Opposing chromatin remodelers control transcription initiation frequency and start site selection

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

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BEING VIEWED BY OTHERS ENHANCER–PROMOTER CONTACT FORMATION REQUIRES RNAPII AND ANTAGONIZES LOOP EXTRUSION Article 03 April 2023 RULER ELEMENTS IN CHROMATIN REMODELERS SET NUCLEOSOME ARRAY

SPACING AND PHASING Article Open access 28 May 2021 RNA POLYMERASE II DYNAMICS SHAPE ENHANCER–PROMOTER INTERACTIONS Article 10 July 2023 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

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references 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. AUTHOR INFORMATION Author notes * These authors contributed equally: Maria Jessica Bruzzone, Drice Challal AUTHORS AND AFFILIATIONS *

Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), Geneva, Switzerland Slawomir Kubik, Maria Jessica Bruzzone, Stefano Mattarocci & David Shore *

Institut Jacques Monod, CNRS-Université Paris Diderot, Paris, France Drice Challal & Domenico Libri * Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland René Dreos 

& Philipp Bucher Authors * Slawomir Kubik View author publications You can also search for this author inPubMed Google Scholar * Maria Jessica Bruzzone View author publications You can

also search for this author inPubMed Google Scholar * Drice Challal View author publications You can also search for this author inPubMed Google Scholar * René Dreos View author publications

You can also search for this author inPubMed Google Scholar * Stefano Mattarocci View author publications You can also search for this author inPubMed Google Scholar * Philipp Bucher View

author publications You can also search for this author inPubMed Google Scholar * Domenico Libri View author publications You can also search for this author inPubMed Google Scholar * David

Shore View author publications You can also search for this author inPubMed Google Scholar 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. CORRESPONDING AUTHOR Correspondence to David Shore. ETHICS DECLARATIONS COMPETING INTERESTS

The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION: Beth Moorefield was the primary editor on this article and managed its editorial process and peer

review in collaboration with the rest of the editorial team. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional

affiliations. 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.

SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figures 1–7 REPORTING SUMMARY SUPPLEMENTARY TABLE 1 _K_-means clustering of ChEC-seq data SUPPLEMENTARY TABLE 2 GO of

clusters SUPPLEMENTARY TABLE 3 Strain list SOURCE DATA SOURCE DATA FIG. 2C SOURCE DATA FIG. 2D RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 01 February 2019 * Accepted: 28 June 2019 * Published: 05 August 2019 * Issue Date: August 2019 * DOI:

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