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. 2021 Jan 1;35(1-2):82-101.
doi: 10.1101/gad.344234.120. Epub 2020 Dec 10.

Argonaute NRDE-3 and MBT domain protein LIN-61 redundantly recruit an H3K9me3 HMT to prevent embryonic lethality and transposon expression

Jan Padeken  1 , Stephen Methot  1 , Peter Zeller  1   2 , Colin E Delaney  1 , Veronique Kalck  1 , Susan M Gasser  1   2
Affiliations

Argonaute NRDE-3 and MBT domain protein LIN-61 redundantly recruit an H3K9me3 HMT to prevent embryonic lethality and transposon expression

Jan Padeken et al. Genes Dev. .

Abstract

The establishment and maintenance of chromatin domains shape the epigenetic memory of a cell, with the methylation of histone H3 lysine 9 (H3K9me) defining transcriptionally silent heterochromatin. We show here that the C. elegans SET-25 (SUV39/G9a) histone methyltransferase (HMT), which catalyzes H3K9me1, me2 and me3, can establish repressed chromatin domains de novo, unlike the SETDB1 homolog MET-2. Thus, SET-25 is needed to silence novel insertions of RNA or DNA transposons, and repress tissue-specific genes de novo during development. We identify two partially redundant pathways that recruit SET-25 to its targets. One pathway requires LIN-61 (L3MBTL2), which uses its four MBT domains to bind the H3K9me2 deposited by MET-2. The second pathway functions independently of MET-2 and involves the somatic Argonaute NRDE-3 and small RNAs. This pathway targets primarily highly conserved RNA and DNA transposons. These redundant SET-25 targeting pathways (MET-2-LIN-61-SET-25 and NRDE-3-SET-25) ensure repression of intact transposons and de novo insertions, while MET-2 can act alone to repress simple and satellite repeats. Removal of both pathways in the met-2;nrde-3 double mutant leads to the loss of somatic H3K9me2 and me3 and the synergistic derepression of transposons in embryos, strongly elevating embryonic lethality.

Keywords: Argonaute; HMT; LIN-61; MBT domain proteins; MET-2; NRDE-3; SET-25; heterochromatin; histone methyltransferases (HMTs); transposon silencing.

