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Histone H3K9 methylation is dispensable for Caenorhabditis elegans development but suppresses RNA:DNA hybrid-associated repeat instability

Abstract

Histone H3 lysine 9 (H3K9) methylation is a conserved modification that generally represses transcription. In Caenorhabditis elegans it is enriched on silent tissue-specific genes and repetitive elements. In met-2 set-25 double mutants, which lack all H3K9 methylation (H3K9me), embryos differentiate normally, although mutant adults are sterile owing to extensive DNA-damage-driven apoptosis in the germ line. Transposons and simple repeats are derepressed in both germline and somatic tissues. This unprogrammed transcription correlates with increased rates of repeat-specific insertions and deletions, copy number variation, R loops and enhanced sensitivity to replication stress. We propose that H3K9me2 or H3K9me3 stabilizes and protects repeat-rich genomes by suppressing transcription-induced replication stress.

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Figure 1: Worms lacking H3K9me were viable but showed stochastically delayed development.
Figure 2: DNA-damage-checkpoint-dependent increase of apoptotic cells in the germ line of met-2 set-25 worms.
Figure 3: Differential enrichment of H3K9me2 and H3K9me3 on repeat element classes and gene types.
Figure 4: Temperature-dependent derepression of subsets of genes and repeat families in embryos and gonads in met-2 set-25 worms.
Figure 5: met-2 set-25 worms accumulate RNA:DNA hybrids at repeat elements.
Figure 6: The met-2 set-25 strain is hydroxyurea sensitive and accumulates mutations in repeat elements and reactivated transposable elements.
Figure 7: Somatic accumulation of indels leading to frameshift mutations in met-2 set-25 mutant larvae.
Figure 8: Transcribed REs in H3K9me-deficient strains can exacerbate replication stress provoking genomic instability.

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Acknowledgements

A number of strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank R. Ciosk and P. Pasero for reagents and materials, I. Katiç and members of the Friedrich Miescher Institute Genomics and Microscopy facilities for advice and discussion, and P. Ginno and L. Constantino for advice on R-loop detection. J.P. is supported by a long-term EMBO fellowship. S.M.G. thanks the Swiss National Science Foundation as well as the Novartis Research Foundation for support.

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P.Z. and J.P. planned and executed most experiments, evaluated results and wrote the paper; S.M.G. planned experiments, evaluated results and wrote the paper; R.v.S. and M.T. helped with evaluation of genome mutations and provided the LacZ mutagenesis assay; and V.K. provided invaluable technical help.

Corresponding author

Correspondence to Susan M Gasser.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Immunofluorescence (IF) confirms absence of H3K9me in met-2 set-25 worms.

IF images of wild-type (wt) and met-2 set-25 worms showing the loss of H3K9me2/me3 at the indicated developmental stages grown at 20 °C (Online Methods). The staining of H4 pan-acetyl (H4ac) served as a positive control. (a) Gonads (bar, 20 μm). (b) Embryos and L2-stage larvae (bar, 20 μm).

Supplementary Figure 2 Phenotypes of strains losing H3K9me in the germ line.

(a) Number of viable progeny of wt and met-2 set-25 per worm at 26 °C, over three generations, starting at generation 3 after transfer of the worms from 20 °C (number of independent experiments (N) = 3, number of worms counted per experiment (n) = 60). (b) Number of viable progeny of wt, met-2 set-25 and mutants of the PIWI pathway per worm at 15 °C, 20 °C and 25 °C by generation 3 after transfer from 20 °C to the indicated temperature (N = 1, n = 25). (c) Percentage of worms developing full gonad arms at 20 °C. mex-5:gfp-h2b was used to visualize gonad cells at all stages (N = 7, n = 2). (d) Analysis of RNA-seq data showing average fold change in expression of apoptosis response genes in the gonads of met-2 set-25 mutant versus wt (N = 3, P < 0.05 adjusted for multiple testing; FDR < 0.05). (e) Percentage of laid eggs hatching at 20 °C. Strains deficient for CEP-1 (p53) additionally expressed the CED-1::GFP apoptosis reporter (N = 2, n = 80). (f) Analysis of RAD-51 foci detected by IF in the mitotic zone of the indicated genotypes to quantify the presence of resected DNA double-strand breaks. The percentage in images is equal to the frequency of mitotic tip cells with detectable RAD-51 foci. The bar graph further segregates positive cells by number of foci per cell (N = 3, n = 40 gonads).

Supplementary Figure 3 H3K9me distribution on genes and repeats.

