Genetics and Genomics

Identification of a somatic H3K23me3 methyltransferase SET-19 in C. elegans

Mingjing Xu
Zixue Fan
Chaoyue Yan
Xiangyang Chen
Xinya Huang
Chengming Zhu
Minjie Hong
Jiewei Cheng
Xinhao Hou
Shuju Li
Mengfeng Li
Yunyu Shi
Meng Huang author has email address
Shouhong Guang author has email address
Xuezhu Feng author has email address

Department of Obstetrics and Gynecology, The First Affiliated Hospital of USTC, The USTC RNA Institute, Ministry of Education Key Laboratory for Membraneless Organelles & Cellular Dynamics, Hefei National Research Center for Physical Sciences at the Microscale, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China
School of Basic Medical Sciences, Anhui Medical University, Hefei, China
School of Food and Biological Engineering, Hefei University of Technology, Hefei, China

https://doi.org/10.7554/eLife.111326.1

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Figures and data

Loss of SET-19 leads to a marked reduction of H3K23me3.
a, Schematic representation of the set-19 and set-32 genes. The set-19(ok1813) allele contains two large deletions and likely functions as a null allele. The set-19(ust644) allele carries a precise deletion of the SET domain, and the open reading frame is retained. The set-32(ok1457) allele contains a deletion that does not disrupt the open reading frame (ORF), whereas set-32(ust737) harbors a large deletion and likely functions as a null allele. SET domains are indicated in purple. b, Heatmap showing relative abundances of histone H3 post-translational modifications (PTMs) measured by mass spectrometry. Values represent log₂(fold change) in set-19(ok1813) mutants relative to wild-type (wt) animals for each biological replicate. c, d, Quantification of H3(9–17) and H3(18–26) methylation states in wild-type and set-19(ok1813) animals at the indicated developmental stages. Methylation levels are shown as relative abundances, calculated as the area under the curve (AUC) of each modified peptide divided by the sum of all detected forms of that peptide. Differences without indicated p values are not statistically significant (two-tailed t test, p < 0.05). N = 3 biological replicates. e, Western blotting analysis of global H3 methylation levels in L3–L4 stage worms of the indicated genotypes. f, Western blotting analysis of H3K23me1/2/3 levels in L3–L4 worms. The fib-1p::gfp::set-19(ustIS338) transgene was used for rescue.

SET-19 specifically catalyzes histone H3K23 methylation in vitro.
a, Schematic diagram of SET-19 and SET-32 proteins. Purple: SET domain, blue: disordered region, pink: coiled-coil region. Recombinant SET-19 SET or SET+CC fragments are shown in green. b, In vitro histone methyltransferase assays using GST-fused SET-19 SET or SET+CC fragments or full-length SET-32 incubated with histone H3 and 80 μM S-adenosylmethionine (SAM). Methylation was detected by western blotting. c, In vitro methylation assays performed as in (b) with increasing concentrations of SAM.

SET-19 is required for genomic H3K23me3 occupancy and transcriptional repression.
a, Heatmaps showing ChIP-seq signal enrichment of heterochromatic marks (H3K9me3, H3K27me3) and euchromatic marks (H3K4me3, H3K36me3) centered on H3K23me3 peak summits. The average signal within ±3 kb regions is shown. b, Genome browser view of H3K23me3 ChIP-seq signals across chromosome III in wild-type and set-19(ok1813) animals. Mean log₂ enrichment over input is shown (N = 2 biological replicates). c, Heatmaps comparing H3K23me3 profiles on H3K23me3 target regions in wild-type and set-19(ok1813) worms. Mean log₂ enrichment over input within ±3 kb of peak summits is shown. d, Heatmaps of H3K23me3 enrichment across gene bodies in wild-type worms. Genes were grouped into quintiles based on RNA-seq expression levels; genes with zero expression were excluded. e, Average H3K23me3 profiles corresponding to the gene groups shown in (d). f, RNA-seq analysis of set-19(ok1813) relative to wild-type worms. Volcano plot showing log₂FoldChange (set-19/wt) versus -log₁₀(adjusted p value [padj]), calculated using DESeq2. Significantly upregulated and downregulated genes are highlighted (padj < 0.01; |log₂FoldChange| > 1). N = 3 biological replicates. g, H3K23me3 enrichment [log₂(IP/Input)] across gene bodies for genes upregulated in set-19 mutants. Heatmaps showing a global reduction in the H3K23me3 signal in set-19(ok1813) relative to wild-type animals, with average enrichment profiles shown above.

