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Opposing chromatin remodelers control transcription initiation frequency and start site selection

Nature Structural & Molecular Biology volume 26, pages 744–754 (2019)Cite this article

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Abstract

Precise nucleosome organization at eukaryotic promoters is thought to be generated by multiple chromatin remodeler (CR) enzymes and to affect transcription initiation. Using an integrated analysis of chromatin remodeler binding and nucleosome occupancy following rapid remodeler depletion, we investigated the interplay between these enzymes and their impact on transcription in yeast. We show that many promoters are affected by multiple CRs that operate in concert or in opposition to position the key transcription start site (TSS)-associated +1 nucleosome. We also show that nucleosome movement after CR inactivation usually results from the activity of another CR and that in the absence of any remodeling activity, +1 nucleosomes largely maintain their positions. Finally, we present functional assays suggesting that +1 nucleosome positioning often reflects a trade-off between maximizing RNA polymerase recruitment and minimizing transcription initiation at incorrect sites. Our results provide a detailed picture of fundamental mechanisms linking promoter nucleosome architecture to transcription initiation.

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Fig. 1: CRs bind in defined combinations.
Fig. 2: CRs display three broad types of activity.
Fig. 3: CRs with similar activities act redundantly.
Fig. 4: Position of +1 nucleosome results from the net activity of multiple cooperating and opposing CRs.
Fig. 5: Changes in +1 nucleosome occupancy are linked to transcriptional down- and upregulation.
Fig. 6: +1 nucleosome shift interferes with transcription start site selection.
Fig. 7: ISW2 and INO80 promote upstream +1 nucleosome movement, leading to suppression of alternative TSSs driven by cryptic TATA elements.

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Data availability

All sequencing data generated in this study were submitted to the GEO database under accession code GSE115412 (for ChEC-seq, MNase-seq and ChIP-seq) and Series GSE114589 (TSS-seq). Source data for Fig. 2c and 2d are available online.

Code availability

Peak-calling software is available at https://gitlab.unige.ch/JLFalcone/peakmatic.

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Acknowledgements

We thank M. Docquier and the iGE3 Genomics Platform (https://ige3.genomics.unige.ch/) at the University of Geneva for high-throughput sequencing services, N. Roggli for expert assistance with data presentation and artwork, B. Albert for help with fluorescence microscopy, and all members of the Shore lab for comments and discussions throughout the course of this work. M.J.B. was supported in part by an iGE3 PhD student fellowship. D.C. was supported by a fellowship from the Ligue Contre le Cancer. D.L. acknowledges support from the Centre National de la Recherche Scientifique (CNRS), the Fondation pour la Recherche Medicale (FRM, programme équipes 2013), l’Agence National pour la Recherche (ANR, grant ANR-16-CE12-0022-01 to D.L.), and the Labex Who Am I? (ANR-11-LABX-0071 et Idex ANR-11-IDEX-0005-02 to D.L.). D.S. acknowledges funding from the Swiss National Science Foundation (grant no. 31003A_170153) and the Republic and Canton of Geneva.

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  1. These authors contributed equally: Maria Jessica Bruzzone, Drice Challal

Authors and Affiliations

  1. Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), Geneva, Switzerland

    Slawomir Kubik, Maria Jessica Bruzzone, Stefano Mattarocci & David Shore

  2. Institut Jacques Monod, CNRS-Université Paris Diderot, Paris, France

    Drice Challal & Domenico Libri

  3. Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

    René Dreos & Philipp Bucher

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Contributions

Conceptualization: S.K., D.C., M.J.B., D.L. and D.S.; formal analysis: S.K., D.C., M.J.B., R.D., P.B., D.L. and D.S.; investigation: S.K., D.C., M.J.B. and S.M.; data curation: S.K., D.C., M.J.B., R.D., D.L. and P.B.; writing of original draft: S.K. and D.S.; funding acquisition: D.S., D.L. and P.B.; resources: D.S., D.L. and P.B.; supervision: D.S., D.L. and P.B.

