Heterochromatic DNA domains have important roles in the regulation of gene expression and maintenance of genome stability by silencing repetitive DNA elements and transposons. From fission yeast to mammals, heterochromatin assembly at DNA repeats involves the activity of small noncoding RNAs (sRNAs) associated with the RNA interference (RNAi) pathway1,2,3,4,5,6,7,8,9. Typically, sRNAs, originating from long noncoding RNAs, guide Argonaute-containing effector complexes to complementary nascent RNAs to initiate histone H3 lysine 9 di- and trimethylation (H3K9me2 and H3K9me3, respectively) and the formation of heterochromatin10,11,12,13,14,15,16,17. H3K9me is in turn required for the recruitment of RNAi to chromatin to promote the amplification of sRNA11,15,18. Yet, how heterochromatin formation, which silences transcription, can proceed by a co-transcriptional mechanism that also promotes sRNA generation remains paradoxical. Here, using Clr4, the fission yeast Schizosaccharomyces pombe homologue of mammalian SUV39H H3K9 methyltransferases, we design active-site mutations that block H3K9me3, but allow H3K9me2 catalysis. We show that H3K9me2 defines a functionally distinct heterochromatin state that is sufficient for RNAi-dependent co-transcriptional gene silencing at pericentromeric DNA repeats. Unlike H3K9me3 domains, which are transcriptionally silent, H3K9me2 domains are transcriptionally active, contain modifications associated with euchromatic transcription, and couple RNAi-mediated transcript degradation to the establishment of H3K9me domains. The two H3K9me states recruit reader proteins with different efficiencies, explaining their different downstream silencing functions. Furthermore, the transition from H3K9me2 to H3K9me3 is required for RNAi-independent epigenetic inheritance of H3K9me domains. Our findings demonstrate that H3K9me2 and H3K9me3 define functionally distinct chromatin states and uncover a mechanism for the formation of transcriptionally permissive heterochromatin that is compatible with its broadly conserved role in sRNA-mediated genome defence.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Gene Expression Omnibus
Holoch, D. & Moazed, D. RNA-mediated epigenetic regulation of gene expression. Nat. Rev. Genet. 16, 71–84 (2015)
Castel, S. E. & Martienssen, R. A. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nat. Rev. Genet. 14, 100–112 (2013)
Allshire, R. C. & Ekwall, K. Epigenetic regulation of chromatin states in Schizosaccharomyces pombe. Cold Spring Harb. Perspect. Biol. 7, a018770 (2015)
Volpe, T. A. et al. Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297, 1833–1837 (2002)
Chan, S. W. et al. RNA silencing genes control de novo DNA methylation. Science 303, 1336 (2004)
Shirayama, M. et al. piRNAs initiate an epigenetic memory of nonself RNA in the C. elegans germline. Cell 150, 65–77 (2012)
Buckley, B. A. et al. A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality. Nature 489, 447–451 (2012)
Aravin, A. A. et al. A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice. Mol. Cell 31, 785–799 (2008)
Sienski, G., Dönertas, D. & Brennecke, J. Transcriptional silencing of transposons by Piwi and Maelstrom and its impact on chromatin state and gene expression. Cell 151, 964–980 (2012)
Verdel, A. et al. RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303, 672–676 (2004)
Motamedi, M. R. et al. Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell 119, 789–802 (2004)
Bühler, M., Verdel, A. & Moazed, D. Tethering RITS to a nascent transcript initiates RNAi- and heterochromatin-dependent gene silencing. Cell 125, 873–886 (2006)
Zhang, K., Mosch, K., Fischle, W. & Grewal, S. I. Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin. Nat. Struct. Mol. Biol. 15, 381–388 (2008)
