Abstract
Cohesin is a DNA translocase that is instrumental in the folding of the genome into chromatin loops, with functional consequences on DNA-related processes. Chromatin loop length and organization likely depend on cohesin processivity, translocation rate and stability on DNA. Here, we investigate and provide a comprehensive overview of the roles of various cohesin regulators in tuning chromatin loop expansion in budding yeast Saccharomyces cerevisiae. We demonstrate that Scc2, which stimulates cohesin ATPase activity, is also essential for cohesin translocation, driving loop expansion in vivo. Smc3 acetylation during the S phase counteracts this activity through the stabilization of Pds5, which finely tunes the size and stability of loops in G2.
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Data availability
Accession number for processed data for all figures is GSE186987.
Sample description and raw sequences for all figures are accessible on SRA database through the following accession number: PRJNA715343.
Reference genomes:
S. cerevisiae W303: https://www.ncbi.nlm.nih.gov/assembly/GCA_002163515.1/;
strains of this study are available from the corresponding authors. Source data are provided with this paper.
Code availability
Programs involved in the study, are listed below. They include custom made, open source programs from the Koszul lab:
HiCstuff (www.github.com/koszullab/hicstuff) version 3.0.1;
Chromosight (www.github.com/koszullab/chromosight)36 version 1.4.1.
As well as programs published by others:
Bowtie2 (version 2.3.4.1 available online at http://bowtie-bio.sourceforge.net/bowtie2/);
Samtools (version 1.9 available online at http://www.htslib.org/download/);
Bedtools (version 2.29.1 available online at https://bedtools.readthedocs.io/en/latest/content/installation.html);
Cooler (version 0.8.7–0.8.11 available online at https://cooler.readthedocs.io/en/latest/);
hicreppy (version 0.0.6 available online at https://github.com/cmdoret/hicreppy);
seaborn (version 0.11.2 available online at https://github.com/mwaskom/seaborn);
pyGenomeTracks (version 3.3 https://github.com/deeptools/pyGenomeTracks);
bamCoverage (version 3.4.1 https://deeptools.readthedocs.io/en/develop/content/tools/bamCoverage.html).
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Acknowledgements
This research was supported by the European Research Council under the Horizon 2020 Program (ERC grant agreement 771813) to R. K., and the Fondation ARC pour la Recherche sur le Cancer (ARCPJA22020060002067) and the Comité de l’Occitanie de la Ligue Nationale contre le Cancer to F. B.. N. B. and L. D. were supported by the Ministère de l’Enseignement Supérieur et de la Recherche. C. C. was supported by Pasteur-Roux-Cantarini fellowship.
We thank all our colleagues from the laboratories régulation spatiale des génomes and organization du noyau, especially C. Matthey-Doret. We also thank M. Houlard, J. P. Javerzat and A. Piazza for helpful comments on the manuscript, K. Nasmyth’s laboratory and B. Albert for strains and K. Shirahige for the Smc3 antibody.
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N. B., C. C., F. B. and R. K. designed the research. N. B. and C. C. performed the experiments, with contributions from L. D. for the early stages of the project. C. C. analyzed the data. All authors interpreted the data. N. B., C. C., R. K. and F. B. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Control map and P(s) related to Fig1, along with HiC maps from cdc20 or nocodazole arrested WT cells.
a, Contact maps of chromosomes III and IV (bin 1kb) from metaphase arrested cells with Smc3 (SMC3 +IAA, yNB54-4). Contact maps are shown as a merge of two independent experiments. b, contact probability curves P(s) representing contact probability as a function of genomic distance (bp) and their respective derivative curves. Two independent replicates are shown. c, Contact maps of a part of chromosome IV (10kb to 510kb, bin 1kb) for cdc20 or nocodazole arrested WT (W303-1A) cells during 2h at 30 °C. Contact maps are shown as a merge of two independent experiments.
Extended Data Fig. 2 Eco1 is required to restrict DNA loop expansion only during S phase.
a, Illustration of the experimental protocol followed to deplete Eco1-AID during metaphase. b, Cell synchronization in G1 and metaphase were confirmed by flow cytometry. c, Western Blot assessing Eco1-PK-AID degradation with anti V5 antibody. Pgk1 was used as loading control. d, Contact maps of chromosomes III and IV (bin 1kb) for metaphase arrested WT (top, FB133-57B) and ECO1-AID (bottom, FB133-20c) cells before (left) and after (right) auxin (IAA) addition. Chromatin interactions along a region (300kb to 510kb) of the chromosome IV are represented in the black square. Contact maps are shown as a merge of two independent experiments. e, Contact probability curves P(s) representing contact probability as a function of genomic distance (bp) and their respective derivative curves for WT and ECO1-AID after auxin treatment. Two independent replicates are shown. f, Mean profile heatmap of loops called by chromosight. Mean score of two independent experiments ± standard deviation. g, Loop spectrum indicating scores in function of loop size. Loops were called on aggregated HiC maps from two independent experiments, subsampled to 20 million reads (see Supplementary Table 1 for replicated values and variance).
