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. 2021 Nov 23;37(8):110050.
doi: 10.1016/j.celrep.2021.110050.

Coordinated maintenance of H3K36/K27 methylation by histone demethylases preserves germ cell identity and immortality

Nico Zaghet  1 , Katrine Madsen  1 , Federico Rossi  2 , Daniel Fernandez Perez  2 , Pier Giorgio Amendola  1 , Samuel Demharter  1 , Ulrich Pfisterer  1 , Konstantin Khodosevich  1 , Diego Pasini  3 , Anna Elisabetta Salcini  4
Affiliations

Coordinated maintenance of H3K36/K27 methylation by histone demethylases preserves germ cell identity and immortality

Nico Zaghet et al. Cell Rep. .

Abstract

Germ cells have evolved unique mechanisms to ensure the transmission of genetically and nongenetically encoded information, whose alteration compromises germ cell immortality. Chromatin factors play fundamental roles in these mechanisms. H3K36 and H3K27 methyltransferases shape and propagate a pattern of histone methylation essential for C. elegans germ cell maintenance, but the role of respective histone demethylases remains unexplored. Here, we show that jmjd-5 regulates H3K36me2 and H3K27me3 levels, preserves germline immortality, and protects germ cell identity by controlling gene expression. The transcriptional and biological effects of jmjd-5 loss can be hindered by the removal of H3K27demethylases, indicating that H3K36/K27 demethylases act in a transcriptional framework and promote the balance between H3K36 and H3K27 methylation required for germ cell immortality. Furthermore, we find that in wild-type, but not in jmjd-5 mutants, alterations of H3K36 methylation and transcription occur at high temperature, suggesting a role for jmjd-5 in adaptation to environmental changes.