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Figures

Figure 1.
Figure 1.
SET-25 is required for de novo establishment of H3K9me2/me3. (A) Immunofluorescence and quantitation of H3K9me2, H3K9me3 and H4 in wt, met-2(n4256), set-25(n5021) and met-2(n4256) set-25(n5021) embryos, hereafter referred to as met-2, set-25, and met-2 set-25. Fluorescence intensity was normalized to H4. Signal quantified by automated image analysis. For H3K9me2, N = 2; wt n = 1917, met-2 n = 1088, set-25 n = 1177, met-2 set-25 n = 1021. For H3K9me3, N = 3; wt n = 1467, met-2 n = 851, set-25 n = 1958, met-2 set-25 n = 1405. Scale bar, 5 µm. (B) Scheme of the heterochromatic reporter (gwIs4) and de novo silencing assay. The reporter gwIs4 [baf-1p::GFP-lacI::let-858 3′UTR; myo-3p::RFP], is integrated in a single site as an array of ∼280 tandem copies. Each copy carries a single lacO site that allows visualization of the array (Meister et al. 2010). To assay maintenance of repression, wild-type hermaphrodites bearing gwIS4 were crossed with met-2 or set-25 mutants and for de novo silencing adult met-2 set-25 double mutants bearing gwIS4 were crossed with either met-2 or set-25 single mutants. Embryos of the F2 generation homozygous for the gwIs4 reporter and either the met-2 or set-25 deletion were scored for colocalization of H3K9me2, H3K9me3, and GFP. (C) Quantitation of the mean H3K9me2 and H3K9me3 signal colocalizing with the GFP signal in wild-type (wt), met-2, and set-25 embryos from B. Yellow shading monitors maintenance and violet shading reflects de novo establishment of the marks on offspring from a met-2 set-25 double mutant. N = 3, n = 68. (****) P < 0.00001 by two-sided Wilcoxon signed-rank test for H3K9me2 met-2 versus set-25 following crosses with the gwIs4 met-2 set-25 parent.
Figure 2.
Figure 2.
Nondegenerated transposons and a subset of genes retain H3K9me3 in met-2 mutant. (A) H3K9me2 and H3K9me3 ChIP-seq was performed on early embryos (up to 100 cells) at 20°C in wt, set-25, and met-2 strains. The mean log2 enrichment over input along a typical autosome, Chr V, is shown (N = 2). (B) Scatter plot correlating the genome-wide mean log2 fold change of H3K9me3 over input to the mean log2 fold change of H3K9me2 over input at 500-bp intervals in the met-2 mutant. (C) Percentage of H3K9me2- or me3-positive loci (log2 IP over input >1) overlapping with pseudo genes, genes, repetitive elements, and intergenic regions in met-2 mutant embryos. (D) Percentage of repetitive element copies per repeat subfamily (DFAM annotation) that retain H3K9me2/me3 in met-2(n4256) mutants at minimum two copies per subfamily. Repeat classification (class.) is annotated by color code at the left. (E) The mean enrichment of H3K9me3 and H3K9me2 IP over input at repetitive elements with a high (>5000), medium (<5000 and >1000), or low (<1000) conservation score (based on DFAM bit score) in wt, met-2, and set-25 mutants. (F) Metaplots of mean log2 fold change H3K9me3 over input in the met-2 mutant at TC1 (Tc1-Mariner-like DNA transposon), LINE2A (LINE [class II] RNA transposon) and MSAT1 (satellite repeat) repetitive elements with a high conservation score (dark blue) and medium/low conservation score (gray). H3K9me3 in met-2 mutant cells is enriched only on conserved elements. Standard deviation between individual regions is indicated as a semitransparent shading.
Figure 3.
Figure 3.
RNAi screen identifies Argonaut NRDE-3 and MBT domain protein LIN-61 as essential for SET-25 targeting to heterochromatin. (A) Scheme describing the RNAi screen for factors essential for SET-25 targeting to the heterochromatic reporter (gwIs4). Wild-type (wt) and met-2 mutant worms expressing gfp::lacI from the heterochromatic reporter (gwIs4) were treated with RNAi against candidate genes, or empty vector (L4440). F1 embryos were screened for loss of colocalization of the mCherry::SET-25 and GFP::LacI. (B) Live-cell fluorescent images of wt and met-2(n4256) embryos, ectopically expressing mcherry::set-25 and gfp::laci from the gwIs4 heterochromatic reporter (Towbin et al. 2012) showing colocalization. Scale bar, 5 µm. (C) Table summarizing the 52 RNAi candidate screen. Effect of knockdown on mCherry::SET-25 foci in wt and met-2 mutants are indicated next to the gene name. (+) Foci present, (+/−) foci absent in a subset of embryos, (−) no foci present. Hits that showed no foci are highlighted in a darker gray. Candidates are grouped according to biological function (source