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.

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, Table S1, Bar: 10 μm), with nuclei enlarged in the inset and at larval stages L1-L4, (strain GW306, Table S1, 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 (Table S1). 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 as in (c). 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.

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.

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 based on the extinction peak height (ExtPH) and the extinction peak width (ExtPW) 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). A, represents the fluorescence of the heterochromatic reporter (GW306) in control RNAi condition and 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 based on the extinction peak height (ExtPH) and the extinction peak width (ExtPW) 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 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 the L3 stage.

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.

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, N=1. 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 (Table S3). Genes upregulated in xrn-2 RNAi treated worms [49] but not upregulated in lsm-8 mutant worms are not significantly enriched for H3K27me3 (Table S2). Statistical source data are provided in Source Data Extended Data Fig. 5.

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. 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 LSM4. 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.

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

Figure 1. LSM proteins silence heterochromatic reporters, but not euchromatic reporters.

a, Sketch of the integrated, high-copy number heterochromatic reporter pkIs1582 from strain GW306 used in the genome-wide screen . The pkIs1582 reporter is integrated as about 300 copies and expresses GFP from the ubiquitously active promoter let-858. b, Here RNAi-based derepression was monitored in progeny of all stages by increased GFP fluorescence in the nuclei. c, Fluorescence microscopy of pkIs1582-encoded GFP in L4 larvae with indicated RNAi versus control (mock/L4440). Bar, 100 μm. These experiments were repeated four times independently with similar results. d, Heterochromatic and euchromatic reporters scored by eye for derepression (+, ++: strong and very strong derepression, respectively) upon LSM RNAi (Table S1). The experiment was repeated twice independently with similar results. e, Quantitation of derepression in L1 larvae by the worm sorter following indicated RNAi. Notched box plots of fluorescence intensity in arbitrary units (a.u), with whiskers = 25^th and 75^th percentiles, min and max 5^th and 95^th percentiles, black circles outliers, thick line: median. The notch around the median represents 95% confidence interval of the median. Quantification and statistical analysis were based on n =2000 (GW306-mock), 1068 (GW306- lsm6), 613 (GW306-lsm7) and 875 (GW1108-mock), 111 (GW1108- lsm6), 1026 (GW1108-lsm7) worms pooled from 3 independent experiments. P values indicated; n.s.= non-significant; two-tailed unpaired t test. f, qPCR analysis of GFP mRNA in L1 larvae as in (e), normalized to his-56 and its-1 mRNA. GFP from GW306 strain is set as 1 (left), and mock RNAi conditions are set as 1 (right). Dots show two independent biological replicates. bars = mean. g, The two main LSM complexes and functions [26, 27]. h, GFP fluorescence of the heterochromatic reporter (pkIs1582; GW306) in L1 larvae after RNAi treatment for indicated genes. Quantification and statistical analysis were based on n =396 worms for each treatment pooled from 3 independent experiments.. P values indicated; two-tailed unpaired t test; p values > 0.05 are 0.82, 0,44 and 0.05 for lsm-1, dcap-2, and xrn-1, respectively. Statistical source data are provided in Source Data fig. 1.

Figure 2. LSM2-8 mediates heterochromatic silencing, and prevents sterility and premature death.

a, Schematic view of the lsm-8 deletion/gene replacement created by CRISPR-Cas9. b, Differential interference contrast (DIC) images of young adults (GW1120) merged with pharynx fluorescence to identify genotypes, as in (d). lsm-8^-/- worms accumulate cavities and vacuoles (black arrows), and protruding vulva (white arrows). Right, enlargement of the vulva region. Bars, 50 μm (left) and 10 μm (right); data shown represent 4 independent experiments. c, Survival assay at 22.5°C after hatching shows premature death of lsm-8^-/- worms. Bars represent mean value derived from four independent experiments, with each experimental value shown as a dot. The four experiments examined 40 worms in total per genotype. d, View of lsm-8^+/- (yellow pharynx in merge) and lsm-8^-/- (red pharynx only) worms carrying the pkIs1582 heterochromatic reporter. Red and green channels are shown separated and merged. Bar, 100 μm. Data shown represent 4 independent experiments. e, Heterochromatic reporter pkIs1582 derepression in lsm-8^-/- background compared to the WT, dcap-2^-/- and lsm-1^-/- background level. Data shown represent 2 independent experiments. Bars, 100 μm, Statistical source data are provided in Source Data fig. 2.

