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Histone deacetylation primes self-propagation of heterochromatin domains to promote epigenetic inheritance

Abstract

Heterochromatin assembly, involving histone H3 lysine-9 methylation (H3K9me), is nucleated at specific genomic sites but can self-propagate across extended domains and, indeed, generations. Self-propagation requires Clr4/Suv39h methyltransferase recruitment by pre-existing H3K9 tri-methylation (H3K9me3) to perpetuate H3K9me deposition and is dramatically affected by chromatin context. However, the mechanism priming self-propagation of heterochromatin remains undefined. We show that robust chromatin association of fission yeast class II histone deacetylase Clr3 is necessary and sufficient to support heterochromatin propagation in different chromosomal contexts. Efficient targeting of Clr3, which suppresses histone turnover and maintains H3K9me3, enables self-propagation of an ectopic heterochromatin domain via the Clr4/Suv39h read–write mechanism requiring methylated histones. The deacetylase activity of Clr3 is necessary and, when inactivated, heterochromatin propagation can be recapitulated by removing two major histone acetyltransferases. Our results show that histone deacetylation, a conserved heterochromatin feature, preserves H3K9me3 that transmits epigenetic memory for stable propagation of silenced chromatin domains through multiple generations.

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Fig. 1: Fusion of Clr3 HDAC to a chromodomain supports efficient sequence-independent localization to heterochromatin.
Fig. 2: Clr3 localization sustains self-templated heterochromatin propagation.
Fig. 3: HDAC chromatin loading is required for self-propagation of extended heterochromatin domains.
Fig. 4: Loss of the HATs Gcn5 and Mst2 enables self-propagation of heterochromatin.
Fig. 5: Effective chromatin association of Clr3 is required for heterochromatin spreading and suppression of histone turnover to preserve epigenetic memory.
Fig. 6: Clr3 localization promotes stable heterochromatin propagation at centromeres.
Fig. 7: CAS-like elements promote epigenetic inheritance of heterochromatin at the ectopic site.
Fig. 8: Direct targeting of Clr3 restores H3K9me3 density and heterochromatin propagation in mutants showing elevated histone turnover.

Data availability

ChIP–chip and ChIP–seq data have been deposited in the GEO under accession number GSE184466. ChIP–seq data for Drosophila HDAC4 and H3K27me were downloaded from the NCBI Sequence Read Archive (GEO records GSE20000 and GSE49490, respectively)62. Source data are provided with this paper.

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Acknowledgements

We thank members of the Laboratory of Biochemistry and Molecular Biology, in particular the Grewal laboratory for helpful discussions, A. Cutter Dipiazza for strain constructions, C. Jayakumar and J. Dhakshnamoorthy for technical help and J. Barrowman for editing the manuscript. This study used the Helix Systems and Biowulf Linux cluster at the National Institutes of Health and the PomBase genome database. This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute.

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Contributions

S.I.S.G. and M.Z. conceived the project. M.Z., R.S. and S.H. performed experiments. R.S. performed Clr3 ChIP–seq. S.H. helped in performing histone turnover experiments. M.Z. performed all other experiments. D.W. performed bioinformatic analyses. All authors contributed to data interpretation. S.I.S.G. and M.Z. wrote the manuscript.

Corresponding author

Correspondence to Shiv I. S. Grewal.

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Nature Structural and Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Carolina Perdigoto, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Chromodomain-coupled chimeric Clr3 protein is functional.

(a) Western blot analysis of HA epitope-tagged Clr3 carrying one or two copies of the Chp1 chromodomain fused at its carboxy-terminus and expressed under the control of the native clr3 promoter. Coomassie staining is shown as a loading control. Representative of two independent analyses is shown. (b) ChIP analysis of Clr3-CDx2 localization at the silent mat region in WT and clr4∆ cells. Quantitative duplex PCR analysis was used to calculate fold enrichment values. The region analyzed by ChIP is marked by the black bar in ‘c’. (c) Serial dilution analysis of heterochromatic repression of the mat2P::ura4+ reporter. The indicated strains were spotted on non-selective (NS), uracil depleted (-Uracil) or counter-selective medium containing FOA (FOA). Colonies grown on PMG minimal medium were stained with iodine vapor to detect haploid meiosis. Note that strains expressing chromodomain-coupled Clr3 are proficient in heterochromatic silencing at the silent mat region. As a control, cells carrying a mutation in the catalytic site of Clr3 (clr3D232N) show defective silencing, which is indicated by growth on medium lacking uracil, loss of viability on FOA medium and dark iodine staining. (d) Serial dilution analyses to test if chromodomain-coupled Clr3 can bypass the Swi6 requirement for heterochromatic silencing. In addition to mat2P::ura4+ expression, haploid meiosis was analyzed by iodine staining. (e) ChIP analysis of Clr3 localization at the silent mat region. Results of quantitative duplex PCR analysis are shown. Mean fold enrichments (MFE) and SEM from 3 or 2 measurements are indicated in b and e.

Source data

Extended Data Fig. 2 Clr3-CD dosage determines the efficacy of self-templated heterochromatin assembly.

(a) Western blot analysis of Clr3-CDx1 expression driven by the native clr3 or adh11 promoter. Extracts from cells expressing untagged Clr3 or Clr3-HA without the chromodomain fusion are included for comparison. Coomassie staining is shown as a loading control. Representative of two independent analyses is shown. (b) ade6+ expression in tetR-clr4ON or tetR-clr4OFF cells carrying the indicated clr3 allele was assayed by serial dilution on low adenine medium (top). Colonies formed by tetR-clr4OFF cells carrying either WT clr3, clr3-CDx1 or adh11::clr3-CDx1 on low adenine medium are shown (bottom). The presence of red colonies indicates stable propagation of ectopic heterochromatin at the ade6+ reporter.