Bayne, E. H. et al. Stc1: a critical link between RNAi and chromatin modification required for heterochromatin integrity. Cell 140, 666–677 (2010)
Gerace, E. L., Halic, M. & Moazed, D. The methyltransferase activity of Clr4Suv39h triggers RNAi independently of histone H3K9 methylation. Mol. Cell 39, 360–372 (2010)
Yu, Y. et al. Panoramix enforces piRNA-dependent cotranscriptional silencing. Science 350, 339–342 (2015)
Sienski, G. et al. Silencio/CG9754 connects the Piwi–piRNA complex to the cellular heterochromatin machinery. Genes Dev. 29, 2258–2271 (2015)
Rangan, P. et al. piRNA production requires heterochromatin formation in Drosophila. Curr. Biol. 21, 1373–1379 (2011)
Collins, R. E. et al. In vitro and in vivo analyses of a Phe/Tyr switch controlling product specificity of histone lysine methyltransferases. J. Biol. Chem. 280, 5563–5570 (2005)
Xiao, B. et al. Structure and catalytic mechanism of the human histone methyltransferase SET7/9. Nature 421, 652–656 (2003)
Al-Sady, B., Madhani, H. D. & Narlikar, G. J. Division of labor between the chromodomains of HP1 and Suv39 methylase enables coordination of heterochromatin spread. Mol. Cell 51, 80–91 (2013)
Li, B., Carey, M. & Workman, J. L. The role of chromatin during transcription. Cell 128, 707–719 (2007)
Ekwall, K., Olsson, T., Turner, B. M., Cranston, G. & Allshire, R. C. Transient inhibition of histone deacetylation alters the structural and functional imprint at fission yeast centromeres. Cell 91, 1021–1032 (1997)
Motamedi, M. R. et al. HP1 proteins form distinct complexes and mediate heterochromatic gene silencing by nonoverlapping mechanisms. Mol. Cell 32, 778–790 (2008)
Sugiyama, T. et al. SHREC, an effector complex for heterochromatic transcriptional silencing. Cell 128, 491–504 (2007)
Schalch, T. et al. High-affinity binding of Chp1 chromodomain to K9 methylated histone H3 is required to establish centromeric heterochromatin. Mol. Cell 34, 36–46 (2009)
Ragunathan, K., Jih, G. & Moazed, D. Epigenetics. Epigenetic inheritance uncoupled from sequence-specific recruitment. Science 348, 1258699 (2015)
Audergon, P. N. et al. Epigenetics. Restricted epigenetic inheritance of H3K9 methylation. Science 348, 132–135 (2015)
Zhang, X. et al. Structural basis for the product specificity of histone lysine methyltransferases. Mol. Cell 12, 177–185 (2003)
Yu, R., Jih, G., Iglesias, N. & Moazed, D. Determinants of heterochromatic siRNA biogenesis and function. Mol. Cell 53, 262–276 (2014)
Zeng, P. Y., Vakoc, C. R., Chen, Z. C., Blobel, G. A. & Berger, S. L. In vivo dual cross-linking for identification of indirect DNA-associated proteins by chromatin immunoprecipitation. Biotechniques 41, 694–698, 696, 698 (2006)
Wong, K. H ., Jin, Y. & Moqtaderi, Z. Multiplex Illumina sequencing using DNA barcoding. Curr. Protoc. Mol. Biol. Chapter 7, Unit 7.11 (2013)
Holoch, D. & Moazed, D. Small-RNA loading licenses Argonaute for assembly into a transcriptional silencing complex. Nat. Struct. Mol. Biol. 22, 328–335 (2015)
Paulo, J. A., O’Connell, J. D. & Gygi, S. P. A triple knockout (TKO) proteomics standard for diagnosing ion interference in isobaric labeling experiments. J. Am. Soc. Mass Spectrom. 27, 1620–1625 (2016)
Huttlin, E. L. et al. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143, 1174–1189 (2010)
Beausoleil, S. A., Villén, J., Gerber, S. A., Rush, J. & Gygi, S. P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 24, 1285–1292 (2006)
Elias, J. E. & Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007)
Elias, J. E. & Gygi, S. P. Target-decoy search strategy for mass spectrometry-based proteomics. Methods Mol. Biol. 604, 55–71 (2010)
McAlister, G. C. et al. Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses. Anal. Chem. 84, 7469–7478 (2012)
Sidoli, S. et al. Sequential window acquisition of all theoretical mass spectra (SWATH) analysis for characterization and quantification of histone post-translational modifications. Mol. Cell. Proteomics 14, 2420–2428 (2015)