Figure 3. The LSM2-8 complex silences endogenous transcripts, and acts both independently and additively to H3K9 methylation.

a, Worm sorting process. L3 worms with the four following genotypes: lsm-8^-/-; met-2^-/- set-25^-/-; triple mutant and WT were sorted and harvested using the same criteria. b, Relative gene expression profiles are shown as scatter plots, with Fold-change (Fc) in log2 for two RNA-seq biological replicas of L3 sorted worms of the indicated genotype versus WT (Table S2). Each dot corresponds to a gene. Deletion of lsm-8 (lsm-8^-/-) derepresses significantly >100 genes (FDR <0.05 and Fc >4, calculated by edgeR, see Methods). c, Scatter plot comparing the relative gene expression between the lsm-8 (x axis) and the met-2 set-25 double mutant (y axis). Common up-regulated genes are shaded yellow; 36% of genes up-regulated in the lsm-8 mutant (FDR <0.05 and Fc >4) are also up-regulated (FDR<0.05 and Fc >4) in the met-2 set-25 mutant. lsm-8^-/--specific up-regulated genes are shaded in pink; met-2^-/- set-25^-/--specific are in blue. d, Comparison of the lsm-8 and met-2 set-25 mutants RNA-seq data, as in (c), overlaid by the set of genes that are up-regulated (FDR <0.05 and Fc >4) in the triple mutant met-2 set-25; lsm-8 (orange dots). The dotted red square highlights genes for which the repression pathways are clearly additive. e, Quantitation of GFP derepression expressed from the gwIs4 heterochromatic reporter in L1 progeny in WT and met-2 set-25 mutant genotypes, respectively from strains GW76 and GW637, after control or lsm-7 RNAi, displayed as in Fig. 1e. P values indicated; two-tailed unpaired t test. Quantification and statistical analysis were based on n= 1460, 2399, 1593, 1189 worms for conditions indicated from left to right, pooled from 3 independent experiments. f, Survival assay. The met-2^-/- set-25^-/-; lsm-8^-/- triple mutants die prematurely compared to the lsm-8^-/- mutant. Bars represent mean value derived from four independent experiments, with the each experimental value shown as a dot. The four experiments examined 40 worms in total per genotype. Statistical source data are provided in Source Data fig. 3.

Figure 4. Over 93% of genes silenced by LSM2-8 carry the Polycomb mark H3K27me3.

a, Scatter plot that compares the average gene expression changes in lsm-8^-/- worms (x-axis in log2, RNA-seq L3 stage) versus enrichment for a histone modification (y-axis in log2, ModEncode data of WT L3 stage). Up-regulated genes (FDR >0.05 and Fc >4 calculated by edgeR, see Methods) in the lsm-8^-/- mutant are in red to the right of the black line, and genes enriched for the histone mark are above the red line (enriched over input). b, Scatter plots as (a), with each dot representing a gene. Upper row, euchromatic marks; lower row, heterochromatin marks. % indicates genes in upper right zone: LSM-8 regulated and enriched genes for indicated mark. c, Scatter plot of absolute gene expression (normalized reads count, log2) of lsm-8^-/- versus WT. Red dots as in (b). Values under 6 (log2) are considered to have very low expression (pink shading). d, Relative gene expression profiles are shown as scatter plots, with Fold-change (Fc) in log2 for two RNA-seq biological replicas of L1 sorted mutant larvae versus WT. Each dot corresponds to a gene. Deletion of lsm-8 (lsm-8^-/-) at the L1 stage leads to a significant increase in expression of >100 genes (FDR <0.05 and Fc >4, calculated by edgeR, see Methods and Table S4) and down-regulates <60 genes. e, Scatter plots as in (b), contrasting the gene expression changes in L1 to the enrichment of the indicated histone mark over input samples. Data shown in a-e are derived from two independent RNA-seq experiments. Statistical source data are provided in Source Data fig. 4.

Figure 5. lsm-7, like the EZH2 homolog mes-2, is required to silence theegl-5 Hox gene

a, On the left, Z-projection of confocal images showing the GFP fluorescence of the egl-5 GFP HOX reporter (bxIs13) under Control (mock/L4440) RNAi, lsm-7 and mes-2 (EZH2 homolog) RNAi conditions, in adult males. On the right, merged images of the Z projection of the GFP signal with the DIC image at the best focal plan to visualize the rays of the male tail. Bar, 50 μm. b, Quantification of the number of expressing egl-5 GFP nuclei/cells under the indicated RNAi conditions per proximal region of worms. P values indicated; two-tailed unpaired t test. Bars represent mean value derived from three independent experiments, with each experimental value shown as a dot. The three experiments examined 18, 19 and 17 worms in total for RNAi conditions indicated from left to right. c, Enlarged male tail inset as in (a) showing the 9 normal rays by arrows and an example of 2 ectopic abnormal rays in mes-2 (asterisks). d, egl-5 GFP derepression is observable mostly in male tail region, as in (a) but a few nuclei (0 up to 4, as shown by the arrowheads) could also exhibit this derepression in other regions of the worm in lsm-7 and mes-2 RNAi conditions. The nucleus indicated by an asterisk express egl-5 GFP in all conditions tested. Bar, 50 μm. Data shown in a, c, and d represent results from 3 independent experiments. Statistical source data are provided in Source Data fig. 5.

