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Cohesin is required for long-range enhancer action at the Shh locus

Nature Structural & Molecular Biology volume 29, pages 891–897 (2022)Cite this article

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Abstract

The regulatory landscapes of developmental genes in mammals can be complex, with enhancers spread over many hundreds of kilobases. It has been suggested that three-dimensional genome organization, particularly topologically associating domains formed by cohesin-mediated loop extrusion, is important for enhancers to act over such large genomic distances. By coupling acute protein degradation with synthetic activation by targeted transcription factor recruitment, here we show that cohesin, but not CTCF, is required for activation of the target gene Shh by distant enhancers in mouse embryonic stem cells. Cohesin is not required for activation directly at the promoter or by an enhancer located closer to the Shh gene. Our findings support the hypothesis that chromatin compaction via cohesin-mediated loop extrusion allows for genes to be activated by enhancers that are located many hundreds of kilobases away in the linear genome and suggests that cohesin is dispensable for enhancers located more proximally.

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Fig. 1: Synthetic Shh activation.
Fig. 2: Auxin-mediated degradation of CTCF and SCC1.
Fig. 3: Depletion of cohesin, but not of CTCF, inhibits distal enhancer-driven gene activation.
Fig. 4: Gene activation from a close enhancer is not affected by cohesin depletion.

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Data availability

The datasets generated during the current study are available from the corresponding author upon reasonable request. Publicly accessible data used were: Ensembl (r 45) (http://jun2007.archive.ensembl.org/Mus_musculus/index.html). Mouse genome assembly: NCBI m37 (mm9). NCBI GEO: GSE98671.Source data are provided with this paper.

Code availability

Analysis of Hi-C data was performed using https://github.com/open2c/distiller-nf

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Acknowledgements

We thank E. Nora (University of San Francisco, US) and J. Rhodes and Rob Klose (University of Oxford, UK) for their generous gifts of CTCF and SCC1-AID cell lines. We are grateful to the IGC FACs and Advanced Imaging facilities for their expert support. We acknowledge the following funding sources: UK Medical Research Council (MRC) University Unit programmes MC_UU_00007/2 (I. W., Y. K. and W. A. B.) and MC_UU_00007/8 (R. E. H. and L. A. L.); L. K. was supported by an MRC PhD studentship.

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Author notes

  1. Ilya M. Flyamer

    Present address: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

Authors and Affiliations

  1. MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK

    Lauren Kane, Iain Williamson, Ilya M. Flyamer, Yatendra Kumar, Robert E. Hill, Laura A. Lettice & Wendy A. Bickmore

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Contributions

W. A. B. and R. E. H. conceived the study. W. A. B., L. K., I. W., Y. K. and L. A. L. designed experiments. L. K. and I. W. performed experiments. I. M. F. analyzed Hi-C data. L. K, I. W and W. A. B. wrote the manuscript.

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Correspondence to Wendy A. Bickmore.

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Nature Structural and Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Carolina Perdigoto, in collaboration with the Nature Structural and Molecular Biology team.

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Extended data

Extended Data Fig. 1 Effect of auxin treatment on mESCs.

(a) Schematic of Shh and Lmbr1 genes showing the position of directly labelled Custom Stellaris® RNA FISH oligo probes used for RNA FISH. Shh probes were labelled with Quasar 670 and Lmbr1 probe with Quasar 570 (b) Images of representative nuclei showing RNA FISH signals for Shh (white) and Lmbr1 (red) probes from wild type mESCs transfected with tShh-VP64 or tShh-Δ, and either untreated (- auxin) or treated with 24 hours of auxin (+ auxin). Shh RNA FISH signal is indicated by white arrow. Scale bars = 5 μm. (c) Quantification of the percent of (left) Shh (pink and red bars) and (right) Lmbr1 – intron 1 (white and grey bars) expressing alleles in mESCs transfected with tShh-VP64, tSBE2-VP64 and tZRS-VP64 and equivalent TALE-Δ controls. Cells were either untreated (- auxin) or treated with 24 hours of auxin (+ auxin). Biological replicate of the data shown in Fig. 1f. The data were compared using a two-sided Fisher’s Exact Test, n = numer of alleles scored, ns – not significant. (d) Table showing two-sided Fisher’s Exact Test p-values for differences in the percent of Shh-expressing alleles in mESCs transfected with TALE-Vp64 or TALE-∆ constructs assayed by RNA FISH in the absence or presence of auxin. Data from Fig. 1f and Extended Data Fig. 1c. p-values in bold are significant (<0.05). (e) Quantification of live cells by DAPI staining during flow cytometry in untreated and auxin-treated wild type (WT), CTCF-AID and SCC1-AID cells after 6, 24 and 48 hours of growth in auxin. Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 Virtual 4 C following auxin mediated degradation of CTCF and SCC1.