Yuan, Z. F. et al. EpiProfile quantifies histone peptides with modifications by extracting retention time and intensity in high-resolution mass spectra. Mol. Cell. Proteomics 14, 1696–1707 (2015)
Recht, J. et al. Histone chaperone Asf1 is required for histone H3 lysine 56 acetylation, a modification associated with S phase in mitosis and meiosis. Proc. Natl Acad. Sci. USA 103, 6988–6993 (2006)
Sidoli, S., Bhanu, N. V., Karch, K. R., Wang, X. & Garcia, B. A. Complete workflow for analysis of histone post-translational modifications using bottom-up mass spectrometry: from histone extraction to data analysis. J. Vis. Exp. (111): (2016)
Oeffinger, M. et al. Comprehensive analysis of diverse ribonucleoprotein complexes. Nat. Methods 4, 951–956 (2007)
We thank M. Kalocsay for help with histone MS analysis, R. C. Kunz (Thermo Fisher Scientific Center for Multiplexed Proteomics, Harvard Medical School) for performing the initial TMT/MS, the Nikon Imaging Center at Harvard Medical School for access to confocal microscopes, K. Connolly for help with imaging, C. K. Wong and Z. Moqtaderi for help with Illumina library construction and data processing, R. Yu for Python scripts, D. Holoch for the Swi6 antibody, R. Behrouzi, D. Holoch and G. Shipkovenska for comments on the manuscript, and members of the Moazed laboratory for discussion. This work was supported by an NIH training grant (T32 GM007226) (G.J.), an EMBO long-term fellowship and a Swiss National Science Foundation postdoctoral fellowship (N.I.), NIH RO1 GM072805 (D.M.), K01 DK098285 (J.A.P.), and RO1 GM110174 (B.A.G.). D.M. is a Howard Hughes Medical Institute Investigator.
The authors declare no competing financial interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Western blot of N-terminal 3 × Flag-tagged Clr4 showing that SET mutations (F449Y or I418P) or a chromodomain mutation (W31G) do not affect Clr4 protein stability (top). The same blot stained with Ponceau dye is shown as a loading control (bottom). Image represents two individual experiments. b, Flag purification of wild-type Clr4 and Clr4(F449Y) showing both proteins are incorporated into the CLRC methyltransferase complex. c, Pull-down assays showing that wild-type Clr4 and Clr4(F449Y) interact with recombinant GST–Swi6 with similar efficiency. d, Coomassie staining (top) and western blot (bottom) of histones enriched for H3K9me using Swi6 affinity pull-down from wild-type and clr4F449Y cells showing that Clr4(F449Y) primarily catalyses H3K9me2. Image represents three individual experiments. e, Quantitative mass spectrometry of histones showing the redistribution of H3K9 methylation states in clr4+ and clr4F449Y cells. The histones were isolated using Swi6 affinity pull-down to increase detection sensitivity. See Extended Data Fig. 6 for quantitative mass spectrometry of H3 tail modifications in total wild-type histones. For gel source data, see Supplementary Fig. 1.
a, Map of the pericentromeric DNA region to the right of centromere 1. Arrowheads indicate the location of primers used for ChIP–qPCR in b and c. b, c, ChIP–qPCR analysis of H3K9me2 (b) and H3K9me3 (c) levels at the dg and dh repeats in cells with the indicated genotypes (clr4dead indicates clr4H410L, C412A). Error bars, s.d.; n = 3 biological replicates. d, Expanded view of H3K9me2 ChIP–seq reads mapped to the pericentromeric repeat regions on the right arm of chromosome 1 in Δclr4, clr4+, clr4F449Y and clr4I418P cells. The location of centromere 1 (cen1), innermost repeats (imr1R), outermost dg and dh repeats, and inverted repeat centromere (irc) sequences are indicated. Chromosome 1 coordinates are indicated above the tracks. Reads were randomly assigned to the dg and dh repeats of each chromosome. e, Same as d but showing H3K9me3 ChIP–seq reads.
