Abstract
Epe1 histone demethylase restricts H3K9-methylation-dependent heterochromatin, preventing it from spreading over, and silencing, gene-containing regions in fission yeast. External stress induces an adaptive response allowing heterochromatin island formation that confers resistance on surviving wild-type lineages. Here we investigate the mechanism by which Epe1 is regulated in response to stress. Exposure to caffeine or antifungals results in Epe1 ubiquitylation and proteasome-dependent removal of the N-terminal 150 residues from Epe1, generating truncated Epe1 (tEpe1) which accumulates in the cytoplasm. Constitutive tEpe1 expression increases H3K9 methylation over several chromosomal regions, reducing expression of underlying genes and enhancing resistance. Reciprocally, constitutive non-cleavable Epe1 expression decreases resistance. tEpe1-mediated resistance requires a functional JmjC demethylase domain. Moreover, caffeine-induced Epe1-to-tEpe1 cleavage is dependent on an intact cell integrity MAP kinase stress signaling pathway, mutations in which alter resistance. Thus, environmental changes elicit a mechanism that curtails the function of this key epigenetic modifier, allowing heterochromatin to reprogram gene expression, thereby bestowing resistance to some cells within a population. H3K9me-heterochromatin components are conserved in human and crop-plant fungal pathogens for which a limited number of antifungals exist. Our findings reveal how transient heterochromatin-dependent antifungal resistant epimutations develop and thus inform on how they might be countered.
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Data availability
The publicly accessible PomBase database (https://www.pombase.org) was used to obtain genome sequence and annotation for the Schizosaccharomyces pombe (fission yeast) genome.
All raw and processed reads from sequencing experiments (ChIP–seq and RNA-Seq) are available at the GEO under accession number GSE190267.
All raw mass spectrometry data are available at PRIDE using project accession number PXD030205. Source data are provided with this paper.
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Acknowledgements
We thank D. Kelly (WCB, Edinburgh) for microscopy and instrumentation support; members of the Allshire Lab for valuable discussions and input; T. Urano for the 5.1.1 (H3K9me2) antibody; A. Fellas for GFP expressing and clr4∆ strains; K. Gull for α-tubulin antibody; K. Sawin for Mto2 antibody; A. L. Marston for the Sgo1-GFP S. cerevisiae strain; J. Svejstrup for provision of the MultiDsk2 expression construct; M. D. Wilson for comments on the manuscript; and colleagues at WCB for support and encouragement during a difficult 2020–21. This research was supported by award of an EMBO Long Term Fellowship to I. Y. (EMBO ALTF 130–2018), Darwin Trust of Edinburgh PhD studentships to S. T.-G. and R. Y., a Wellcome 4 Year iCM program PhD studentship to E. G. (218470), a Wellcome Instrument grant to J. R. (108504), a Wellcome Investigator award to M. E. K. (205008), a Wellcome Principal Research Fellowship to R. C. A. (200885; 224358) and core funding for the Wellcome Centre for Cell Biology (203149). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. For the purpose of Open Access, the author has applied a CC-BY public copyright licence to any author-accepted manuscript version arising from this submission.
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I. Y. and S. A. W. designed and performed experiments. S. T.-G. contributed to ChIP, ChIP–seq and RNA-seq experiments and CRISPR design. M. L. carried out ChIP–seq and RNA-seq analyses and data visualization. E. G. performed Halo-tag experiments with advice on microscopy and experimental design from M.E.K. R. Y. contributed to antifungal-resistance experiments. C. S. ran samples and performed mass spectrometry analysis with J. R. A. L. P. advised on experimental design, performed ChIP and constructed strains. I. Y., S. A. W., A. L. P. and R. C. A. prepared figures and wrote the manuscript.
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Extended data
Extended Data Fig. 1 Caffeine-induced alteration in Epe1-GFP mobility does not result from transcriptional changes in 5’ region of epe1+ gene or Epe1-Myc phosphorylation.
a. Epe1-GFP or α-tubulin western from cells untreated (-)/treated (+) with 14 mM caffeine for 16 h. b. qRT-PCR measurement of steady-state Epe1 transcript levels in cells untreated (-)/treated (+) with 14 mM caffeine for 16 h. Locations of primers used indicated. Data shown represent 3 technical replicates + /− SEM. c. Scheme to detect caffeine-induced Epe1-GFP phosphorylation by mass spectrometry (top). Representation of phosphorylated residues detected on Epe1-GFP from cells untreated or treated with 14 mM caffeine for 16 h (middle). Spectra showing detection of phosphorylated serine S791, S795 and S803 residues after caffeine treatment (bottom). Full data, Supplementary Table 4. d. Epe1-Myc and Mto2 westerns from cells treated with 14 mM caffeine for 16 h before (-)/after (+) lambda protein phosphatase addition. Mto2 mobility is known to be Lambda Phosphatase sensitive1.