Virtual 4 C plots obtained by extracting Hi-C interactions using the Shh promoter as a viewpoint (grey dashed line) from untreated (- auxin) and treated (+ auxin) (a) CTCF-AID mESCs (data are from ref. 6) or (b) SCC1-AID mESCs (data are from ref. 25). Gene track is shown above and yellow dashed lines indicate the position of enhancers SBE6, SBE2 and ZRS. The lowest panel shows a subtraction of untreated and treated cells with gain of interactions indicated in red and loss of interactions indicated in blue.

Extended Data Fig. 3 Replicate data for effect of CTCF or cohesin depletion on distal enhancer driven gene activation.

(a) Quantification of the percentage of (left axis) Shh and (right axis) Lmbr1 expressing alleles, assayed by RNA FISH, in TALE-transfected wild type mESCs (parental cell line used to generate the CTCF-AID cell line) and in CTCF-AID cells either untreated (- auxin) or treated with 24 hours of auxin (+ auxin). Cells were transfected with tShh-VP64, tSBE2-VP64 and tZRS-VP64 and equivalent TALE-Δ controls. Data shown are from an indepenent biological replicate of the experiment shown in Fig. 3a. The data were compared using a two-sided Fisher’s Exact Test, n = numer of alleles scored, ns – not significant. Source Data ED Fig. 3. (b) As for (a) but for SCC1-AID with 6 hours of auxin (+ auxin). Data shown are from an indepenent biological replicate of the experiment shown in Fig. 3b. **p < 0.01. Source Data ED Fig. 3. (c) Table showing two-sided Fisher’s Exact Test p-values for differences in the percent of Shh-expressing alleles in TALE-transfected CTCF-AID cells assayed by RNA FISH in the absence or presence of auxin. Cells were transfected with Shh-VP64, SBE2-VP64, ZRS-VP64, and equivalent TALE-∆ controls. Data from Fig. 3a and Extended Data Fig. 3a. p-values in bold are significant (<0.05). (d) As (c) but for SCC1-AID cells. Data from Fig. 3d and Extended Data Fig. 3b. (e) Table showing the two-sided Mann-Whitney U p-values for differences in FISH inter-probe distances, for Shh-SBE2, SBE2-ZRS and Shh-ZRS probe pairs, between the data from TALE-Vp64 transfected CTCF-AID or SCC1-AID ESCs with or without the addition of auxin. No of alleles scored is indicated (in parentheses). Data are from Fig. 3c and f. Median and inter-quartile distances are shown. p-values in bold are significant (<0.05).

Source data

Extended Data Fig. 4 Replicate data showing gene activation from a close enhancer is not affected by cohesin depletion.

(a) Percentage of (left axis) Shh and (right axis) Lmbr1 expressing alleles, assayed by RNA FISH, in TALE-transfected SCC1-AID cells either untreated (- auxin) or treated with 6 hours of auxin (+ auxin). Cells were transfected with tSBE6-VP64 or tSBE6-VP64 -Δ. Data shown are from one biological replicate. Data from an independent biological replicate are shown in Fig. 4a. The data were compared using a two-sided Fisher’s Exact Test, n = numer of alleles scored, ns – not significant. Source Data ED Fig. 4. (b) Table showing two-sided Fisher’s Exact Test p-values for differences in the percent of Shh-expressing alleles in TALE-transfected wild type ESCs and SCC1-AID cells assayed by RNA FISH in the absence or presence of auxin. Cells were transfected with SBE6-VP64 and SBE6-∆. Data from Fig. 4a and Extended Data Fig. 4a. p-values in bold are significant (<0.05). (c) Table showing the two-sided Mann-Whitney U p-values for differences in the Shh-SBE6 inter-probe distances between the data from SCC1-AID ESCs with or without the addition of auxin and transfected with either tSBE6-Vp64 or tSBE6-Δ. No of alleles scored is indicated (in parentheses). p-values in bold are significant (<0.05). Also shown are the median and interquartile distances of each data set. Data are from Fig. 4c.

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Kane, L., Williamson, I., Flyamer, I.M. et al. Cohesin is required for long-range enhancer action at the Shh locus. Nat Struct Mol Biol 29, 891–897 (2022). https://doi.org/10.1038/s41594-022-00821-8

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