SET-19 is dispensable for feeding RNAi and its inheritance.
a, Representative fluorescence images of animals expressing pie-1_p::gfp::h2b subjected to gfp RNAi or control treatment. F1 progeny were maintained on control bacteria without feeding RNAi. b, The percentages of the indicated P0 and F1 animals expressing GFP were quantified. At least 30 animals were scored per genotype and generation. c, Representative fluorescence images of animals expressing sur-5p::gfp following gfp RNAi treatment. d, Quantification of relative GFP fluorescence intensity for animals shown in (c). Data are presented as the mean ± SD (N = 3 biological replicates; n > 18 worms per genotype). Statistical significance was assessed using a two-tailed t test.

SET-19 is expressed in somatic cells and required for developmental timing.
a, Brood size of the indicated genotypes maintained at 20°C. Individual L4 animals were transferred to fresh NGM plates, and total progeny were counted. b, Hatch rate of embryos laid by synchronized adult hermaphrodites at 20°C. The hatch rate was calculated as the ratio of hatched larvae to total embryos. c, Developmental progression of the indicated genotypes at 20°C (n > 50). d, The expression levels of set-19 mRNA at different developmental stages. Data were downloaded from WormBase (version WS297). EE, early embryos; LE, late embryos; YA, young adults. e, The expression levels of set-19 mRNA in different tissues at the L2 stage ⁶⁵. f, Fluorescence images of L2 animals expressing GFP::SET-19. g, Fluorescence images of L3 animals expressing GFP::SET-19 and H2B::mCherry. GFP::SET-19 shows enrichment in somatic cells. h, Subcellular localization of GFP::SET-19 and H2B::mCherry in embryos.

SET-19 mediates H3K23me3 predominantly in somatic cells.
a, Representative immunofluorescence images of H3K23me3 staining in L2-stage worms. b, Representative H3K23me3 immunostaining in embryos. c, Representative images (left) and quantification of H3K23me3 immunostaining in the intestinal nuclei of young adult worms (right). H3K23me3 levels were calculated as nuclear fluorescence intensity after background subtraction, with three nuclei averaged per worm. H3K23me3 (green); DAPI (blue). Signal quantification was performed using ImageJ. A two-tailed t test was performed to determine statistical significance. For intestinal nuclei: wild type, n = 60 worms, N = 3 biological replicates; set-19(ok1813), n = 60 worms, N = 3 biological replicates; set-19(ust644), n = 60 worms, N = 3 biological replicates; set-32(ok1457), n = 51 worms, N = 2 biological replicates; set-32(ust737), n = 60 worms, N = 3 biological replicates. d, Representative images (left) and quantification of H3K23me3 immunostaining in the germline nuclei of young adult worms (right). H3K23me3 levels were calculated as nuclear fluorescence intensity after background subtraction, with three nuclei averaged per worm. H3K23me3 (green); DAPI (blue). Signal quantification was performed using ImageJ. A two-tailed t test was performed to determine statistical significance. For germline nuclei: wild type, n = 46 worms, N = 3 biological replicates; set-19(ok1813), n = 44 worms, N = 3 biological replicates; set-19(ust644), n = 42 worms, N = 2 biological replicates; set-32(ok1457), n = 29 worms, N = 2 biological replicates; set-32(ust737), n = 18 worms, N = 2 biological replicates.