(a) H3K9me2 and H3K9me3 ChIP-seq were performed on early embryos at 20 °C (N = 2) in a wild-type (N2) strain. Distribution along chr. I in relation to repetitive elements (REs) is shown. Quantification to the right shows the ratio of the coverage of the indicated histone modification on chromosome arms (outer two-thirds) versus the center (inner one-third). H3K27me3 and H3K4me3 mapping data are from the modENCODE project based on ChIP performed on mixed-population embryos, 20 °C. (b) Metaplot and heat map showing log2 enrichment of H3K9me2 and H3K9me3 (IP versus input) over gene bodies. Each row represents one gene, displaying the binned coding region plus 1 kb upstream of the transcription start site (TSS) and 1 kb downstream of the transcription termination sites (TES). Blue is most enriched, red is least enriched. (c) High-density scatterplot showing H3K9me2 and me3 enrichment over muscle-specific genes and pseudogenes. The upper number indicates the percentage of genes that are H3K9me3 positive (including K9me2 positive and negative), and the lower number indicates the percentage of genes that are H3K9me2 positive but H3K9me3 negative (d,e) Distribution of H3K9me2 and H3K9me3 determined by ChIP-seq over the gene body of a gene with tissue-spec expression (d) neuronal unc-54, and a cluster of pseudogenes (e).

Supplementary Figure 4 Gene and repeat element derepression in the absence of H3K9me.

(a,b) H3K9me2 and H3K9me3 ChIP-seq was performed on early embryos at 20 °C (N = 2) in a wt strain, and gene expression was determined by two replicas of RNA-seq from embryos (20 °C and 25 °C) and from isolated gonads (20 °C) from wt and met-2 set-25 strains. Scatterplots show H3K9me2 and H3K9me3 enrichment over the genes that are derepressed in early embryos at 20 °C and 25 °C, and in the gonads of met-2 set-25 animals, versus wt. Number indicates the percentage of derepressed genes enriched for either H3K9me2 or H3K9me3 in wt embryos. (b) Scatterplot of H3K9me2 and H3K9me3 enrichment over repeat subfamilies. Red dots mark repeat subfamilies that are derepressed in the indicated tissue and conditions; black dots represent non-affected repeat subfamilies. The red number indicates the percentage of derepressed repeat subfamilies that are either H3K9me2 or H3K9me3 positive. (c) qPCR verification of a subset of REs that were detected as derepressed (>2 fold, met-2 set-25/wt) in met-2 set-25 embryos at 20 °C. Expression of the same REs was additionally analyzed in gonads and L1-stage larval RNA. REs of all three main classes are detected (N = 3), but clearly there are strong stage-specific expression differences, with larvae and embryos showing more similarity.

Supplementary Figure 5 R-loop accumulation on repeat elements (REs).

(a) RNA:DNA hybrids were detected on dot plots of genomic DNA isolated from the indicated genotypes grown at 20 °C or 25 °C (three blots at each temperature were quantified by scanning for Fig. 5a). Equal amounts of DNA (determined by OD260/280) extracted from adult worms were spotted with or without RNase H treatment in decreasing concentrations (4, 2 and 1 μg). The nitrocellulose membrane was probed with the RNA:DNA-specific S9.6 antibody (n(20 °C) = 3, n(25 °C) = 3). Quantification on the right side with signals normalized to the background of each blot. (b) DRIP-seq signals in wt embryos are shown for genes grouped on the basis of their transcriptional activity. The upper box blot shows the level of transcription of the separate groups (N = 1). This enhancement of R loops on very highly expressed genes has been observed in many organisms. (c,d) Graphs show the accumulation of RNA:DNA hybrids in wt and met-2 set-25 embryos relative to the distribution of H3K9me2 and me3 over the rDNA cluster (c) or the right telomere of chr. I (d), in wt embryos. (e) DRIP-seq examples showing the R-loop signal over two RE clusters. The ChIP signal from antibody S9.6, which is specific for RNA:DNA hybrids, was normalized to input, and the RNase H control values were subtracted.

Supplementary Figure 6 Germline mutations in met-2 set-25 worms.

Further verification and characterization of the mutations detected in the genome sequencing experiment described in Figure 6. (a) Number and distribution of single-nucleotide variants (SNVs) or polymorphisms observed in the sequencing experiment described in Figure 6a. No dinucleotide preferences were found among SNVs. (b) Sketch of a complex rearrangement involving a Tc3 transposon, found exclusively in the met-2 set-25 genome. The rearrangement was identified by genome sequencing. The graph to the left indicates the precise site of Tc3 insertion. Below are H3K9me2 and H3K9me3 ChIP-seq tracks for the region around the Tc3 transposon that provoked the inversion: it bears high levels of H3K9me3 and showed roughly a twofold change in expression in met-2 set-25 over wt gonads at 20 °C. (c) Southern blotting with a probe against Tc3 shows a novel band detected in the met-2 set-25 mutant. (d) Depiction of the rearrangement shown in b indicating the position of the primer pairs used to verify the rearranged genomic context by PCR. PCR confirmation of the rearrangement is shown to the right. (e) Copy number ratios of met-2 set-25 worms relative to wt for the entire telomeric repeat subfamily, and for single telomere repeats. (f) Copy number ratios of met-2 set-25 worms relative to wt for rDNA repeats.

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Zeller, P., Padeken, J., van Schendel, R. et al. Histone H3K9 methylation is dispensable for Caenorhabditis elegans development but suppresses RNA:DNA hybrid-associated repeat instability. Nat Genet 48, 1385–1395 (2016). https://doi.org/10.1038/ng.3672

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