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Correspondence to David Shore.

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Integrated supplementary information

Supplementary Figure 1 Characterization of remodeler binding by ChEC-seq.

a, Growth assays (serial dilution “spot assays”) of the indicated MNase-tagged chromatin remodeler strains on YPAD medium, compared to the wild-type parent strain (WT). Plates were photographed following 24 or 48 hrs incubation at 30oC. b, Distribution of identified remodeler binding sites between different genomic features. c, Heatmaps displaying ChEC signal for every CR centered on the +1 nucleosome of 5,040 protein-coding genes, sorted (top to bottom) by decreasing signal calculated in a region -250 to -50 bp from +1 nucleosome dyad. d, Grid representing Pearson correlation coefficients between ChEC signal for different CRs calculated at all identified remodeler binding sites. e, Plots displaying average normalized ChEC signal for each CR calculated for all clusters. f, Average nucleosome occupancy in each cluster. g, Boxplots displaying expression of genes belonging to each cluster measured either as RNAPII ChIP-seq signal in the gene body or NET-seq signal. h, Fraction of genes in each cluster which contain a canonical TATA-box, dashed line indicates genomic average (~17%).

Supplementary Figure 2 Verification and characterization of remodeler depletion and effects on nucleosome occupancy and stability.

a, Fluorescence microscopy of cells bearing FRB-GFP fusions of Sth1, Snf2, Isw2 and Chd1; cells were treated with rapamycin for indicated times, fixed and stained with DAPI. b, Western blotting (anti-myc antibodies) of cell lysates from an untagged strain and strains bearing Ino80-AID*-myc and Isw1-AID*-myc fusions treated with auxin for indicated amount of time. c, Growth assays (serial dilution “spot assays”, as in Supplementary Fig. 1a) of the indicated anchor-away or AID depletion strains on YPAD medium. “WT” indicates the parental anchor-away and/or AID strains background. d, Pearson correlations for all pairwise comparisons of genome-wide nucleosome occupancy change over 100 bp windows for the indicated CR depletion strains in the absence of depletion (mock-treated). e, Screenshots of sample regions in which nucleosomes become stabilized (marked with a red rectangle) upon depletion of RSC (top) or SWI-SNF (bottom). f, Average plots of nucleosome occupancy for all nucleosomes becoming stabilized upon depletion of RSC (top) or SWI-SNF (bottom). g, Screenshots of sample regions in which nucleosomes become destabilized (marked with a red rectangle) upon depletion of ISW2 (top) or INO80 (bottom). h, Average plots of nucleosome occupancy for all nucleosomes destabilized upon depletion of ISW2 (top) or INO80 (bottom). i,j, Average plots of nucleosome occupancy with (red) or without (blue) depletion of ISW1 (i) or CHD1 (j), plotted separately for genes with the lowest (top) or highest (bottom) binding by the relevant CR (average binding profiles shown in green).

Supplementary Figure 3 Remodeler redundancy in nucleosome positioning.

a, Nucleosome occupancy change upon depletion of RSC (left) or SWI-SNF (right) at sites bound by each remodeler and displaying varying binding signal (+, +/-, -) of the other one. b, Nucleosome occupancy change upon depletion of ISW2 at sites bound by this remodeler and displaying varying binding signal of INO80 (as in (a)). c, Nucleosome occupancy change upon depletion of INO80 at sites bound by this remodeler and displaying varying binding signal of ISW2 (as in (a)). d, Boxplot of INO80 binding signal at INO80-bound sites displaying varying binding signal of ISW2. In (a-d), asterisks indicate significant differences (p<0.05, Mann-Whitney test). e, Snapshot of a sample genomic region displaying nucleosome occupancy change upon depletion of ISW1, CHD1 or both remodelers simultaneously.