Keywords: H3K27 methylation; H3K36 methylation; germ cell identity; germ cell immortality; histone demethylases; temperature.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
jmjd-5 mutant germline defects at high temperatures (A) Genomic structure of jmjd-5 gene. The H-shaped bar specifies the deletion in jmjd-5(tm3735) allele. Asterisks indicate the two amino acids (H484 and D486) mutagenized in jmjd-5(zr1234) allele. The position of the JmjC domain is indicated by a black line. (B) Mortal germline assay at 25°C, using indicated strains. Dots designate the number of fertile lines at the indicated generation. n = 8. (C) Brood size of indicated strains at 20°C and at indicated generations at 25°C. n > 10. (D) Percentage of germlines with endomitotic eggs. Data are from three biological replicas. n > 95. (E) Representative images of DAPI-stained endomitotic eggs, marked by asterisks, in animals of the indicated genotypes grown at 25°C for five generations. Scale bar: 20 μm. (F) Length of germlines (normalized to N2) at early L4 stage, measured in jmjd-5 mutants grown at 25°C at the indicated generations. n > 17. (G) Representative images of DAPI-stained germlines of N2 and jmjd-5 mutants grown at 25°C for five generations. Scale bar: 10 μm. (H) Reversibility of the mortal germline phenotype. jmjd-5(zr1234) 25°C: n = 14; jmjd-5(tm3735) 25°C: n = 12; jmjd-5(zr1234) 20°C: n = 12; jmjd-5(tm3735) 20°C: n = 7. Data are obtained from two biological replica. In (C), (D) and (H), bars indicate SEM. ∗∗∗∗p < 0.0001, ∗∗p < 0.01, ∗p < 0.05, with two-tailed, unpaired t test. See also Figure S1.
Figure 2
Figure 2
jmjd-5 controls transcription in germ cells (A) Gene Ontology (GO) analysis of differentially expressed (DE) genes in jmjd-5(zr1234) mutant animals identified by comparing the germline transcriptomes of N2 and jmjd-5(zr1234) animals grown for three generations at 25°C (p < 0.01). Selected GO terms are presented as −log10(p-adjusted). (B) Expected and observed number of germline-expressed and germline-enriched genes in upregulated (left) and downregulated (right) genes identified as in (A). ∗∗∗∗p < 0.0001, ∗∗p < 0.01, ∗p < 0.05 with Fisher’s exact test. (C) Chromosomal distribution of jmjd-5(zr1234) DE genes identified at 15°C and 25°C, compared with N2 animals raised in similar conditions (p < 0.01). The graphic reports the ratio between jmjd-5(zr1234) DE genes at 15°C and 25°C, located on the X chromosome and autosomes.
Figure 3
Figure 3
jmjd-5 protects germ cells from trans-differentiation at high temperature (A) Representative images of GFP expression in extracted germlines of unc-75::GFP and jmjd-5(zr1234);unc-75::GFP grown at 15°C or at 25°C at indicated generations. A magnification of a GFP-positive cell is shown in the square. Scale bar: 50 μm. Genotypes and numbers of germlines analyzed are presented in Table S2. (B) Quantification of PGL-1 signal in PGCs of N2 and jmjd-5(zr1234) at the indicated temperature and generations. Each dot represents an embryo (n > 23). ∗∗∗∗p < 0.0001, with one-way ANOVA. (C) Representative images N2 and jmjd-5(zr1234) PGCs, from animals grown at the indicated temperatures and generations, stained with PGL-1 and DAPI. Scale bar: 5 μm. ns, not significant.
Figure 4
Figure 4
jmjd-5 controls H3K36me2 levels across the entire genome (A) Venn diagram of H3K36me2 peaks. Left: 20°C (N2 versus jmjd-5(zr1234)); right: 25°C (N2 versus jmjd-5(zr1234)). (B) Boxplots of normalized H3K36me2 ChIP-seq signal across H3K36me2 consensus peaks of N2 samples and jmjd-5 mutants at 20°C (left) and 25°C (right). (C) Heatmaps of H3K36me2 ChIP-seq signal across H3K36me2 consensus peaks in N2 and jmjd-5(zr1234) at 20°C and 25°C. Two replica are shown. (D) Scatterplots of H3K36me2 5-kb genome-wide bins signal. N2 versus jmjd-5(zr1234) at 20°C (left) and N2 versus jmjd-5(zr1234) at 25°C (right). See also Figure S2.
Figure 5
Figure 5
jmjd-5 regulates the cross-talk with H3K27me3 (A) Venn diagram of H3K27me3 peaks. Left: 20°C (N2 versus jmjd-5(zr1234)); right: 25°C (N2 versus jmjd-5(zr1234)). (B) Boxplots of normalized H3K27me3 ChIP-seq signal across H3K27me3 consensus peaks of N2 samples and jmjd-5 mutants at 20°C (left) and 25°C (right). (C) Heatmaps of H3K27me3 ChIP-seq signal across H3K27me3 consensus peaks in N2 and jmjd-5 mutants at 20°C and 25°C. Two replicates are shown. (D) H3K27me3 scatterplots of 5-kb genome-wide bins signal. N2 versus jmjd-5 at 20°C (left) and N2 versus jmjd-5 at 25°C (right). See also Figure S3.
Figure 6
Figure 6
Temperature effects on H3K36/K27 methylation and transcription (A) Heatmap of H3K36me2 and H3K27me3 ChIP-seq signal on gene bodies enriched in H3K36me2 and/or H3K27me3 in N2 at 25°C. For each sample, ChIP-seq signal was transformed into Z scores to represent relative in-sample abundances of H3K36me2 and H3K27me3. K-means clustering (k = 5) was used to split H3K36me2+/H3K27me3+ genes in five different groups (C1–C5), annotated on the left of the heatmap. On the right, mRNA-seq expression −log2(RPKM+1) of the corresponding genes in N2 25°C samples. Data for two biological replicates are shown. (B) Normalized H3K36me2 and H3K27me3 ChIP-seq signal across gene bodies in N2 and jmjd-5 samples at 20°C and 25°C. One replica is shown. (C) Scatterplots of H3K36me2 (left) and H3K27me3 (right) 5-kb genome-wide bins signal. N2 25°C versus N2 20°C (top) and jmjd-5(zr1234) 25°C versus jmjd-5(zr1234) 20°C (bottom) are shown. (D) Heatmaps of H3K36me2 and H3K27me3 ChIP-seq signal on gene bodies from clusters identified in (A). For each histone modification, clusters with low, medium, and high enrichments are shown. (E) GO analysis of DE genes (p < 0.01) identified by RNA sequencing of isolated germlines of N2 grown at 15°C or at 25°C for three generations (F3). Selected GO terms are presented as −log10(p-adjusted). TES, transcription end site; TSS, transcription start site.
Figure 7
Figure 7
Loss of H3K27me2/3 demethylases ameliorates jmjd-5 defects (A) Brood size of indicated strains grown at 25°C, at the indicate generations. Bars represent SEM; n > 15. (B) Alluvial diagram of differentially expressed genes from jmjd-5 versus N2 (25°C) and 4M versus N2 (25°C). 4M indicates jmjd-5;jmjd-3.1/2/3 quadruple mutant. (C) Venn diagrams showing the overlap of upregulated (left) and downregulated (right) genes (p-adjusted < 0.01 and Log2FC ± 1) identified in indicated strains grown at 25°C for three generations (F3). See also Figures S4 and S5.
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