A working model for somatic and germline H3K23 methylation in C. elegans.
(Left) In somatic cells, SET-19 acts as the major H3K23 methyltransferase. SET-19-catalyzed H3K23me3 coexists with H3K9me3 and H3K27me3 in heterochromatic regions and represses transcription. (Right) In the germ line, SET-32 and SET-21 participate in the maintenance of H3K23me3 downstream of the HRDE-1-mediated RNAi pathway and contribute to the transgenerational inheritance of RNAi.

Mass spectrometry analysis of global H3 methylation in set-19(ok1813) mutants.
Quantification of H3 methylation levels in wild-type and set-19(ok1813) mutants by quantitative mass spectrometry. H3 methylation levels were quantified as relative abundances, calculated by dividing the area under the curve (AUC) of each modified peptide by the sum of the AUCs of all observed forms of that peptide and multiplying by 100. Differences without indicated p values are not statistically significant (two-tailed t test, p < 0.05). N = 3 biological replicates.

The loss of set-19 reduces H3K23 methylation levels.
a, Western blotting analysis of global H3 methylation levels in embryos of the indicated genotypes. b, Quantification of H3K23me1/2/3 levels in L3–L4 worms, related to Fig. 1f. N = 3 biological replicates; two-tailed t test. c, Western blotting analysis of H3K23me1/2/3 levels in embryos. The fib-1p::gfp::set-19(ustIS338) transgene was used for rescue.

SET-19 specifically catalyzes histone H3K23 methylation in vitro.
a, Multiple sequence alignment of histone H3 proteins across species, highlighting the conserved lysine 23 residue. b, c, Independent replicates of in vitro methylation assays corresponding to Fig. 2b and Fig. 2c, respectively.

The loss of set-19 alters genomic H3K23me3 occupancy.
a, Genome-wide distribution of ChIP-seq peaks for H3K4me3, H3K36me3, H3K9me3, H3K27me3, and H3K23me3, as identified by MACS2. b, Distribution of H3K23me3 ChIP-seq peaks across chromosomes in wild-type worms. Peak positions identified by MACS2 from one representative biological replicate (replicate 1) are shown along each chromosome. Chromosome arms and centers are indicated. c, Comparison of H3K23me3 ChIP-seq signal distributions across chromosomes in wild-type and set-19(ok1813) worms. Mean log₂ enrichment over input is shown (N = 2 biological replicates). d, Differential H3K23me3 peak analysis between set-19(ok1813) and wild-type worms. Volcano plot showing log₂FoldChange (set-19/wt) versus -log₁₀(adjusted p value [padj]) from DiffBind ⁵⁴ analysis. Differentially enriched peaks are highlighted (|log₂FoldChange|>1 and padj < 0.01). N = 2 biological replicates.

SET-19 is dispensable for feeding RNAi responses.
a, b, Quantification of embryonic lethality following feeding RNAi targeting pos-1 (a) or mex-3 (b). Synchronized adult hermaphrodites of the indicated genotypes were exposed to dsRNA-expressing bacteria, and the ratio of hatched offspring was scored. c, Analysis of RNAi efficiency using dpy-11 feeding RNAi. The indicated animals were exposed to dpy-11 dsRNA until adulthood, and the relative body length of F1 progeny was measured. Individual data points represent single animals. d, Quantification of paralysis phenotypes following unc-15 RNAi in the indicated genotypes. The percentage of paralyzed animals was scored. e, Animals of the indicated genotypes were exposed to dpy-11 RNAi in the P0 generation and transferred to control bacteria. Representative images of P0 and F1 animals are shown (left). The relative body length of P0 and F1 animals was quantified (right). f, Larval arrest analysis following lir-1 RNAi in the eri-1(mg366) background. Representative images of larvae are shown (left), and the percentage of animals exhibiting larval arrest was quantified (right). g, Analysis of the RNAi response to dpy-13 feeding RNAi in the eri-1(mg366) background. The indicated animals were exposed to dsRNA until adulthood, and the relative body length was measured.

Expression pattern of SET-32.
a, Fluorescence images of L4-stage animals expressing GFP::SET-32. b, Subcellular localization of GFP::SET-32 in the germline at different developmental stages. c, Subcellular localization of GFP::SET-32 in embryos.

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