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LSM2-8 and XRN-2 contribute to the silencing of H3K27me3-marked genes through targeted RNA decay

Abstract

In fission yeast and plants, RNA processing and degradation contribute to heterochromatin silencing, alongside conserved pathways of transcriptional repression. It has not been known whether similar pathways exist in metazoans. Here, we describe a pathway of silencing in Caenorhabditis elegans somatic cells, in which the highly conserved RNA-binding complex LSM2-8 contributes selectively to the repression of heterochromatic reporters and endogenous genes bearing the Polycomb mark, histone H3K27me3. This acts by degrading selected transcripts through the XRN-2 exoribonuclease. Disruption of the LSM2-8 pathway leads to mRNA stabilization. Unlike previously described pathways of heterochromatic RNA degradation, LSM2-8-mediated RNA degradation does not target nor require H3K9 methylation. Intriguingly, loss of this pathway coincides with a localized reduction in H3K27me3 at lsm-8-sensitive loci. We have thus uncovered a mechanism of RNA degradation that selectively contributes to the silencing of a subset of H3K27me3-marked genes, revealing a previously unrecognized layer of post-transcriptional control in metazoan heterochromatin.

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Fig. 1: LSM proteins silence heterochromatic reporters, but not euchromatic reporters.
Fig. 2: LSM2-8 mediates heterochromatic silencing and prevents sterility and premature death.
Fig. 3: The LSM2-8 complex silences endogenous transcripts and acts both independently and additively to H3K9 methylation.
Fig. 4: Over 93% of genes silenced by LSM2-8 carry the Polycomb mark H3K27me3.
Fig. 5: lsm-7, like the EZH2 homologue mes-2, is required to silence the egl-5 Hox gene.
Fig. 6: LSM2-8 and XRN-2 work on the same silencing pathway.
Fig. 7: LSM2-8 mediates silencing primarily through RNA degradation.

Data availability

RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE92851. Previously published ChIP-seq data that were re-analysed here are available from ModEncode (http://data.modencode.org/) for L3_H3K9me1/2/3 (5036, 5050, 5037, 5040), L3_H3K27me3 (5045, 5051), L3_H3K27ac (5054), L3_H3K4me2/3 (5055, 3576). Source data for Figs. 1–7 and Extended Data Figs. 1, 2 and 48 are presented with the paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

The accession number for the RNA-seq data in the NCBI Gene Expression Omnibus is GSE92851. Some strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank I. Katiç, the FMI Genomics and Microscopy facilities, P. Zeller and M. Fukushima for technical help, advice and discussions, and T.S. Miki for access to data and discussions. We thank M. Bühler, H. Großhans and W. Filipowicz for discussions and proofreading of the text. We acknowledge support from the Novartis Research Foundation, as well as a Marie Curie Intra-European grant (#PIEF-GA-2010-276589) and Swiss National Science Foundation Marie-Heim Vögtlin grant (#PMPDP3_151381, # PMPDP3_168717) to A.M., an SNF grant (#310030B_156936) to S.M.G. and support from NCCR RNA & Disease to H. Großhans (to F.A.). This project received funding from the European Research Council under the European Union’s Horizon 2020 Research and Innovation programme (Epiherigans; grant no. 743312 to S.M.G.).

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Authors and Affiliations

Authors

Contributions

A.M. planned and executed most experiments, evaluated results and wrote the paper. S.M.G. planned experiments, evaluated results and wrote the paper. D.G., with A.M., analysed the RNA-seq data and other genome-wide data. C.S. analysed the L1 RNA-seq data. J.P. performed the H3K27me3 ChIP-qPCR experiment and analysis. V.K. performed the gonad staining and analysis. F.A. helped generate the lsm-8 mutant strain.

Corresponding author

Correspondence to Susan M. Gasser.

