SET-9 and SET-26 are H3K4me3 readers and play critical roles in germline development and longevity
- Cornell University, United States
Figures
Figure 1
set-26 but not set-9 is important for longevity.
(A) Schematic of the set-9(rw5) and set-26(tm2467) mutants. Red star indicates the position of the sgRNA (single guide RNA) targeting the set-9 gene. Premature stop codons caused by deletions of 38 …
Figure 2 with 1 supplement
set-9 and set-26 act redundantly to maintain fertility.
(A) The set-9(rw5) set26(tm2467) double mutant worms derived from heterozygous parents (F1) displayed a mild fertility defect. The double mutant worms displayed a much more severe fertility defect …
Figure 2—figure supplement 1
Fertility defects of the set-9 set-26 double mutant.
(A) The set-9(rw5) set-26(tm2467) double mutant exhibited variable percentage of sterility at the different generations. N = 155–517. (B) Representative DAPI staining images showing the germline of …
Figure 3 with 2 supplements
SET-26 is broadly expressed and SET-9 is only detectable in the germline.
(A, B) Fluorescent micrographs of worms carrying gfp knock-in at the C-terminus of set-9 or set-26 gene (set-9::gfp and set-26::gfp). GFP-fused SET-26 was detected in all cells and GFP-fused SET-9 …
Figure 3—figure supplement 1
GFP-tagged SET-9 and SET-26 are largely functional.
(A–B) Worms carrying a gfp knock-in at the C-terminus of set-26 exhibited mild lifespan extension and heat resistance phenotypes, but the phenotypes were much weaker than those of the …
Figure 3—figure supplement 2
GFP-tagged SET-9 is only detectable in the germline.
Fluorescent micrographs of worms carrying gfp knock-in at the C-terminus of set-9 gene (set-9::gfp) is shown. ‘GFP’ indicates signals under GFP channel and ‘Autofluorescence’ indicates signals under …
Figure 4 with 1 supplement
DAF-16-dependent somatic SET-26 regulated genes are enriched for lifespan determinant genes.
(A) Venn diagrams show the overlap between up-regulated genes in glp-1(e2141); set-26(tm2467) (comparing with glp-1(e2141)) and down-regulated genes in daf-16(mgDf47); glp-1(e2141); set-26(tm2467) …
Figure 4—figure supplement 1
Transcriptional profile of glp-1(e2141); set-26(tm2467) mutant.
(A) GO term analysis of genes with expression change in glp-1(e2141); set-26(tm2467) (comparing with glp-1(e2141)). (B) Venn diagram shows the overlap between set-26 regulated genes in glp-1(e2141); …
Figure 5 with 1 supplement
Transcriptional profiles of set-9(rw5), set-26(tm2467), and F1 set-9(rw5) set-26(tm2467) mutants.
(A) Venn diagrams show the overlap among set-9(rw5), set-26(tm2467), and set-9(rw5) set-26(tm2467) down-regulated (left) and up-regulated (right) gene sets. Hashtag indicates genes that only show …
Figure 5—figure supplement 1
Transcriptional profile comparison between F1 and F3 set-9(rw5) set-26(tm2467) double mutants.
(A) GO term analysis of down-regulated and up-regulated genes in F1 set-9(rw5) set-26(tm2467) double mutant. (B) GO term analysis of down-regulated and up-regulated genes in F3 set-9(rw5) …
Figure 6 with 5 supplements
SET-9 and SET-26 bind to H3K4me3.
(A) PHD domain of SET-9 and SET-26 specifically pulled down H3 in vitro. Coomassie-blue stained gel showing pulldown results using GST–SET-9/26PHD and GST control. (B) Binding intensity of …
Figure 6—figure supplement 1
SET-9 and SET-26 are not the major enzymes for H3K9me3 in vivo.
(A) Clustal Omega alignment of the SET domains of SET-9 and SET-26 from C. elegans, SETD5, MLL5, SET7 and SUV91 from human, UpSET from Drosophila melanogaster and SET1 from Saccharomyces. Red boxes …
Figure 6—figure supplement 2
SET-9 and SET-26 are not the major enzymes for H3K9me3 in vivo (part 2).
(A) Heatmaps showing the H3K9me3 profiles in wild-type and the F3 set-9(rw5) set-26(tm2467) double mutant for all H3K9me3 peaks.
Figure 6—figure supplement 3
SET-9 and SET-26 share similar genomic binding profiles in vivo.
(A) Genome browser view showing ChIP z-scores (standardized log2 ratios of ChIP/Input or ChIP/H3ChIP signals) for SET-9, SET-26 and SET-9/26 at a representative region. (B) A large proportion of …
Figure 6—figure supplement 4
SET-9 and SET-26 bind to H3K4me3 in vivo.