Figure 6. LSM2-8 and XRN-2 work on the same silencing pathway

a, Scatter plot comparing relative gene expression changes of lsm-8^-/- L3 larvae (this study) and xrn-2 RNAi treated L4 . Common up-regulated genes are shaded yellow; 71% of genes up-regulated in the lsm-8 mutant (FDR <0.05 and Fc >4) are also up-regulated to some extent (50% increase) in xrn-2 depleted worms. lsm-8^-/--specific up-regulated genes are shaded pink; xrn-2 RNAi-specific are shaded green. Data shown are derived from two independent RNA-seq experiments (Table S2). b, Experimental flow for testing the involvement of additional factors in LSM2-8 mediated silencing. RNAi experiments were performed in parallel in WT (b-c) and lsm-8 mutant (d-f) backgrounds, from strains GW306 and GW1119, respectively, both carrying the same heterochromatic reporter pkIs1582. Derepression assay in WT background confirming derepression following RNAi of indicated factors and RNAi efficiency. c, Quantitation of GFP expression from the heterochromatic reporter pkIs1582, scored in L1 progeny under different RNAi conditions. Fluorescence intensities are displayed as in Fig. 1e. P values are indicated; two-tailed unpaired t test. Quantitation and statistical analysis were based on n= 500 worms for each condition except for the xrn-2 RNAi where n = 295 worms. Samples were pooled from 3 independent experiments. d, Scheme for analysis of epistasis of RNAi targets with lsm-8 mutant worms bearing the reporter pkIs1582. e, Fluorescence microscopy of L4 larvae showing same/ non-additive (+) and additive (++) derepression of the reporter pkIs1582 in lsm-8^-/- worms under indicated RNAi conditions. Bar, 50 μm. Data shown in e-f represent two independent experiments. f, Quantitation of GFP intensity by semi-automated analysis of microscopic images as in e, displayed as box plots overlaid with dots showing the individual sample values. Quantification and statistical analysis were based on n = 55, 45, 22, 11, 10, 23, 25, 25, 85 worms for RNAi conditions indicated from left to right, pooled from two independent experiments. P values were assessed with a two-tailed unpaired t test, and are 0.42, 0.38, 0.11, 0.76, 0.59; 0.0034, 0.0023 and 0.0001 respectively. Statistical source data are provided in Source Data fig. 6.

Figure 7. LSM2-8 mediates silencing primarily through RNA degradation.

a, H3K27me3 ChIP-qPCR on lsm-8-target genes in WT and lsm-8^-/- worms. Bars represent mean value derived from three independent experiments, with each experimental value shown as a dot, except for otpl-8 and irdl-59, for which two independent experiments were done. b, WT lsm-8^-/- and mes-2^-/- worms treated with 50 µg/ml of the transcriptional inhibitor α-amanitin for indicated times. Levels of transcripts from 3 genes regulated by LSM-8 (see Extended Data Figure 7) were tested by RT-qPCR and normalized to 18S rRNA levels. 0h was defined as 100%. c, RNA levels of the pre-mRNA and mRNA of GFP from the heterochromatic reporter pkIs1582 from the strain GW306 were determined by RT-qPCR and normalized to pmp-3 mRNA. The levels on mock RNAi conditions are defined as 1. mes-2 RNAi depletes MES-2/PRC2-like and H3K27me3 levels; set-25 RNAi depletes SET-25 and H3K9me3 levels. Bars in b and c represent mean value derived from three independent experiments, with each experimental value shown as a dot. d, LSM2-8 complex and XRN-2 silence transcripts arising from heterochromatic H3K27me3-enriched domains through RNA degradation. The LSM-8 mediated silencing pathway makes use of XRN-2 ribonuclease, and may involve other transcript binding factors, such as PABP-2 (HsPABPN1, see Discussion). We hypothesize that RNA arising from H3K27me3 genomic regions that are controlled by the LSM2-8 complex may acquire a specific feature during transcription (e.g. a specific structure, RNA modification, 3'UTR, poly-A/U tail, or specific RNA binding protein(s)), that allows recognition and processing by LSM2-8. LSM2-8-mediated silencing also feeds back to regulate H3K27me3 levels on LSM-8-regulated genes, although it is unclear if the interaction with PRC2 or H3K27me3 is direct (dotted arrow). The LSM-2-8-mediated silencing of H3K27me3-bound loci defines a selective post-/co-transcriptional silencing through RNA decay, beyond the transcriptional repression attributed to facultative heterochromatin. Statistical source data are provided in Source Data fig. 7.