Extended Data Figure 3 Clr4 mutants have reduced H3K9me spreading at mating type and telomeric regions.
a–g, ChIP–seq data showing changes in H3K9me2 and H3K9me3 levels outside of RNAi-dependent nucleation regions (indicated by solid black bars below tracks) at mating type (mat) and telomeric DNA regions (tel1L, tel1R, tel2L, tel2R, tel3L and tel3R) in Δclr4, clr4+, clr4F449Y and clr4I418P cells. tel3L and tel3R represent reads from the rDNA repeats. H3K9me2 reads were randomly assigned to repeated sequences. The reads at cenH are therefore shared with those at the pericentromeric dg and dh repeats (with which cenH shares 98% sequence identity); H3K9me2 reads that map uniquely to the mating type locus are shown (a, right panel). When only unique reads are mapped, a large fraction of total reads corresponding to repeated sequences at centromeres, telomeres, and the mat locus, are removed. This changes the normalized peak heights, which are affected by fewer total mapped reads. Data are presented as reads per million (y axis).
Extended Data Figure 4 ChIP–qPCR analysis showing increased Pol II levels at pericentromeric DNA repeats of clr4-mutant cells.
a, Map showing the location of the heterochromatin reporter ura4+ inserted to the right of cen1. b, ChIP–qPCR data showing changes in the association of RNA Pol II with the dg and dh pericentromeric DNA repeats in clr4 mutant cells. qPCR primer locations are indicated by arrowheads in a. Error bars, s.d.; n = 3 biological replicates.
Extended Data Figure 5 Clr4 mutant cells have increased levels of Chp1 and activating histone marks at pericentromeric DNA repeats.
a, ChIP–qPCR showing increased Chp1 recruitment to pericentromeric DNA repeats. Error bars, s.d.; n = 3 biological replicates. b, ChIP–seq data showing increased Chp1 reads mapping to pericentromeric regions of chromosome 2 in clr4F449Y and clr4I418P mutants compared to wild-type (wt) cells. c, Same as b, but showing pericentromeric regions of chromosome 3. d, ChIP–qPCR analysis of Flag–Ago1 recruitment to pericentromeric DNA repeats in wild-type clr4 and clr4F449Y cells. Error bars, s.d.; n = 3 biological replicates. e, f, ChIP–qPCR analysis of H3K4me3 (e) and H3K36me3 (f) levels at pericentromeric DNA repeats in wild-type clr4 and clr4-mutant cells. dg2 primer location is indicated by empty arrowhead. Error bars, s.d.; n = 3 biological replicates. g, ChIP–seq data showing increased H3K14ac mapped reads at pericentromeric regions of chromosome 1 in clr4F449Y cells. h, ChIP–seq data showing increased H4K16ac mapped reads at pericentromeric regions of chromosome 1 in clr4F449Y cells. Data are presented as reads per million (y axis).
a, Steps for the isolation of chromatin-bound histones and their analysis by liquid chromatography and tandem mass spectrometry (LC–MS/MS). b, Results of quantitative mass spectrometry analysis of modifications associated with the indicated H3 tail tryptic peptide in clr4+ and Δclr4 cells.
a, Tenfold serial dilution of cells plated on non-selective (N/S) and FOA-containing (+ FOA) medium to evaluate re-establishment of otr1R::ura4+ silencing at 0, 3, 5, 7 h after TSA removal. Untreated and Δclr4 cells serve as positive and negative control for otr1R::ura4+ silencing, respectively. Image represents three individual experiments. b, H3K9me2 ChIP–seq reads mapped to pericentromeric repeats to the right of cen1 in untreated and TSA-treated cells at the indicated time points following TSA removal. The highlighted region (darker blue) displayed the greatest loss of H3K9 methylation resulting from TSA treatment. Read ratio (indicated on the right) was obtained by normalizing the sum of reads mapping to the highlighted region for TSA-treated compared to untreated cells. c, Same as b, but showing H3K9me3 ChIP–seq reads. See Fig. 2g for ChIP–qPCR analysis.