http://www.wormbase.org). N = 3, nuclei scored per RNAi event = 25. (D) Scheme of siRNA pathway from ERGO-1, NRDE-2/-3/-4, to transcriptional (trx.) silencing and H3K9me3 in the somatic small RNA pathway. Dotted lines indicate genetic links, solid lines reflect molecular evidence, and “?” indicates putative mechanisms. (E) Live-cell images of embryos expressing mcherry::set-25 and gfp::lacI from the gwIs4 heterochromatic reporter in wt and met-2 embryos treated with RNAi against lin-61 and nrde-3 or the vector control. Scale bar, 5µm. (F) Quantitation of mean fluorescence signal intensity from mCherry::SET-25 at the GFP::LacI foci normalized to mean mCherry::SET-25 signal in the nucleoplasm, from selected RNAi knockdowns in wt and met-2 embryos, ruling out roles for Polycomb (mes-2 and mes-6) and HP1 homologs (hpl-1 and hpl-2). RNAi targets resulting in a significant reduction of the mCherry::SET-25 enrichment on the gwIs4 array are shaded in gray. P-values were calculated using one-sided Anova and are indicated above box plots. (****) P < 0.00001; (***) P < 0.0001; (**) P < 0.001. N = 3, n = 25.
Figure 4.
Figure 4.
RNAi or deletion of lin-61 results in a loss of SET-25 targeting and a reduction in H3K9me3. (A) Live cell imaging of foci of endogenously tagged SET-25::FLAG::mCherry (SET-25::mCherry) upon RNAi of the HP1 homologs hpl-1 and hpl-2 and the MBT domain protein lin-61. Synchronized L1 were exposed to RNAi and embryos of the F1 generation were imaged. A typical nucleus for each is enlarged. Scale bar, 5 µm. (B) Quantitation of SET-25::mCherry foci number per nucleus in the embryos shown in panel A using automated image analysis. N = 2, n(vector):236, n(hpl-1):445, n(hpl-2):288, n(lin-61):308. P-values were calculated using two-sided ANOVA and are indicated above violin plots. (****) P-value<0.00001; (n.s.) not significant). (C) H3K9me2 and H3K9me3 immunostaining of embryos isolated from wild-type (wt), met-2, lin-61(tm2649) (hereafter labeled lin-61), and lin-61(tm2649);met-2 mutants. Scale bar, 5 µm. (D) Mean immunofluorescence signal of H3K9me2 and H3K9me3 normalized to H4 using automated image analysis. For H3K9me2, N = 2; wt n = 1917, met-2 n = 1088, lin-61 n = 1147, lin-61;met-2 n = 1027. For H3K9me3, N = 3; wt n = 1467, met-2 n = 851, lin-61 n = 2455, lin-61;met-2 n = 1813. P-values were calculated using a two sided ANOVA; H3K9me3: P(wt vs. lin-61) < 0.00001, P(met-2 vs. lin-61;met-2) < 0.00001. (E) H3K9me3 ChIP-seq was performed on early embryos at 20°C in wild-type (wt), met-2, lin-61, and lin-61;met-2 strains. The mean log2 enrichment over input along a typical autosome, Chr III, is shown (N = 3). Asterisks mark regions that show H3K9me3 in met-2 and a reduction in lin-61;met-2. (F) Scatter plot showing the correlation of the genome-wide H3K9me3 enrichment over input (log2) counted >500-bp bins in wt and lin-61, as well as met-2 and lin-61;met-2 mutants. r = Pearson correlation coefficients for both correlations. (G) Metaplots compare mean enrichment of H3K9me3 over input in domains retained in either met-2 single (red) or lin-61;met-2 double (blue) mutants. Plots are anchored at the start (right panel) or the center (left panel) of the domains. See Supplemental Figure S4A for H3K9me3 signal at individual domains.
Figure 5.
Figure 5.
Loss of lin-61 is epistatic with set-25 for the derepression of genes and repetitive elements. (A) Scatter plot of transcripts from genes as log2 fold change over wt in early embryos of lin-61 mutants (two replicas shown, N = 3) at genes (left column) and from repetitive elements (right column). Loci significantly changed are in cyan (genes: FDR 0.01 and log2 fold change >2 or <−2; repetitive elements: FDR 0.05 and fold change >2 or <−2). (B) Venn diagrams showing the overlap between genes and repetitive elements derepressed in lin-61, met-2, and set-25 single mutants (genes: FDR 0.01 and log2 fold change >2 or <−2; repetitive elements: FDR 0.05; and fold change >2 or <−2). (C) Correlation between the fold change (log2) over wt for gene and repetitive element transcripts between met-2 or set-25 single and lin-61;met-2 and lin-61;set-25 double mutants. Loci significantly changed compared with wt (genes: FDR 0.01 and log2 fold change >2 or <−2; repetitive elements: FDR 0.05; and fold change >2 or <−2) are colored according to the genotype and loci significantly changed in both of the two compared genotypes are colored in dark purple. Pearson correlation coefficients (r) are in figures. (D) Percentage of derepressed copies per repeat subfamily of the 50 most derepressed repeat subfamilies from set-25, analyzed in met-2, set-25, lin-61, lin-61;set-25, and lin-61;met-2 mutants, as indicated. Repeat name and classification according to DFAM are indicated next to heat map. (TIR) Terminally inverted repeat, (Har.) harbinger TIR DNA transposon, (Tc1-M.) Tc1-Mariner-like DNA transposon, (R.C.) rolling circle DNA transposon, (SINE) short interspersed nuclear elements, (Gypsy) Gypsy endogenous retrovirus, (LINE) long interspersed nuclear elements (class II), (Bel-Pao) Bel-Pao endogenous retrovirus, (Sat.) satellite repeat.