Source data

Extended Data Fig. 3 Enhanced chromatin association of Clr3 HDAC stabilizes heterochromatin at endogenous loci.

(a) Schematic showing the silent mat region with the RNAi-targeted cenH element replaced by the ura4+ reporter (K∆::ura4+). In the absence of RNAi targeted cenH, de novo heterochromatin assembly is compromised, and heterochromatin propagation requires the read-write mechanism involving Clr4Suv39h recruitment by the parental H3K9me3. Cells carrying the silenced K∆::ura4+ OFF state are passaged for 10 or 30 generations in non-selective media and then serial dilutions are plated onto non-selective, -Uracil and FOA media to assay propagation of the heterochromatic state. Cells expressing chromodomain-coupled Clr3, in particular Clr3-CDx2, propagated heterochromatin more stably as compared to WT Clr3. (b) ChIP analysis of the indicated Clr3 proteins at facultative heterochromatin islands. (c) ChIP analyses of H3K9me levels at facultative heterochromatin islands in cells expressing WT Clr3 or Clr3 fused to one or two chromodomains. Relative ChIP enrichment values at the indicated loci, as compared to control leu1, that are shown in ‘b’ and ‘c’ were determined using quantitative duplex PCR. Mean fold enrichments (MFE) and SEM of two experiments are indicated.

Source data

Extended Data Fig. 4 Sequence-independent epigenetic inheritance of an ectopic heterochromatin domain in cells lacking histone acetyltransferases.

(a) Schematic showing the strategy used to assay epigenetic inheritance of an ectopic heterochromatin domain in cells lacking Mst2 and Gcn5. The self-propagation of ectopic heterochromatin domains assembled by TetR-Clr4 (tetR-clr4ON) was investigated upon the release of tethered Clr4 (tetR-clr4OFF) in the indicated single and double mutant strains. Heterochromatin persistence was measured by plating cells on low adenine medium to assay tetO-ade6+ expression. Red colony coloration indicates ade6+ repression, while white color indicates expression. (b) Close-up of colonies formed by WT and mst2∆ gcn5∆ cells on low adenine medium. The maintenance of the heterochromatic state at tetO-ade6+ in tetR-clr4OFF cells lacking Mst2 and Gcn5 is indicated by red-colored colonies, as compared to white colonies formed by WT background cells.

Extended Data Fig. 5 H3K9me and Clr3-CDx2 distribution across the ectopic heterochromatin domain.

Results of ChIP-chip analyses of H3K9me3 and Clr3-CDx2 are plotted. A strain expressing untagged Clr3 is included as a control.

Extended Data Fig. 6 Chromodomain-coupled Clr3 affects heterochromatin domains at pericentromeric repeats and at telomeres.

(a) Clr3-Myc distribution along S. pombe chromosomes as determined by ChIP-seq analysis. Note prominent peaks of Clr3 mapping to the mat locus and rDNA, in addition to its localization at centromeres, telomeres and other loci including Tf2 retroelements. (b) Clr3-CDx2 expression suppresses histone turnover at pericentromeric regions. ChIP-chip analysis of H3K9me distribution and incorporation of pulse-expressed H3-T7 at centromere 1 in cells carrying either WT clr3 or clr3-CDx2 allele. Replication-independent turnover of H3 was measured in cells blocked at the G2/M boundary. The central core domain (cnt) of centromere that contains Cenp-A does not incorporate histone H3. Note that cells expressing Clr3-CDx2 show lower histone turnover as compared to WT. (c) ChIP-chip analysis of Clr3-CDx2 and H3K9me3 distribution at telomere 1R. Cells expressing Clr3-CDx2 show enhanced spreading of H3K9me3 compared to WT cells.

Extended Data Fig. 7 Effective chromatin association of Clr3 HDAC, or prevention of histone acetylation, restores heterochromatic silencing in mutants showing high histone turnover.

The enhanced recruitment of chromodomain-coupled Clr3, or loss of both Mst2 and Gcn5 HATs, mitigates defective heterochromatic silencing at the silent mat region in cells lacking Amo1, involved in the nuclear peripheral tethering of heterochromatin, or the FACT histone chaperone component Pob3, implicated in suppression of histone turnover. Serial dilutions on non-selective (NS) and FOA-containing medium (FOA) were used to assay expression of the mat2P::ura4+ reporter gene at the silent mat region.

Extended Data Fig. 8 Drosophila class II HDAC, HDAC4, preferentially localizes to H3K27me3-coated domains silenced by the Polycomb proteins.

Results of ChIP-seq analysis of HDAC4 and H3K27me3 performed by Negre et al62 (GEO accession number: GSE49490 and GSE20000 respectively) are plotted. Note that major HDAC4 peaks map to the polycomb response elements (PREs; indicated by red triangles) located in the center of individual H3K27me3 domains. HDAC4 distribution resembles Clr3 class II HDAC localization at the silent mat region in S. pombe (see Fig. 6a). Similar to PRE elements in Drosophila, CAS elements, including the CAS element bound by ATF-CREB family proteins Atf1/Pcr1 implicated in heterochromatin assembly20, show prominent Clr3 HDAC peaks.

Supplementary information

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Supplementary Tables 1–2

List of strains and primers used in this study.

Source data

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Zofall, M., Sandhu, R., Holla, S. et al. Histone deacetylation primes self-propagation of heterochromatin domains to promote epigenetic inheritance. Nat Struct Mol Biol 29, 898–909 (2022). https://doi.org/10.1038/s41594-022-00830-7

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