Lactate regulates major zygotic genome activation by H3K18 lactylation in mammals

Abstract

Lactate is present at a high level in the microenvironment of mammalian preimplantation embryos in vivo and in vitro. However, its role in preimplantation development is unclear. Here, we report that lactate is highly enriched in the nuclei of early embryos when major zygotic genome activation (ZGA) occurs in humans and mice. The inhibition of its production and uptake results in developmental arrest at the 2-cell stage, major ZGA failure, and loss of lactate-derived H3K18lac, which could be rescued by the addition of Lac-CoA and recapitulated by overexpression of H3K18R mutation. By profiling the landscape of H3K18lac during mouse preimplantation development, we show that H3K18lac is enriched on the promoter regions of most major ZGA genes and correlates with their expressions. In humans, H3K18lac is also enriched in ZGA markers and temporally concomitant with their expressions. Taken together, we profile the landscapes of H3K18lac in mouse and human preimplantation embryos, and demonstrate the important role for H3K18lac in major ZGA, showing that a conserved metabolic mechanism underlies preimplantation development of mammalian embryos.

Keywords: epigenetic remodeling; histone lactylation; lactate; mammalian preimplantation development; zygotic genome activation.

Figure 1.

Lactate is essential for the preimplantation development of early embryos of mice. (A) Lactate levels during the development of preimplantation embryos in mice. Three experimental replicates were performed. Error bars represent SEM. (B) Fluorescence images of mouse embryos from different stages expressing FiLa in nucleus. Scale bars, 20 μm. (C) Interconversion of pyruvate and lactate. (D and E) Development rates of embryos in the groups. Three experimental replicates were performed. (F) Fluorescence images of mouse embryos at the 2-cell stage from different groups expressing FiLa in the nuclei at the 2-cell stage. Scale bars, 20 μm. (G) Overview of the experimental designs. (H and I) Development rates of embryos deprived of lactate at the late zygote and 4-cell stages. DMSO, mKSOM culture medium plus DMSO; G&N, mKSOM culture medium plus Gne140 and NMN; G&N-L, mKSOM culture medium without lactate plus Gne140 and NMN; GSKA&N, mKSOM culture medium plus GSK2837808A and NMN; GSKA&N-L, mKSOM culture medium without lactate plus GSK2837808A and NMN; Error bars are SEM. n.s. means not significant. **P < 0.01, ***P < 0.001. Differences between means were calculated using two-tailed Student's t-test.

Figure 2.

Loss of lactate results in failure of major ZGA in early embryos of mice. (A) RNA-seq analysis of late 2-cell embryos from the G&N and G&N-L groups. Volcano plot showing changes in gene expression. Yellow and purple dots indicate upregulated and downregulated genes, and red dots indicate major ZGA genes. (B) Gene set enrichment analysis (GSEA) of ZGA genes shows preferential downregulation in late 2-cell embryos from the G&N-L group. (C) Fractions of minor ZGA, major ZGA, and other coding genes among the downregulated genes in (A). (D) Violin plot showing the significant downregulation of major ZGA genes in the G&N-L group. (E) RNA-seq analysis of early 2-cell embryos from the G&N and G&N-L groups. Volcano plot showing changes in gene expression. Yellow and purple dots indicate upregulated and downregulated genes. (F) Violin plot showing that the expressions of minor ZGA genes had no significant change between the G&N and G&N-L groups. (G) 5ʹ-Ethynyluridine (EU) staining showing a remarkable decrease in total de novo transcripts in late 2-cell embryos of the G&N-L group. Scale bars, 50 μm. (H) IF of Pol II Ser2 of late 2-cell embryos showing a remarkable decrease in the G&N-L group. Scale bars, 50 μm. DMSO, mKSOM culture medium plus DMSO; G&N, mKSOM culture medium plus Gne140 and NMN; G&N-L, mKSOM culture medium without lactate plus Gne140 and NMN. (I) Fluorescence images of mouse 2-cell embryos expressing FiLa in the nucleus. Scale bars, 20 μm. Differences between means were calculated using two-tailed Student's t-test.

Figure 3.

