A team of heterochromatin factors collaborates with small RNA pathways to combat repetitive elements and germline stress
- University of Cambridge, United Kingdom
Figures
Figure 1 with 1 supplement
Heterochromatin proteins have redundant roles in fertility.
(A) Genetic interactions in fertility assayed using RNAi. Indicated RNAi of wild-type, let-418(n3536), or lin-13(n770) was conducted by feeding at 20°C as described in the methods. Results are a …
Figure 1—figure supplement 1
Heterochromatin mutants display abnormal oogenesis.
(A) Strains of the indicated genotype were shifted to 25°C at the L4 stage and imaged by Nomarksi microscopy 48 hr later. Oocyte quality was determined and deemed ‘abnormal’ when oocytes appeared …
Figure 2 with 8 supplements
HPL-2, LET-418, LIN-13, LIN-61, and MET-2 show extensive co-binding and are enriched at repetitive elements.
(A) Distribution of the indicated proteins and histone modifications over each C. elegans chromosome. z-scored ChIP-seq tracks are shown for HPL-2 (red), LET-418 (blue), LIN-13 (green), LIN-61 …
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Figure 2—source data 1
Peak calls.
- https://doi.org/10.7554/eLife.21666.005
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Figure 2—source data 2
Enrichment of factors at repeat families.
- https://doi.org/10.7554/eLife.21666.006
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Figure 2—source data 3
Alignment Statistics for ChIP and RNA sequencing.
- https://doi.org/10.7554/eLife.21666.007
Figure 2—figure supplement 1
Correlation of HPL-2, LET-418, LIN-13, LIN-61, and MET-2 ChIP-seq tracks and enrichment on chromosome arms, repetitive elements, and telomeres.
(A) Distribution of HPL-2, LET-418, LIN-13, LIN-61, MET-2, All5, or Any5 ChIP-seq peaks between chromosome arms and centers (left) and between genes (introns and exons) and intergenic regions on the …
Figure 2—figure supplement 2
Enrichment of HPL-2 at repeat families.
Boxplots show HPL-2 levels measured as log2 fold change relative to the genome median for each repeat family with at least 10 individual repeat elements; boxplots are colored by repeat class. For …
Figure 2—figure supplement 3
Enrichment of LIN-61 at repeat families.
Boxplots show LIN-61 levels measured as log2 fold change relative to the genome median for each repeat family with at least 10 individual repeat elements; boxplots are colored by repeat class. For …
Figure 2—figure supplement 4
Enrichment of MET-2 at repeat families.
Boxplots show MET-2 levels measured as log2 fold change relative to the genome median for each repeat family with at least 10 individual repeat elements; boxplots are colored by repeat class. For …
Figure 2—figure supplement 5
Enrichment of LIN-13 at repeat families.
Boxplots show LIN-13 levels measured as log2 fold change relative to the genome median for each repeat family with at least 10 individual repeat elements; boxplots are colored by repeat class. For …
Figure 2—figure supplement 6
Enrichment of LET-418 at repeat families.
Boxplots show LET-418 levels measured as log2 fold change relative to the genome median for each repeat family with at least 10 individual repeat elements; boxplots are colored by repeat class. For …
Figure 2—figure supplement 7
Enrichment of H3K9me2 at repeat families.
Boxplots show H3K9me2 levels measured as log2 fold change relative to the genome median for each repeat family with at least 10 individual repeat elements; boxplots are colored by repeat class. For …
Figure 2—figure supplement 8
Enrichment of H3K9me3 at repeat families.
Boxplots show H3K9me3 levels measured as log2 fold change relative to the genome median for each repeat family with at least 10 individual repeat elements; boxplots are colored by repeat class. For …
Figure 3 with 3 supplements
Repetitive elements are upregulated in hpl-2, let-418, lin-13, lin-61, met-2 set-25, prg-1, nrde-2, and nrde-2; let-418 mutants.
(A) Venn diagram of elements upregulated in hpl-2, let-418, lin-13, lin-61, and met-2 set-25 mutants. The seven elements upregulated in all five strains are MIRAGE1 elements. (B) Distribution of …
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Figure 3—source data 1
Analysis of repeats.
- https://doi.org/10.7554/eLife.21666.017
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Figure 3—source data 2
Repeats upregulated in any mutant strain.
- https://doi.org/10.7554/eLife.21666.018
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Figure 3—source data 3
Analysis of genes.
- https://doi.org/10.7554/eLife.21666.019
Figure 3—figure supplement 1
Examples of unique and co-regulated repeat elements in various heterochromatin mutants.