(A) Metagene plots showing the average H3K4me3 z-score (standardized log2 ratios of H3K4me3ChIP/H3ChIP signals) for the H3K4me3 peaks bound (shown in blue) and not bound by SET-9 and SET-26 (shown …
Figure 6—figure supplement 5
SET-9 and SET-26 bind to H3K4me3 in vivo (part 2).
(A) 28% of the SET-9 and SET-26 peaks overlapped with H3K9ac peaks. (***p<0.001 indicates overlapping more than expected) (B) Scatter plot of H3K4me3 z-score and H3K9ac z-score for all H3K4me3 peak …
Figure 7 with 1 supplement
Binding of SET-9 and SET-26 regulate the RNA expression of specific target genes.
(A) Venn diagram showing comparisons of the genes with increased and decreased expression changes in the F1 set-9(rw5) set-26(tm2467) double mutant and the SET-9 and SET-26 target genes. (B) GO term …
Figure 7—figure supplement 1
SET-9 and SET-26 target genes.
(A) Venn diagrams showing comparisons of the genes with increased and decreased expression changes in the set-9(rw5) mutant. (B) GO term analyses of the up-regulated genes in set-9(rw5) bound by …
Figure 8 with 4 supplements
SET-9 and SET-26 restrict the spreading of H3K4me3.
(A) Western blotting of H3K4me3 levels in three independent replicates of wild type and F3 set-9(rw5) set-26(tm2467) double mutant (left). Synchronized L4 worms for N2 and the F3 set-9(rw5) …
Figure 8—figure supplement 1
SET-9 and SET-26 restrict the spreading of H3K4me3.
(A) H3K4me3 and H3K9me3 levels in N2, set-9(rw5) and set-26(tm2467) mutants. Synchronized L4 worms were used. Quantification showed results from three independent experiments. (B) Western blotting …
Figure 8—figure supplement 2
SET-9 and SET-26 restrict the spreading of H3K4me3 (part 2).
(A) Representative genome browser views of H3K4me3 profiles in N2 (black) and the F1 (blue) and F3 (purple) set-9(rw5) set-26(tm2467) double mutant surrounding SET-9 and SET-26 binding sites (red). …
Figure 8—figure supplement 3
SET-9 and SET-26 restrict the spreading of H3K4me3 (part 3).
(A) Metagene plots showing the average H3K4me3 z-score (standardized log2 ratios of ChIP/H3ChIP signals) in N2 (black) and the F1 (blue) and F3 (purple) set-9(rw5) set-26(tm2467) double mutants. …
Figure 8—figure supplement 4
Germline-specific genes show correlated changes in H3K4me3 and RNA expression.
(A) Venn diagram shows the overlaps between SET-9/26 target genes with expression change in F1 set-9(rw5) set-26(tm2467) and germline-specific genes. (B) Average H3K4me3 z-score in N2 (black) and …
Figure 9
SET-9 and SET-26 and SET-2 act synergistically to regulate fertility.
(A) Ratio of eggs laid by F1 set-9(rw5) set-26(tm2467) double mutant worms fed dsRNA of C. elegans potential histone modifiers (mainly methyltransferases and demethylases) or empty vector (L4440) …
Figure 10
SET-9 and SET-26 regulate H3K4me3 and target gene expression.
In the germline, loss of set-9 and set-26 results in the broadening of H3K4me3 domains surrounding most SET-9 and SET-26 binding regions and up-regulation of germline genes. In the soma, loss of set-…
Additional files
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Supplementary file 1
Quantitative data for experiments reported.
Table S1 (A) Lifespan data for Figure 1B were shown. (B) Heat stress resistance data for Figure 1C were shown. Table S2 (A) Brood size data for Figure 2A were shown. (B) Mortal germline assay data for Figure 2B were show. (C) Maternal and paternal effect on brood size for Figure 2C were shown. (D) Mitotic germ cell number for Figure 2D were shown. Table S3 (A) Wild type lifespan data with RNAi treatment for Figure 3B were shown. (B) rrf-1(pk1417) mutant lifespan data with RNAi treatment for Figure 3C were shown. Table S4 (A) Lifespan data for Figure 3—figure supplement 1A were shown. (B) Heat stress resistance data for Figure 3—figure supplement 1B were shown. (C) Brood size data for Figure 3—figure supplement 1C were shown. Table S5 (A) Histone peptide array binding assay for Figure 6B were shown. Table S6 (A) qPCR RNA-seq validation for set-26(tm2467) compare with wild type data were shown. (B) qPCR RNA-seq validation for F3 set-9(rw5) set-26(tm2467) compare with wild type data were shown.
- https://doi.org/10.7554/eLife.34970.028
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Supplementary file 2
Gene lists for comparison indicated gene list comparisons were shown.
- https://doi.org/10.7554/eLife.34970.029
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Transparent reporting form
- https://doi.org/10.7554/eLife.34970.030