H3K18lac is reduced by deprivation of lactate and affects major ZGA. (A) IF of late 2-cell embryos shows that H3K18lac is remarkably decreased in the G&N-L compared to the other groups. DMSO, mKSOM culture medium plus DMSO; G&N, mKSOM culture medium plus Gne140 and NMN; G&N-L, mKSOM culture medium without lactate plus Gne140 and NMN. Scale bars, 50 μm. (B) IF of H3K18lac during the development of preimplantation embryos developing in vivo in mice. Representative images are shown. Scale bars, 50 μm. (C and D) Development rates of embryos with overexpressions of wide type (WT) and mutated H3 mRNAs (H3K18R). Error bars are SEM. n.s. means not significant. *P < 0.05. (E) IF of late 2-cell embryos showing that H3K18lac is decreased by overexpressing H3K18R mRNAs. Scale bars, 50 μm. (F) RNA-seq analysis of late 2-cell embryos with overexpressions of wide type (WT) and mutated H3 mRNAs (H3K18R). Volcano plot shows changes in gene expression. Yellow and purple dots indicate upregulated and downregulated genes, and red dots indicate major ZGA genes. (G) Violin plot showing the significant downregulation of major ZGA genes in the H3K18R mRNAs-overexpressed group. ***P < 0.001. (H) Comparative analysis of the RNA-seq data of the lactate-deprived and H3K18R-mutated embryo. Yellow and purple dots indicate commonly upregulated and downregulated genes, respectively. Red dots represent the major ZGA genes. Differences between means were calculated using two-tailed Student's t-test.

Figure 4.

H3K18lac genome-wide distribution in preimplantation embryos of mice. (A) Unsupervised clustering of H3K18lac signals among the embryos at the stages indicated. (B) MDS analysis of H3K18lac profiles. Different colors indicate different developmental stages and lineages. ICM, inner cell mass; TE, trophoblast. (C) Fraction of the mouse genome covered by H3K18lac reads at different developmental stages, and percentages of H3K18lac peaks assigned to the promoter, intron, exon, and intergenic regions. (D) Metaplot of H3K18lac enrichment at the promoters of minor and major ZGA, and other coding genes at the late 2-cell stage. (E) Pearson correlation analysis of gene expression and H3K18lac enrichment at the gene promoters in mouse preimplantation embryos. Red, green and grey dots represent major ZGA, minor ZGA, and coding genes, respectively. (F) Genome browser view of H3K18lac signals at the Usp29 locus during the development of the preimplantation embryo.

Figure 5.

Lac-CoA can rescue the abnormal phenotypes caused by deprivation of lactate. (A) IF of H3K18lac in late 2-cell embryos from the groups indicated. Scale bars, 50 μm. (B) RNA-seq analysis showing the expressions of major ZGA genes rescued by the addition of 1 mM Lac-CoA. Purple dots indicate the rescued major ZGA genes. (C and D) Development rates of embryos from the groups indicated. Three experimental replicates were performed. Error bars are SEM. n.s. means not significant. *P < 0.05; **P < 0.01; ***P < 0.001. (E) Fraction of the mouse genome covered by H3K18lac reads in the groups indicated, and percentages of H3K18lac peaks assigned to the promoter, intron, exon, and intergenic regions. (F) Heatmap (left panel) of H3K18lac signals ranked by their relative changes among the groups indicated. Heatmap (right panel) showing the expression changes of major ZGA genes between the groups indicated. (G) Genome browser view of H3K18lac signals at the Usp29 locus of late 2-cell embryos from the groups indicated. G&N, mKSOM culture medium plus Gne140 and NMN; G&N-L, mKSOM culture medium without lactate plus Gne140 and NMN; G&N-L + Lac-CoA, zygotes injected into 1 mM Lac-CoA and cultured in mKSOM medium without lactate plus Gne140, NMN. (H) Violin plot showing the significantly upregulated expressions of major ZGA genes with H3K18lac enrichment. (I) Metaplot showing the higher Pol II loading on the locus of major ZGA genes with H3K18lac enrichment. Differences between means were calculated using two-tailed Student's t-test.

Figure 6.

H3K18lac-enriched ZGA markers in human 8-cell embryos. (A) Fluorescence images of human embryos from different stages expressing FiLa in nuclei. (B) IF of H3K18lac of human 4-cell, 8-cell, 16-cell, and morula embryos. Scale bars, 50 μm. (C) Unsupervised clustering of H3K18lac signals among the embryos at the stages indicated. (D) MDS analysis of samples in the indicated stages. Different colors indicate different developmental stages. (E) Ratio of fraction of the human genome covered by H3K18lac reads in the indicated developmental stages, and percentages of H3K18lac peaks assigned to the promoter, intron, exon, and intergenic regions. (F) Heatmaps of H3K18lac enrichment (right panel) in 8-cell signature genes indicated in human embryos; left panel, corresponding RNA levels. (G) Lactate in nuclei when major ZGA occurs in humans and mice during preimplantation development and a model of lactate-derived H3K18lac regulating major ZGA.