IGV screenshots of repeat elements upregulated in a subset of heterochromatin mutants. (A) HELITRON1 element only upregulated in lin-13 mutants. (B) PALTTAA2 and CELE46B elements only upregulated in …
Figure 3—figure supplement 2
Heterochromatin factors and H3K9 methylation show enriched association with upregulated genes and repeats.
(A) Percent overlap of heterochromatin factor peaks or >1.5 fold enrichment for H3K9me2 or H3K9me3 on upregulated (left) or downregulated (right) genes (−500 bp to gene end). Parentheses give number …
Figure 3—figure supplement 3
MIRAGE1 RNA is upregulated in the germ lines of hpl-2, lin-13, let-418, and prg-1 mutants.
(A) Single molecule RNA-FISH signals of MIRAGE 1 RNA in the adult germ line (left panels; zoomed in on the right). Middle panels show sqv-1 (positive control) RNA-FISH and DAPI to counterstain …
Figure 4
Phenotypic suppression of hpl-2, let-418 and lin-13 by inhibition of MIRAGE1, cep-1/p53, or spo-11.
(A) RNAi of MIRAGE1, cep-1/p53 or spo-11 partially suppresses sterility of hpl-2, let-418, and lin-13. Average number of progeny per worm for control empty vector RNAi (EV) or the indicated RNAi …
Figure 5 with 1 supplement
Heterochromatin mutants have increased germline apoptosis.
CED-1::GFP (bcIs39 [Plim-7::ced-1::gfp]), expressed in gonad sheath cells, marks engulfed apoptotic cells in the pachytene region of the adult gonad. (A) CED-1::GFP images for wild type, hpl-2, and m…
Figure 5—figure supplement 1
hpl-2 mutants are hypersensitive to ionizing radiation-induced DNA damage.
(A) Phospho-CHK-1 staining of adult germ lines 1 hr post IR treatment. Bars are 10 µm. (B) Quantification of phospho-CHK-1 staining. hpl-2 mutants showed significantly more phospho-CHK-1 positive …
Figure 6 with 1 supplement
Heterochromatin factors interact with the piRNA pathway.
(A) Venn diagram showing extent of overlap between repeats upregulated in prg-1 mutants and repeats upregulated in any of the five heterochromatin factor mutant strains (hpl-2, let-418, lin-13, …
Figure 6—figure supplement 1
Quantification of piRNAs and dependent 22G RNAs in prg-1 and hpl-2 mutants.
(A) Unique piRNAs, repeat elements targeted by a piRNA, number of unique 22G RNAs mapping near piRNA target sites, and number of repeat elements hit by a piRNA-associated 22G RNA. The number in each …
Figure 7 with 3 supplements
nrde-2 and let-418 show functional redundancy.
(A) nrde-2 and let-418 mutants show genetic interaction in fertility. Brood sizes of nrde-2; hpl-2, nrde-2; let-418, and nrde-2; lin-61 double mutants were compared to those of single mutants. …
Figure 7—figure supplement 1
Overlap of nrde-2, prg-1, and heterochromatin targets.
(A) Repeat families with members upregulated in nrde-2, prg-1, and any of the five heterochromatin mutants (let-418, hpl-2, lin-13, lin-61, and met-2 set-25). Families in bold are specific to a …
Figure 7—figure supplement 2
H3K9me3 levels on repeats in nrde mutants.
(Top) H3K9me3 log2FC of average signal at indicated regions in hrde-1, nrde-2, nrde-4, and nrde-3 mutants relative to wild-type. Control repeats have > 1.5 fold average signal in wild-type relative …
Figure 7—figure supplement 3
H3K9me3 levels on genes in nrde mutants.
(Top) H3K9me3 log2FC of average signal at indicated regions in hrde-1, nrde-2, nrde-4, and nrde-3 mutants relative to wild-type. nrde-2, genes upregulated in nrde-2 mutants. For each of the other …
Figure 8
Heterochromatin proteins collaborate with small RNAi pathways to maintain fertility.
(A) Pathways of transposable element silencing in C. elegans. Heterochromatin factors participate in repetitive element silencing together with the piRNA and nuclear RNAi pathways, as well as …
Author response image 1
Quantification of piRNA sensor expression in wild type and heterochromatin mutants hpl-2, let-418 and lin-61.
Animals were fed on indicated RNAi bacteria from L1 at 20°C and scored as 1 day old adults, similar to Figure 6D. A minimum of 15 worms were scored over 2 independent experiments. None of the RNAi …
Additional files
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Supplementary file 1
LET-418, LIN-13, HPL-2, LIN-61, and MET-2 are required for normal fertility.
- https://doi.org/10.7554/eLife.21666.033
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Supplementary file 2
Strains used in this study.
- https://doi.org/10.7554/eLife.21666.034
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Supplementary file 3
Antibodies used in this study.
- https://doi.org/10.7554/eLife.21666.035
