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Measurement of histone replacement dynamics with genetically encoded exchange timers in yeast

Abstract

Histone exchange between histones carrying position-specific marks and histones bearing general marks is important for gene regulation, but understanding of histone exchange remains incomplete. To overcome the poor time resolution of conventional pulse–chase histone labeling, we present a genetically encoded histone exchange timer sensitive to the duration that two tagged histone subunits co-reside at an individual genomic locus. We apply these sensors to map genome-wide patterns of histone exchange in yeast using single samples. Comparing H3 exchange in cycling and G1-arrested cells suggests that replication-independent H3 exchange occurs at several hundred nucleosomes (<1% of all nucleosomes) per minute, with a maximal rate at histone promoters. We observed substantial differences between the two nucleosome core subcomplexes: H2A-H2B subcomplexes undergo rapid transcription-dependent replacement within coding regions, whereas H3-H4 replacement occurs predominantly within promoter nucleosomes, in association with gene activation or repression. Our timers allow the in vivo study of histone exchange dynamics with minute time scale resolution.

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Fig. 1: A genetically encoded timer for histone exchange.
Fig. 2: Histone timers reveal promoter H3-H4 exchange versus exchange in gene body for H2A-H2B.
Fig. 3: Differential behavior of H2A-H2B and H3-H4 replacement with gene expression.
Fig. 4: Nucleosome subcomplex dynamics during transcriptional reprogramming.
Fig. 5: Induced and repressed genes display high continuous promoter H3 replacement.
Fig. 6: Scheme of main results.

Data availability

All figures were generated from the raw sequencing data accessible at accession number GSE157402. Source data are provided with this paper.

Code availability

Analysis code is available at https://doi.org/10.5281/zenodo.4746166 and https://github.com/barkailab/Yaakov2021.

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Acknowledgements

We thank all lab members for critical comments throughout and careful reading of the manuscript. We also thank Y. Voichek, R. Bar-Ziv, E. Metzl-Raz and B. Shilo for comments on the manuscript. We are very grateful to R. Diskin for structural insights during the method development. This work was funded by the Israel Science Foundation FIRST Program (grant No. 966/19), ERC and the Minerva Foundation.

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Authors

Contributions

G.Y. and N.B. conceived, developed and designed the sensors. G.Y. performed all experiments. F.J. and N.B. developed and performed all analyses. All authors designed the experiments, discussed the results and wrote the manuscript.

Corresponding authors

Correspondence to Gilad Yaakov or Naama Barkai.

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The authors declare no competing financial interests.

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Peer review information Nature Biotechnology thanks Frederic Berger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Supporting figure for Fig. 1.

a, Pearson correlation across all nucleosomes for HA and myc ChIP-seq levels in strains with the HHT1 allele (median of 17 repeats), the alternate HHT2 H3 allele (3 repeats), or both H3 alleles (4 repeats) tagged with the sensor. b, Left: Pearson correlation between histone mark signal intensity on nucleosomes profiled from exponentially growing cells bearing the HHT1 H3 exchange sensor in this study (rows) with Weiner et al. (columns 21). Right: Mean histone mark (H3K79-trimethyl, H3K4-trimethyl and H3K9-acetyl) traces around the TSS of all genes in the H3 sensor strain. c, Ponceau staining corresponding to Western-blot in Fig. 1b. Three repeats were performed giving similar results. d, Myc and HA level of all nucleosomes for the non-cleavable (median of 3 repeats) and the slowly cleaved variant with a point mutation in the 7AA cleavage site (2 repeats). Rho indicates the Pearson correlation between both intensities. e, Comparison of measured H3 exchange (myc/HA) per nucleosome with an additional external dataset 18. f, Quantitative ChIP-qPCR measurements for absolute exchange (log2(myc/HA)) are plotted versus the relative ChIP-seq counterparts for alpha factor arrested cells (left) or asynchronously growing cells with both H3 alleles tagged with the sensor (right). Analysis was on selected nucleosomes as in Fig. 1f. 2 biological ChIP-qPCR repeats were performed, each consisting of 4 technical ChIP repeats and 2 technical qPCR repeats. The mean values with standard of error are shown.

Extended Data Fig. 2 Supporting figure for Fig. 2.

a, Comparison between HA occupancy signal of all nucleosomes in the H2A, H2B or H4 reporter strains with the H3 reporter strain. rho indicates Pearson correlation. b, Genome-wide Pearson correlation between myc and HA levels per nucleosome of all reporter strains. Median of all ChIP repeats is shown with the number of repeats ‘n’ for each strain indicated on the right. a indicates a strain with G133A in the HHT1 allele (see Methods), b indicates a strain in which TEV is fused to H2B rather than H2A, * indicates a strain in which the alternate allele was tagged (HHT2 for H3 and HHF1 for H4) and # indicates the H2B reporter in which both TEV and the sensor are fused to H2B alleles (HTB2 and HTB1 respectfully). c, Mean myc and HA signal around the TSS for the indicated sensors, binned by expression level (top) or plasticity (bottom). Note that both leftmost systems equally report on H3 dynamics, with fusing TEV to either H2A or H2B. myc/HA signal (log) is shown in the corresponding Fig. 2c. d, As in (c) for strains in which the H2A.Z variant (Htz1) has been deleted in the H2A (median of 4 repeats) or H3 (8 repeats) sensors.