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Extended data

Extended Data Fig. 1 LSM proteins are highly conserved and silence heterochromatic, but not euchromatic reporters.

a, LSM protein comparison between C. elegans and H. sapiens. b, Heterochromatic reporters derepression at all developmental stages. lsm-7 RNAi is compared to control RNAi. Derepression monitored by GFP live imaging was assessed at the embryonic stage (strain GW566, Supplementary Table 1, Bar: 10 μm), with nuclei enlarged in the inset and at larval stages L1-L4, (strain GW306, Supplementary Table 1, Bar: 50 μm, Bar: 100 μm for gravid adults). These observations were repeated ten times independently with similar results. c, Quantitation of derepression assays. In L1 progeny under gut-2/lsm-2, lsm-5, lsm-6 and control RNAi conditions (mock: negative control and mes-4: positive control), the GFP fluorescence intensity of the heterochromatic reporter pkIS1582 was measured by the worm sorter. F2: second generation. Quantification and statistical analysis were based on n =375 worms for each condition pooled from three independent experiments. Data are displayed as in Fig. 1e. P values indicated were calculated with a two-tailed unpaired t test. d, Quantitation of derepression of different heterochromatic reporters (Supplementary Table 1). P values indicated were calculated with a two- tailed unpaired t test. Quantification and statistical analysis were based on n= 1460, 2399, 2631, 3850, 634, 1855 worms for conditions indicated from left to right, pooled from two independent experiments. e, Confirmation of lsm-1 and lsm-7 knockdown by RNAi. qPCR analysis of lsm-7 and lsm-1 mRNA in L1 worms upon mock, lsm-7 or lsm-1 RNAi treatments. lsm-7 and lsm-1 mRNA are expressed relative to the levels in mock RNAi condition. Bars represent mean value derived from three (lsm-7 RNAi) and two independent experiments (lsm-1 RNAi), with the value of each experiment shown as dots. f, Quantitation of fluorescence intensity of the euchromatic reporter (GW849, gain2) in L1 progeny. P values calculated as in (c). Quantification and statistical analysis were based on n =375 worms for each condition pooled from three independent experiments. g, Same as in (f), with a gain=1 for the fluorescence of both the heterochromatic (GW306) and euchromatic (GW849) reporters. P values as in (c). Quantification and statistical analysis were based on n =370 worms for each condition, pooled from two independent experiments. Statistical source data are provided in Source Data. Extended Data Fig. 1.

Source data

Extended Data Fig. 2 lsm-8-/- mutant worms are 100% sterile but developing gonads resemble WT through L3 and L4 stages.

a, Z-projection of confocal images showing fixed DAPI staining of a WT (N2) worm, at L3 stage. Gonad arms are highlighted by the red dashed line and same to right with a lsm-8-/- L3 larva (GW1125). b, Quantification of the length, width and gonad nuclei count. Bars represent mean value derived from two independent experiments, with the value of each experiment shown as the dot. The two experiments examined 4 worms in total per genotype. c, DIC image of a WT young adult (YA) with a normal anatomy and normal gonad (red dashed line) with oocytes (pink shading). The white arrow indicates the vulva as in YA. DIC image of lsm-8-/-, lsm-2-/- and lsm- 5-/- YA. The gonad (red dashed line) has no forming oocytes and has an abnormal composition of cells at that stage. Black arrows indicate the presence of vacuoles. d, Heterochromatic reporter (pkIs1582) derepression in WT (GW306) background following lsm-7 RNAi in a L4 larva. The enlargement to the right shows the gonad (red dashed line) with germ cells which are not derepressed. e, Merge DIC and live GFP microscopy of lsm-8 mutant (GW1119) carrying the heterochromatic reporter pkIs1582, at the L4 larvae stage as confirmed by the vulva in the inset. The derepression of the reporter in the gonad is not detectable in germ cells, but is in the somatic gonad cells marked with asterisks: DTC (distal tip cells), gonadal sheath, spermathecal cells. f, Z-projection of confocal images showing the nuclear GFP derepression of the heterochromatic reporter pkIs1582 (GW1119) in nearly all if not all somatic cells of an lsm8-/- worm. g, GFP and DIC merged images at a single focal plan showing the optimal view of germ cells (inside dashed red line), which are not derepressed in lsm8-/- worm (GW1119) even treated with RNAi against piRNA factors such as csr-1. Scale Bar in a, c-g, 50 μm. Data in a and c-g represent results from three independent experiments, except for a and g, where the experiments have been performed twice with similar results. Statistical source data are provided in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 Worm sorting and quantification settings based on gating region.