Extended Data Fig. 2 Deletions beyond residue 150 in Epe1-GFP do not hinder caffeine-induced processing and peptides within the first 100 residues are not detected following caffeine treatment.
a. Schematic of indicated 20 residue deletion mutants in the N-terminal coding region of the endogenous epe1 gene expressed as GFP fusions (Epe1∆150–170-GFP, Epe1∆171–190-GFP, Epe1∆191–210-GFP; left). Western detecting indicated mutant Epe1-GFP fusion proteins or α-tubulin from cells untreated (-) or treated (+) with 14 mM caffeine; right). b. Epe1-Myc peptides detected following immunoprecipitation from cells untreated (-) or treated (+) with 14 mM caffeine and analysis by mass spectrometry. Top: schematic showing position of peptides detected relative to Epe1 (residues 1–948). Bottom: Epe1 peptides detected in Epe1-Myc immunoprecipitates from treated (+) or untreated (-) with 14 mM caffeine. Of the eighteen peptides detected from Epe1-Myc in untreated (-) samples three were not detected (n.d.; red) in the caffeine-treated sample. The three peptides not detected in the presence of caffeine are derived from within the first 100 residues on the N-terminus. Analysis was performed on three independent immunoprecipitates. Full data, Supplementary Table 5.
Extended Data Fig. 3 A caffeine-induced N-terminal processing product is not detectable but Epe1-associated ubiquitination increases and Epe1 processing is sensitive to loss of specific E3 ligases which influence resistance.
a. Anti-Myc western of cells untreated (-)/treated (+) with 14 mM caffeine showing that a predicted N-terminal 20 kDa Myc-Epe11–150 processing product is not detectable. b. Western showing detection of both isoforms of the histone acetyltransferase Mst2-Myc or α-tubulin from wild-type or mst2–1 proteasome defective cells untreated (-)/treated (+) with 14 mM caffeine for 16 h. This control demonstrates that other known stress-induced changes still occur in mst2–1 cells. c. Western showing detection of both isoforms of the histone acetyltransferase Mst2-Myc or α-tubulin from wild-type cells incubated without (-)/with (+) with the 26 S proteasome inhibitor Bortezomib (BTZ) prior to no caffeine (-)/14 mM caffeine (+) treatment for 16 h. This control demonstrates that other known stress-induced changes still occur in the presence of BTZ. d. Immunolocalization of Epe1-GFP in wild-type or mts2–1 cells untreated (-)/ treated(+) with 14 mM/16 h caffeine. No tag, negative control. e. Quantification of anti-GFP/Epe1-GFP nuclear signals of cells in d. Data are represented as individual measurements from ≥ 20 cells per sample. Bars represent mean, with error bars + /− SD. The significance of the difference between samples was evaluated using a two-tailed Student’s t-test. (*) P < 0.033, (**) P < 0.002; (***) P < 0.0002; (****) P < 0.0001; (n.s). not significant. f. Western detecting Epe1-GFP or α-tubulin in wild-type or ddb1Δ, cells untreated (-)/treated (+) with 14 mM caffeine for 16 h. Numbers below tracks: levels of full-length FL-Epe1 and truncated tEpe1 normalised to no treatment and adjusted relative to α-tubulin loading control, measurement average of 2 biological replicates. g. Number of resistant colonies formed/1×104 viable cells by wild-type or ddb1Δ cells plated on caffeine or fluconazole plates. Error bars are from independent measurements from 3 biological replicates. Data are presented mean values +/− SD, with dots representing independent plate counts. The significance of the difference between samples was evaluated using a two-tailed Student’s t-test. (*) P < 0.033, (**) P < 0.002; (***) P < 0.0002; (****) P < 0.0001; (n.s). not significant.