Extended Data Fig. 3 Supporting figure for Fig. 3.

a, Calculated myc and HA nucleosome intensities across a broad kon versus koff parameter space (leftmost, kclv=1.5 and f = 1 as in Fig. 2a). Varying the fraction of myc in unbound histones (f = 0.5, second from left), or myc cleavage rate (kclv*=0.15, second from right) were further tested. HA levels in all cases remain unchanged (rightmost). All axes and color are in log-scale. b, HA signal intensity distribution of nucleosomes on A/T-rich (75 + /-2%) and A/T-poor (50 + /-2%) regions in the genome for H2A and H3 sensor strains. The average A/T content in yeast is 63%. c, Gene body (+2) nucleosome myc signal in H2A versus its respective HA signal (left) or versus H3 myc (right). Color indicates expression level of the gene corresponding to the given nucleosome. d, Mean myc and HA intensity as in Fig. 3c for nucleosomes -2 to +3 (n = 2561, 3500, 4778, 4768 and 4637 for the different positions) in the various reporter strains. For H2B, analyses are shown for strains in which TEV was fused to either H3 or the other allele of H2B as indicated. For H3, TEV was fused to either H2A or H2B. Moving average is calculated over 0.5 expression levels. Shaded area is standard error. e, Fold change in H2A (y-axis) and H3 myc (x-axis) nucleosome levels in Spt6 over-expressing versus wild type cells. f, Antisense transcription was ordered by expression level32. Mean myc and HA intensity are shown for nucleosomes -2 to +2. Shaded area is standard error. g, Candidate screen for chaperones involved in exchange. As in (d) for indicated histone chaperone mutants alongside wildtype for promoter (-1) and gene body (+2) nucleosomes. DAmP; Decreased Abundance by mRNA Perturbation 52, OE; overexpression by TEF1 promoter substitution of native promoter. Number of repeats for H2A/H3 systems correspondingly: OE-Spt6 n = 3/3, spt16DAmP n = 2/3, spt6DAmP 2/3, hir1 n = 2/3. Shaded area is standard error.

Extended Data Fig. 4 Supporting figure for Fig. 4.

a, HA and myc intensity dynamics for the indicated sensor at gene body nucleosomes (+2) during H2O2 exposure. Each column is a single (repressed or induced) gene that corresponds to the given nucleosome. Color indicates the signal intensity change relative to its median (log) at each nucleosome. b, Median gene expression change (log2) of annotated functional gene groups (stress genes, ribosomal biogenesis53) or internally derived expression clusters (induced, repressed). Corresponding mean myc and HA levels around the TSS of stress genes and ribosomal biogenesis genes. c, As in (a) for promoter nucleosomes (-2) in the H3 reporter system. d, Top: Yap1 and Msn2 ChEC binding signal on gene promoters from54. The dashed line indicates the chosen binding threshold for Yap1 and Msn2 target genes. Color indicates the gene’s maximal expression level change during H2O2 exposure. 9 Yap1 targets that do not induce expression are marked in red squares. Bottom: Median Msn2 and Yap1 ChEC signal around the determined Msn2 and Yap1 binding sites. e, Median intensities around the Yap1 and Msn2 binding sites on target gene promoters during H2O2 exposure in the H2A reporter system. The number in brackets indicates the number of target genes for each transcription factor.

Extended Data Fig. 5 Supporting figure for Fig. 5.

a, Gene state (induced or repressed, Fig. 5a) was defined by the median expression change (Y-axis) versus plasticity (standard deviation, X-axis) in a deletion collection 51. Dashed lines indicate the threshold (delta median over 2-fold standard error) used to define high confidence genes with increased (red) or decreased (green) mRNA in exponentially growing wild type cells. b, Histogram of plasticity scores derived in (a) for TATA and TATA-less gene groups55. c, Expression plasticity vs. expression level for promoter (-2) and gene body (+2) nucleosomes. Color indicates myc intensity in the H3 or H2A reporter system respectively. d, Mean exchange signals for all reporter strains at promoter nucleosomes (-2 and -1, n = 1503 and 2028), as a function of gene expression plasticity defined in (a). Shaded area is standard error. e, Mean HA intensity on promoters in Hir1-deleted versus wild-type cells, corresponds to Fig. 5b. Rho indicates the Pearson correlation, and cyan diamonds highlight histone promoters. f, HA and myc for Asf1-deleted cells as in (e). g, Mean HA intensity for wild type, Asf1, Hir1 and Hir2-delted cells around two histone loci, corresponds to Fig. 4c. Refer to main figure for number of repeats in (e-g).

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Source Data Fig. 1

Unprocessed anti-myc western blot in Fig. 1b

Source Data Fig. 2

Unprocessed anti-HA western blot in Fig. 1b

Source Data Fig. 3

Unprocessed Ponceau stain for western blot in Fig. 1b and Extended Data Fig. 1c

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Yaakov, G., Jonas, F. & Barkai, N. Measurement of histone replacement dynamics with genetically encoded exchange timers in yeast. Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-00959-8

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