Supplementary Figure 4 Multiple concordant and opposing remodeler activities control promoter nucleosome occupancy.

a, Nucleosome occupancy change upon RSC depletion at sites bound by RSC (left) and change upon SWI-SNF depletion at sites bound by SWI-SNF (right). In both cases comparisons are made between sites co-bound by ISW2 (+) or not (-). Asterisk indicates significant difference (p<0.05, Mann-Whitney test). b, Nucleosome occupancy change upon INO80 depletion at sites bound by INO80 and co-bound by RSC and/or SWI-SNF or not, as indicated below. c, Average plots of nucleosome occupancy for all nucleosomes destabilized upon depletion of ISW2, comparing wild-type cells and cells depleted of RSC, ISW2, or both remodelers simultaneously. d, Average plots of nucleosome occupancy for all nucleosomes destabilized upon depletion of INO80, comparing for wild-type cells and cells depleted of RSC, INO80, or both remodelers simultaneously. e, Average plots of nucleosome occupancy for all nucleosomes stabilized upon depletion of RSC, comparing wild-type cells and cells depleted of RSC, RSC and ISW2, RSC and INO80, or all three remodelers simultaneously.

Supplementary Figure 5 Links between nucleosome occupancy and transcriptional regulation.

a, Scatterplot showing relationship between TBP binding change at gene promoters and RNAPII binding change in corresponding gene bodies following SWI-SNF depletion, for all genes with a well-defined TSS; Pearson R value shown. b, Scatterplot showing relationship between TBP binding and nucleosome occupancy changes following SWI-SNF depletion at down-regulated genes. c, Average plots displaying nucleosome occupancy, RNAPII and TBP ChIP signals, in the presence and absence of SWI-SNF, for those genes down-regulated upon SWI-SNF depletion. d-f, Average plots displaying nucleosome occupancy together with RNAPII and TBP ChIP-seq signals for genes up-regulated by INO80 depletion (d), down-regulated by INO80 depletion (e), or up-regulated by ISW2 depletion (f), in each case with or without (+/-) the indicated CR. g, Scatter plot displaying the relationship between TBP binding and nucleosome occupancy changes following double “puller” depletion at genes where transcription was affected (see Fig. 5b, c). h, Scatterplot displaying the TBP - RNAPII ChIP-seq signal relationship at all genes following double “puller” depletion. i-k, Average plots displaying nucleosome occupancy together with RNAPII and TBP ChIP-seq signals for genes down-regulated upon ISW1 depletion (i), up-regulated upon ISW1 depletion (j), or up-regulated upon CHD1 depletion (k), in each case with or without (+/-) the indicated CR.

Supplementary Figure 6 Effects of remodeler depletion on TSS selection.

a, Snapshot of genomic region showing 5’-RACE signal for the Watson (w) and the Crick (c) strands as well as nucleosome occupancy in the presence and absence of RSC; upon RSC depletion the RPO49 gene transcription initiates more upstream comparing to wild-type conditions. b, As in (a) but for SWI-SNF depletion; upon SWI-SNF depletion the SNQ2 gene transcription initiates more frequently downstream comparing to wild-type conditions; additionally, there is more initiation events in the opposite strand just downstream from the upstream-most SNQ2 TSS. c, As in (a) but for ISW2 and INO80 simultaneous depletion; upon “pullers” depletion the FRP6 gene transcription initiates at a downstream position comparing to wild-type conditions. d, Average 5’-RACE signal at genes upregulated upon simultaneous depletion of ISW2 and INO80. e, As in (d) but for downregulated genes.

Supplementary Figure 7 Effect of “puller” remodelers on nucleosome occupancy.

Heatmaps showing nucleosome occupancy, centered at TSS positions determined in cells depleted of ISW2 and INO80, shown in wild type cells and cells depleted of both remodelers.

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Supplementary Table 1

K-means clustering of ChEC-seq data

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GO of clusters

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Strain list

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Kubik, S., Bruzzone, M.J., Challal, D. et al. Opposing chromatin remodelers control transcription initiation frequency and start site selection. Nat Struct Mol Biol 26, 744–754 (2019). https://doi.org/10.1038/s41594-019-0273-3

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