a, COPAS Biosort conditions optimised for the quantification of the heterochromatic reporter fluorescence. The COPAS Biosort (Union Biometrica) machine is an adapted flow cytometry version that can be used in order to quantify and collect worms according to their size and fluorescence criteria. The upper panel reflects the gating region (black diamond) based on the optical density of the object (optical extinction, EXT in the y axis) and the axial length (time of flight, TOF, in the x axis) of the object selecting the L1 worm population, as determined empirically in pilot experiments by verifying the stage through microscopic examination of sorted worms with this gate criteria. The same criteria gating was identical for every quantification of the heterochromatic reporter fluorescence. The lower panel shows the worm distribution of the size-selected worms based on green parameters (green peak height (green PH) and green peak width (green PW). This lower panel in a, represents the fluorescence of the heterochromatic reporter (GW306) in control RNAi condition and in b, in lsm-7 RNAi conditions. c, COPAS Biosort conditions optimised for the sorting of homozygous lsm-8 mutant at the L3 stage. The upper panel reflects the gating region (black diamond) based on the optical density of the objects (optical extinction, EXT in the y axis) and the axial length (Time of Flight, TOF, in the x axis) of the objects selecting the L3 worm population. The lower panel shows the worm distribution based on green parameters (green peak height (green PH) and green peak width (green PW)), the second gating region (black window) shown in that panel selects here the non-green worms, homozygous for lsm-8. The gating strategies were determined empirically in pilot experiments by verifying the size, shape gonad and vulva developmental stage by microscopic examination. Morphological validations during the sorting process were also performed. Sorting of the homozygous animals was done by selecting non- GFP pharynx animals, and the gating was also determined stringently by examining the two populations and by verifying the different criteria with fluorescent microscopy.

Extended Data Fig. 4 Control of the developmental timing of the RNA-seq samples within L3 stage larvae.

a, Gene expression data were collected over larval development at 25 °C and the average expression of somatic genes that were found to increase during this time course (rising somatic genes) is plotted in the left part ([35], see Methods). This analysis allowed us to compare the average expression of somatic genes that increase naturally during development to the average expression in our RNA-seq samples within the L3 larval stage. Samples from the four biological replicates of the four different genotypes that were the closest by developmental timing were selected accordingly and assigned to replica 1 and replica 2. Those two matched replicates (developmentally timed) were used for the main bioinformatics analysis, but the other samples were used for additional validations, and confirmed the main findings. b, Relative gene expression profiles as scatter plots. Fold-change (log2) in gene expression of two biological replicas of RNA-seq from sorted L3 worms of lsm-8-/-, met-2-/- set-25-/- and the triple (lsm-8-/-, met-2-/- set-25-/-) mutant versus WT. Each dot corresponds to a gene. Red dots here are rising genes, genes with increased expression level during the time course described [35], which do not change significantly in any of the mutant strains. Statistical source data are provided in Source Data Extended Data Fig. 4.

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Extended Data Fig. 5 Genes silenced by LSM2-8 have a low steady-state expression and are not enriched on chromosome arms.

a, Scatter plots comparing the mean of log2(Fc) in lsm-8-/- vs WT (x axis) from two independent RNA-seq with the H3K9me1 mark ChIP-seq data and with additional ChIP- seq data for H3K9me3 and H3K27me3 (y axis) from ModEncode using different antibodies than those used in Figure 4. b, Scatter plots comparing absolute transcript abundances (log2 of normalized reads count) of annotated genes in lsm-8-/-, met-2-/- set-25-/- and the triple (lsm-8-/-, met-2-/- set-25-/-) mutant versus WT from the two biological replica 1 and 2. Boxes with pink background indicate low abundance values smaller than 6 in log2 scale for genes considered to be repressed in WT. This corresponds to <64 normalized RNA-seq reads per gene, in contrast to 1024 reads per gene represented by a value of 10. Note the large proportion among the genes upregulated in the assessed mutants (above the diagonal), which are repressed or very poorly expressed in WT. c, Distribution of upregulated genes in lsm-8-/- along chromosomes. LEM-2 ChIP enrichment plotted over chromosomes (embryonic WT data from [76] is in grey, indicating proximity to the nuclear periphery. Up- regulated genes in lsm-8-/- (FDR <0.05 and Fc >4) represented by the red dots are plotted over autosomes and X chromosome. Data shown represent two RNA-seq experiments. d, Comparison between our RNA-seq and other available RNA-seq datasets [49, 74, 75] in L3 stage C. elegans, for the percentage of H3K27me3-enriched genes among misregulated genes. Average of two replicas. We classify a gene as enriched for H3K27me3, if it has positive reproducible enrichment of H3K27me3 over input from two ChIP-seq datasets from ModEncode (Supplementary Table 3). Genes upregulated in xrn-2 RNAi treated worms [49] but not upregulated in lsm-8 mutant worms are not significantly enriched for H3K27me3 (Supplementary Table 2). Statistical source data are provided in Source Data Extended Data Fig. 5.

