SPR-5 and MET-2 function cooperatively to reestablish an epigenetic ground state during passage through the germ line

Abstract

The Caenorhabditis elegans LSD1 H3K4me2 demethylase SPR-5 reprograms epigenetic transcriptional memory during passage through the germ line. Here we show that mutants in the H3K9me2 methyltransferase, met-2, result in transgenerational epigenetic effects that parallel spr-5 mutants. In addition, we find that spr-5;met-2 double mutants have a synergistic effect on sterility, H3K4me2, and spermatogenesis expression. These results implicate MET-2 as a second histone-modifying enzyme in germ-line reprogramming and suggest a model in which SPR-5 and MET-2 function cooperatively to reestablish an epigenetic ground state required for the continued immortality of the C. elegans germ line. Without SPR-5 and MET-2, we find that the ability to express spermatogenesis genes is transgenerationally passed on to the somatic cells of the subsequent generation. This indicates that H3K4me2 may act in the maintenance of cell fate. Finally, we demonstrate that reducing H3K4me2 causes a large increase in H3K9me2 added by the SPR-5;MET-2 reprogramming mechanism. This finding suggests a novel histone code interaction in which the input chromatin environment dictates the output chromatin state. Taken together, our results provide evidence for a broader reprogramming mechanism in which multiple enzymes coordinately regulate histone information during passage through the germ line.

Fig. 1.

SPR-5 spermatogenesis targets acquire MET-2-dependent H3K9me2 in the embryo. (A) ChIP-qPCR-measured enrichment of H3K9me2 at four germ-line-expressed genes in mixed-stage wild-type (N2) and met-2 mutant strains. (B) Chip-qPCR measured enrichment of H3K9me2 at the indicated genes in embryos from wild-type (N2) and met-2 animals. The fold enrichment of H3K9me2 Ab over no Ab is shown. The individual measurements from which the fold enrichments were calculated are shown in Fig. S2.

Fig. 2.

Heritable accumulation of H3K4me2 and the expression of spermatogenesis genes across generations in met-2 mutants. (A) ChIP-qPCR-measured enrichment of H3K4me2 at four spermatogenesis genes in mixed-stage met-2 mutants from generations 2, 12, and 24. The fold enrichment of H3K4me2 Ab over no Ab is shown. The individual measurements from which the fold enrichments were calculated are shown in Fig. S3. (B) Quantitative RT-PCR showing the relative expression of 12 spermatogenesis genes in mixed-stage met-2 mutants over 24 generations. The expression is normalized to actin (act-1).

Fig. 3.

spr-5;met-2 synthetic sterile phenotype. The average brood size of spr-5, met-2, and spr-5;met-2 strains in progressive generations. spr-5;met-2 mutants display complete sterility in one generation.

Fig. 4.

Synergistic accumulation of H3K4me2 and expression of spermatogenesis genes in spr-5;met-2 mutants. (A) ChIP-qPCR-measured enrichment of H3K4me2 at four spermatogenesis genes in mixed-stage met-2, spr-5, and spr-5;met-2 mutants. Data from three independent biological replicates (series 1, 2, and 3) are shown. The fold enrichment of H3K4me2 Ab over no Ab is shown. The individual measurements from which the fold enrichments were calculated are shown in Fig. S5. (B) Average fold enrichment from series 1, 2, and 3 (from A) of H3K4me2 Ab over no Ab. (C) qRT-PCR analysis of 12 spermatogenesis genes was performed on mixed-stage met-2, spr-5, and spr-5;met-2 mutants. Data from two independent biological replicates (series 1 and 2) are shown. The expression is normalized to actin (act-1). The exact value and error for the two highest values are shown to accommodate scale. ZK546.7 and W01B6.4 are inset because of scale. The error bars represent SEM. (D) qRT-PCR showing the relative expression of two spermatogenesis genes in wild-type (N2) and spr-5;met-2 mutant L1 larvae. The expression is normalized to ama-1, which is unchanged in spr-5;amx-1 mutants. The error bars represent SEM.

Fig. 5.

Synergistic accumulation of H3K4me2, but not expression, at nonspermatogenesis targets. (A) ChIP-qPCR-measured enrichment of H3K4me2 at an oocyte (rme-2), gut (ges-1), and muscle (unc-98) gene, but no enrichment at an intergenic region 3′ of the spermatogenesis gene C25G4.6, in mixed-stage spr-5;met-2 mutants compared with met-2 and spr-5 mutants. The fold enrichment of H3K4me2 Ab over no Ab is shown. The individual measurements from which the fold enrichments were calculated are shown in Fig. S6. (B) qRT-PCR analysis of rme-2, ges-1, unc-98, and C25G4.6 was performed on mixed-stage met-2, spr-5, and spr-5;met-2 mutants. C25G4.6 was included as a positive control and for comparison with Fig. 4C. The expression is normalized to actin (act-1).

Fig. 6.

Decreased H3K4me2 and increased H3K9me2 in wdr-5 mutants. (A) ChIP-qPCR-measured enrichment of H3K4me2 at four germ-line-expressed genes in mixed-stage wild-type (N2) and wdr-5 mutant strains. Data from two independent biological replicates (series 1 and 2) are shown. The fold enrichment of H3K4me2 Ab over no Ab is shown. The individual measurements from which the fold enrichments were calculated are shown in Fig. S7. (B) ChIP-qPCR-measured enrichment of H3K9me2 at four germ-line-expressed genes in mixed-stage wild-type, met-2 and wdr-5 mutant strains. The fold enrichment of H3K9me2 Ab over no Ab is shown. (C) ChIP-qPCR-measured enrichment of H3K9me2 at an oocyte (rme-2), gut (ges-1), and muscle (unc-98) gene in mixed-stage wild-type, met-2, and wdr-5 mutant strains. The fold enrichment of H3K9me2 Ab over no Ab is shown. The individual measurements from which the fold enrichments were calculated are shown in Fig. S7. (D) SPR-5;MET-2 model.