Figure 6.
Figure 6.
NRDE-3 targets SET-25 to conserved DNA and RNA transposons in a manner that is parallel to and independent of MET-2. (A) Live-cell imaging and quantitation of endogenous set-25 fused to flag::mcherry (set-25::mcherry) in wt, nrde-3(tm1116) (hereafter nrde-3), met-2, and met-2;nrde-3(tm1116) mutant embryos. Scale bar, 5 µm. Quantitation of SET-25::mCherry foci per nucleus using automated image analysis. N = 3, n(wt):540, n(met-2):769, n(nrde-3):590, n(met-2;nrde-3):1020 ; P-values were calculated using two-sided ANOVA and are indicated above violin plots. (****) P-value < 0.00001, (n.s.) not significant. (B). Mean counts of 22-nt RNA in counts per million (cpm) in wt and met-2 (for nrde-3 and met-2;nrde-3) (see Supplemental Fig. S6C) early embryos over domains that either retain H3K9me3 in met-2 mutants (met-2-independent), or lose H3K9me3 in a met-2 mutant, but retain/gain H3K9me2 in set-25 (met-2-dependent), split byDNA strand. Solid lines indicate the mean signal and shaded regions the standard deviation. (C) H3K9me3 ChIP-seq performed on early embryos at 20°C in wt, nrde-3, met-2, and met-2;nrde-3 strains. Mean log2 enrichment over input along a typical autosome, Chr III, is shown (N = 2). (D) Scatter plot correlating the H3K9me3 signal over input (log2) in met-2;nrde-3 met-double mutants with met-2 single mutants at repetitive elements and genes. Red indicates loci enriched for H3K9me3 in met-2, but not in met-2;nrde-3 (log2 fold change >1 in met-2 and <1 in met-2;nrde-3). These are marked with a red rectangle. Colored in dark purple are loci common to both genotypes (log2 fold change >1 in met-2 and >1 in met-2;nrde-3 and genes exclusively H3K9 trimethylated in met-met-2;nrde-3 are colored in brown. (E) ChIP qPCR of H3K9me2 and H3K9me3 in wt, met-2, set-25(n5021) (hereafter set-25), nrde-3, met-2;nrde-3, met-2 set-25, and set-25;nrde-3 mutant embryos using primers spanning a domain on Chr III that retains H3K9me3 in met-2 mutants (N = 3; bars indicate mean, error bars indicate SD) (for control loci, see Supplemental Figs. S2A, S6E).
Figure 7.
Figure 7.
met-2 and nrde-3 loss leads to synergistic derepression of transposons and embryonic lethality. (A) Scatter plot of gene expression as log2 fold change over wt in early embryos of indicated mutants (two replicas shown, N = 3) at genes (left column) and repetitive elements (right column). Loci significantly changed are in color (genes: FDR 0.01 and log2 fold change >2 or <−2; repetitive elements: FDR 0.05 and fold change >2 or <−2). (B) Correlation between the fold change (log2) over wt for gene and repetitive element expression between met-2 or set-25 single and met-2;nrde-3 and set-25;nrde-3 double mutants. Loci significantly changed versus wt (genes: FDR 0.01 and log2 fold change >2 or <−2; repetitive elements: FDR 0.05 and fold change >2 or <−2) are colored according to the genotype. In dark purple are loci significantly changed in both genotypes. Genes and repetitive elements up-regulated only in met-2;nrde-3 are boxed in red. (C) The mean log2 fold change in the indicated mutants over wt in expression of repetitive elements with a high, medium or low conservation score (based on DFAM bit score) in met-2, or nrde-3 single and met-2;nrde-3 double mutants. (D) Representative bright-field images of wt embryos showing normal development and met-2;nrde-3, set-25;nrde-3, and lin-61;met-2 mutant embryos showing terminally disrupted development (red asterisk) and dead embryos (blue triangles). (E) Quantitation of percentage of dead embryos and embryos with a terminal disrupted development in wt, met-2, set-25, met-2 set-25, nrde-3, met-2;nrde-3, set-25;nrde-3, lin-61, lin-61;met-2 and lin-61;set-25 mutants at the right (N = 3, n = 500; error bars indicate SD).
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