Extended Data Fig. 4 Chromatin-associated nuclear proteins are enriched with full length Epe1 whereas more cytoplasmic proteins are enriched with constitutively truncated Epe1ΔN150-GFP.
a. Volcano plot of proteins enriched with full-length Epe1-GFP extracted from cells detected by proteomic analysis (top), specific proteins labelled (red dots). Table with names and functions of proteins enriched (bottom). Full data, Supplementary Table 6. b. Volcano plot of proteins enriched with constitutively processed Epe1ΔN150-GFP extracted from cells and detected by proteomic analysis (top), specific proteins labelled (red dots). Table with names and functions of proteins enriched (bottom). Full data, Supplementary Table 7.
Extended Data Fig. 5 Altered silencing and gene expression in epe1∆ and Epe1-∆N150-GFP cells.
a. Wild-type, epe1∆, Epe1-∆N150, Epe1-GFP/wt and Epe1-∆N150-GFP cells harbouring ura4+ marker gene insertions at either site 1 or site 2 as indicated (top) were serially diluted and plated on non-selective (N/S), selective (-URA) or counter-selective (FOA) plates. b. Heat-map generated from processed data of RNA-seq two biological replicates from Epe1∆N150-GFP or epe1Δ cells compared with that of wild-type Epe1-GFP cells. Genes with higher (turquoise) or lower (brown) expression relative to wild-type Epe1-GFP cells are shown as a Log2 scale. The position of specific affected genes along S. pombe chromosomes Chr I, II, III are shown with arrowheads indicating telomeres on Chr I and ChrII. The annotated S. pombe genome shows only rDNA arrays (rectangles) at both ends of Chr III. c. Heat-map showing that RNA-seq data (used in b.) resulting from two Epe1∆N150-GFP and epe1Δ biological replicates are more similar to each other than they are to wild-type Epe1-GFP cells.
Extended Data Fig. 6 Cells expressing predicted catalytically inactive Epe1-GFP and Epe1-∆N150-GFP exhibit decreased resistance.
a. Schematic showing position of H297A and K314A mutations predicted to reduce association of the essential iron/Fe(2) and 2-Oxoglutarate (α-ketoglutarate) cofactors, respectively, with the JmjC demethylase domain of Epe1 (Top). Westerns detecting Epe1-GFP, Epe1-cd-GFP, Epe1∆N150-GFP and Epe1∆N150-cd-GFP (cd; catalytically dead) or α-tubulin (bottom). b. Number of resistant colonies formed/1×104 viable cells by Epe1-GFP, Epe1-cd-GFP, Epe1∆N150-GFP and Epe1∆N150-cd-GFP cells plated on caffeine or fluconazole plates Error bars are from independent measurements from 3 biological replicates. Data are presented mean values+/−SD, with dots representing independent plate counts. The significance of the difference between samples was evaluated using a two-tailed Student’s t-test. (*) P < 0.033, (**) P < 0.002; (***) P < 0.0002; (****) P < 0.0001; (n.s). not significant.
Extended Data Fig. 7 The cell integrity/Pmk1 but not the TORC2/Gad8 stress signalling pathway regulates Epe1 processing and resistance.
a. Diagram of part the S. pombe TORC2/Tor1-dependent signalling pathway. b. Western detecting Epe1-GFP or α-tubulin from wild-type or gad8Δ cells untreated (-)/ treated (+) with 14 mM/16 h caffeine. c. Western detecting Epe1-Myc or α-tubulin from wild-type, pek1Δ, pmk1Δ, atf1Δ or pmp1Δ cells untreated (-)/treated (+) with 0.5 mM/16 h fluconazole (FLC). d. Number of resistant colonies formed/1×104 viable cells plated by wild-type, epe1Δ, sty1Δ, atf1Δ and gad8Δ on plates containing 16 mM caffeine (CAF). e. Number of resistant colonies formed/1×104 viable cells plated by wild-type, epe1Δ, sty1Δ, atf1Δ and gad8Δ on plates containing 0.5 mM fluconazole (FLC). Plots d and e; Error bars are from independent measurements from 3 biological replicates. Data are presented mean values + /− SD, with dots representing independent plate counts. The significance of the difference between samples was evaluated using a two-tailed Student’s t-test. (*) P < 0.033, (**) P < 0.002; (***) P < 0.0002; (****) P < 0.0001; (n.s). not significant.
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Yaseen, I., White, S.A., Torres-Garcia, S. et al. Proteasome-dependent truncation of the negative heterochromatin regulator Epe1 mediates antifungal resistance. Nat Struct Mol Biol 29, 745–758 (2022). https://doi.org/10.1038/s41594-022-00801-y
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DOI: https://doi.org/10.1038/s41594-022-00801-y