Extended Data Fig. 3 scc2-45 allele fully inactivates establishment of DNA loop (related to Fig. 4).
a, Schematic representation of the protocol followed to inactivate Scc2 during S phase. b, Cell synchronization was monitored by flow cytometry. c, Western blotting showing that inactivation of Scc2 prior to S phase induces loss of Smc3 acetylation. d, Contact maps of a part of chromosome V (100kb to 400kb) for WT (W303-1A) and scc2-45 (KN20751) cells arrested in mitosis at permissive temperature during 2h or arrested at restrictive temperature during 1h or 2h. Contact maps derived from 30 °C cells are shown as a merge of two independent experiments. e, Intrachromosomal contact probability as a function of genomic distance and their respective derivative curves for WT and scc2-45 cells arrested in mitosis at permissive temperature during 2h or arrested at restrictive temperature during 1h or 2h. Two independent replicates are shown. f, Mean profile heatmap of loops called by chromosight. Mean score of two independent experiments ± standard deviation. g, Loop spectrum indicating scores in function of loop size. Loops were called on aggregated HiC maps from two independent experiments, subsampled to 20 million reads (see Supplementary Table 1 for replicated values and variance).
Extended Data Fig. 4 scc2-45 allele inactivates cohesin loading at restrictive temperature in G2 (related to Fig. 4), along with HiC maps from double scc2-45 PDS5-PK-AID or triple scc2-45 PDS5-PK-AID scc3-K404 cells (related to Fig. 5).
a, Illustration of the experimental protocol followed to inactivate Scc2-45 at 37 °C and induce Scc1-HA expression in G2 by Oestradiol addition b, Western blotting showing Scc1-HA expression after temperature shift at 37 °C in G2 c, Calibrated ChIP seq profiles showing effect of scc2-45 on the distribution of Scc1-HA on chromosomes I and IV at 30 or 37 °C and after oestradiol induced expression of Scc1-HA in G2 for 40min. d, Hi-C contact matrices of a part (20kb to 520kb) of chromosome VII (bin = 2kb) for double scc2-45 PDS5-PK-AID or triple scc2-45 PDS5-PK-AID scc3-K404 cells at permissive 25 °C temperature.
Extended Data Fig. 5 Effect of Hos1 inactivation on chromatin interactions and effect of smc3-RR on Pds5 binding to DNA (related to Fig. 6).
a, Schematic representation of the protocol followed to synchronize Δhos1 cells and degrade Pds5 in G2. b, Cell synchronization in G1 and metaphase were confirmed by flow cytometry. c, Hi-C contact matrices of a part (10kb to 510kb) of chromosome IV (bin = 1kb) of WT (W303-1A) and hos1Δ (yNB40-1c) cells. Contact maps derived from untreated cells with IAA are shown as a merge of two independent experiments. d, Mean profile heatmap of loops called by chromosight. Mean score of two independent experiments ± standard deviation. e, Illustration of the experimental protocol used to process yeast cells containing either Scc1-PK or Pds5-PK from G1 to metaphase in the presence of Smc3-RR f, Calibrated ChIP seq profiles showing effect of smc3-RR on the distribution of Scc1-PK and Pds5-PK on chromosome I (two independent experiments). Ratios representing differences in protein occupancy are given.
Extended Data Fig. 6 HiC data reproducibility.
Cluster map showing the stratum adjusted correlation coefficients between contact maps and HiC replicates of a, Pds5-AID, b, Eco1-AID, c Smc3-AID, d, scc2-45 conditions.
Supplementary information
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Bastié, N., Chapard, C., Dauban, L. et al. Smc3 acetylation, Pds5 and Scc2 control the translocase activity that establishes cohesin-dependent chromatin loops. Nat Struct Mol Biol 29, 575–585 (2022). https://doi.org/10.1038/s41594-022-00780-0
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DOI: https://doi.org/10.1038/s41594-022-00780-0
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