b, Genome-wide saddle plots of Hi-C data binned at 100kb resolution for HAP1-CTCFdegron-TIR1 cells without/with auxin (NT and IAA) treated with siRNA (siCTRL, siDDX55 and siTAF5L) (left) and CTRL, DDX55 and TAF5L clones (right). The compartment strengths are indicated in the corners. c, Number of Differentially Expressed genes in HAP1-CTCFdegron-TIR1 cells without/with auxin (NT and IAA) treated with siRNA (siCTRL, siDDX55 and siTAF5L) and CTRL, DDX55 and TAF5L clones. Gray bars indicate the CTCF depletion, blue bars indicate the siRNA depletions and red bars indicate the clones. d, Number of alternatively spliced genes in HAP1-CTCFdegron-TIR1 cells in absence and presence of auxin treated with siRNA (siCTRL, siDDX55 and siTAF5L) and CTRL, DDX55 and TAF5L clones. Gray bars indicate the CTCF depletion, blue bars indicate the siRNA depletions and red bars indicate the clones.

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Supplementary Methods and Supplementary Fig. 1.

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Supplementary Tables 1–7

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Valton, AL., Venev, S.V., Mair, B. et al. A cohesin traffic pattern genetically linked to gene regulation. Nat Struct Mol Biol 29, 1239–1251 (2022). https://doi.org/10.1038/s41594-022-00890-9

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