Extended Data Figure 8 Chp2 and Swi6 are not required for the formation of H3K9me2 or me3 domains at pericentromeric DNA repeats.
a, b, ChIP–seq data showing that unlike Chp1, Chp2 and Swi6 are not required for RNAi-mediated H3K9 methylation, as indicated by similar levels of H3K9me2 (a) and H3K9me3 (b) mapped reads at pericentromeric regions of chromosome 1 in wild-type (wt), Δchp2, and Δswi6 cells. Δclr4 serves as a control for specificity of the anti-H3K9me antibodies. Data are presented as reads per million (y axis).
a, Swi6 ChIP–qPCR analysis at dg and dh. Error bars, s.d.; n = 3 biological replicates. b, Live cell imaging using confocal microscopy showing the localization of GFP–Swi6 and Cut11–mCherry in clr4+, clr4F449Y and ∆clr4 cells. In wild-type clr4+ cells, GFP–Swi6 foci representing centromeres, telomeres, and the mating type locus are predominantly localized at the nuclear periphery (marked by Cut11–mCherry nuclear pore component). These peripheral GFP–Swi6 foci are lost in ∆clr4 cells, but in clr4F449Y cells, in which H3K9me2 is restricted primarily to pericentromeric repeats, one fluorescent focus corresponding to centromeres (indicated by white arrows), which cluster at the nuclear periphery independently of H3K9me, is maintained. The peripheral GFP–Swi6 focus in clr4F449Y cells is weaker than that in clr4+ cells, which is probably a results of the lower affinity of Swi6 for H3K9me2 relative to H3K9me3. Correspondingly, diffuse GFP–Swi6 signals are observed throughout the nucleoplasm in clr4F449Y cells. Three representative cells for each genotype are presented. 18 of 18 clr4+, 15 of 16 clr4F449Y, and 0 of 10 ∆clr4 cells had perinuclear localization of GFP–Swi6. c–g, Flag–Chp2 (c), Flag–Clr3 (d), H3K9me3 (e), H3K9me2 (f), and Flag–Clr4 (g) ChIP–qPCR analysis at dg and dh in clr4 wild-type and mutant cells. Error bars, s.d.; n = 3 biological replicates.
a, b, ChIP–qPCR analysis showing loss of H3K9me2 at the 10xtetO-ade6+ reporter gene (a) and the adjacent mug135+ gene (b) upon the release of tethered TetR–Clr4-I by the addition of tetracycline (−tet versus +tet). c, ChIP–qPCR analysis showing loss of H3K9me2 at pericentromeric dg and dh DNA repeats in Δago1; clr4I418P double-mutant cells. Arrowheads indicate the location of primers used for ChIP–qPCR. Error bars, s.d.; n = 3 biological replicates.
About this article
Cite this article
Jih, G., Iglesias, N., Currie, M. et al. Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription. Nature 547, 463–467 (2017). https://doi.org/10.1038/nature23267
Nucleic Acids Research (2021)
The Journal of Biochemistry (2021)
Trends in Cell Biology (2021)
The Catalytic-Dependent and -Independent Roles of Lsd1 and Lsd2 Lysine Demethylases in Heterochromatin Formation in Schizosaccharomyces pombe
Mechanistic Insights into the Allosteric Regulation of the Clr4 Protein Lysine Methyltransferase by Autoinhibition and Automethylation
International Journal of Molecular Sciences (2020)