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Extended Data Fig. 6 LSM-8 ablation does not alter transcription termination accuracy, strand specificity nor splicing.

a, UCSC genome browser view showing wiggle tracks from positive (+) or negative (-) strands show the differential expression of the col-2 gene, which is upregulated in lsm-8-/- compared to WT (y axis in log2). Data shown are derived from the two independent biological RNA-seq replicas. The expression level of the neighboring genes is not affected and termination defects are not observed. All introns were as efficiently spliced in lsm-8-/- as in WT. b, G browse view showing the ModEncode ChIP-seq tracks for H3K27me1, H3K27me3 (two different antibodies) and H3K27Ac at the same genomic locus (IV:10,082,495..10, 087, 496) around the col-2 gene, as shown in (a). The col-2 gene is upregulated in lsm-8-/- compared to WT and enriched for H3K27me3, as 95% of the genes upregulated in lsm-8-/-. Statistical source data are provided in Source Data Extended Data Fig. 6.

Source data

Extended Data Fig. 7 lsm-8 deletion does not affect splicing globally.

a, RNA IP-qPCR. LSM-4-FLAG RNA IP analysis in native conditions. RNA levels were normalized to input and U1snRNA levels. ZK970.7 is upregulated in lsm-8-/- (lsm-8 target gene) and associate with LSM4 (>1), whereas F08G2.8 is not (non-target gene) and do not associate with LSM-4. Those two examples suggest that the LSM-8 complex can bind to the RNAs it regulates. Bars represent mean value derived from two independent experiments, with the value of each experiment shown as a dot. b, Reads which align on exon-exon junctions were counted in lsm-8-/- and WT worms. Scatter plot compares exon-exon junction mapped reads (log2) normalized to their intrinsic gene level in WT (x-axis) and lsm-8-/- worms (y-axis). r: Pearson correlation coefficient. c, List of genes including the 18 exon-exon junctions reproducibly affected in lsm-8-/- worms as in (b). Statistical source data are provided in Source Data Extended Data Fig. 7.

Source data

Extended Data Fig. 8 LSM2-8 promotes the degradation of specific transcripts.

a, Scheme of the RNA decay assay. WT and lsm-8 -/- worms were sorted, re-fed with OP50 in liquid culture for 1h at room temperature and treated with 50 μg/ml final concentration of α-amanitin, which inhibits Pol II and Pol III transcription. RNA was isolated at time 0, 4.5h and at 6h, as indicated for each independent experiment. b, RNA levels of three transcripts affected by LSM-8 (upper graph) and two control transcripts (expression not affected by LSM-8, lower graph) were determined by RT- qPCR and normalized to 18S rRNA levels which are insensitive to α-amanitin. The value at 0h is defined as 100%. Bars represent mean value derived from four independent experiments for eft-3, from three independent experiments for far-3, ZK970.7 and F08G2.8 and two independent experiments for grl-23, with the value of each experiment shown as the dot. Statistical source data are provided in Source Data Extended Data Fig. 8.

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Supplementary information

Supplementary Information

Supplementary Note and reference associated with Extended Data Figures.

Reporting Summary

Supplementary Table 1

List of strains and primers used in this study.

Supplementary Table 2

Processed RNA-seq data for genes in lsm-8, met-2set-25 and lsm-8, met-2set-25 triple mutant versus WT at the L3 stage.

Supplementary Table 3

Enrichment of histone marks in (ModEncode Data)

Supplementary Table 4

Processed RNA-seq data for genes in lsm-8 mutant versus WT at the L1 stage.

Supplementary Table 5

Lists of biological processes affected in the different mutants, GO analysis.

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Mattout, A., Gaidatzis, D., Padeken, J. et al. LSM2-8 and XRN-2 contribute to the silencing of H3K27me3-marked genes through targeted RNA decay. Nat Cell Biol 22, 579–590 (2020). https://doi.org/10.1038/s41556-020-0504-1

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