Maternal vitamin C regulates reprogramming of DNA methylation and germline development

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Abstract

Development is often assumed to be hardwired in the genome, but several lines of evidence indicate that it is susceptible to environmental modulation with potential long-term consequences, including in mammals1,2. The embryonic germline is of particular interest because of the potential for intergenerational epigenetic effects. The mammalian germline undergoes extensive DNA demethylation3,4,5,6,7 that occurs in large part by passive dilution of methylation over successive cell divisions, accompanied by active DNA demethylation by TET enzymes3,8,9,10. TET activity has been shown to be modulated by nutrients and metabolites, such as vitamin C11,12,13,14,15. Here we show that maternal vitamin C is required for proper DNA demethylation and the development of female fetal germ cells in a mouse model. Maternal vitamin C deficiency does not affect overall embryonic development but leads to reduced numbers of germ cells, delayed meiosis and reduced fecundity in adult offspring. The transcriptome of germ cells from vitamin-C-deficient embryos is remarkably similar to that of embryos carrying a null mutation in Tet1. Vitamin C deficiency leads to an aberrant DNA methylation profile that includes incomplete demethylation of key regulators of meiosis and transposable elements. These findings reveal that deficiency in vitamin C during gestation partially recapitulates loss of TET1, and provide a potential intergenerational mechanism for adjusting fecundity to environmental conditions.

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Fig. 1: Maternal vitamin C promotes female germ-cell development.
Fig. 2: Gestational vitamin C deficiency has a long-term effect on female fecundity.
Fig. 3: Vitamin C deficiency induces a Tet1−/−-like expression profile in the developing germline.
Fig. 4: Vitamin C deficiency leads to incomplete loss of DNA methylation at meiosis regulators and transposable elements in the embryonic germline.

Code availability

Custom codes used for data analysis were deposited in Github (https://github.com/Santosi/VitC-PGCs) and are also available upon request.

Data availability

RNA-seq, RRBS and CUT&RUN data have been deposited in Gene Expression Omnibus (GEO) under accession number GSE109747.

Change history

  • 13 November 2019

    An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank M. Conti, R. Blelloch, P. Rinaudo, M. Lorincz, S. Fisher, L. Selleri and members of the Santos laboratory for input and critical reading of the manuscript. We thank E. Chow and members of the UCSF Center for Advanced Technology for assistance with sequencing; B. Soygur for meiotic spread protocol and reagents; Y. Zhang and L. Shen for technical advice on RRBS. We are grateful to S. Henikoff for providing the pA-MN and yeast tRNA spike-ins, and to S. Henikoff and T. Fazzio for providing technical help with performing CUT&RUN experiments. Flow cytometry data were generated in the UCSF Parnassus Flow Cytometry Core, which is supported by a Diabetes Research Center grant and NIH grant P30 DK063720. S.P.D. was supported by the National Science Foundation Graduate Research Fellowship Program under grant no. 1650113. Any opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. G.L. was partly supported by Institut Universitaire de France. This work was supported by National Institutes of Health (NIH) grants R21ES023297 and R01ES028212 to D.J.L., and NIH grants R01OD012204 and R01GM123556, and a Canada 150 Research Chair to M.R.-S.

Author information

S.P.D. and M.R.-S. conceived the project. S.P.D. designed, performed and analysed most experiments with following exceptions. M.P. performed and analysed CUT&RUN experiments and somatic cell RNA-seq. M.P. collaborated with E.C., S.M. and M.A. to quantify vitamin C levels in embryos. M.-J.G. performed histology, immunohistochemistry and meiosis analysis on E14.5 fetal ovaries and testes under the supervision of G.L. E.W. performed and analysed whole-mount imaging on day 7 ovaries under the supervision of D.J.L. E.C. performed hmC quantifications. K.T.E. and K.M. set up the initial conditions for analysis of the mouse gestational vitamin C deficiency model. M.R.-S. supervised the project. S.P.D. and M.R.-S. wrote the manuscript with feedback from all authors.

Correspondence to Miguel Ramalho-Santos.

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The authors declare no competing interests.

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A correction to this article is available online at https://doi.org/10.1038/s41586-019-1699-9.

Extended data figures and tables

Extended Data Fig. 1 Validation of vitamin C depletion and the downregulation of TET1-dependent germline genes in a mouse model of gestational vitamin C deficiency.

a, Expression of vitamin C transporters (SVCT1, SVCT2 and GLUT8, encoded by Slc23a1, Slc23a2 and Glut8, respectively) in developing female (F) germ cells. Data are from Seisenberger et al.3. b, Kinetics of vitamin C depletion from the serum of pregnant Gulo−/− mice after withdrawal from their drinking water. Pregnant female mice were removed from vitamin C supplementation at E3.5 and circulating blood serum was tested over the time course indicated. It takes five days of withdrawal for the circulating vitamin C levels to be <25%, and seven days to be essentially undetectable. Line plot connects the average blood serum values of three biological replicates. c, Vitamin C levels measured by mass spectrometry in E13.5 embryonic tissue with and without gestational vitamin C removal. E13.5 female head or liver were normalized by tissue weight. Samples with non-detectable levels of vitamin C were set to zero. n = 5 biological replicates per condition. d, The reduction in E13.5 female germ cells numbers after vitamin C deficiency is confirmed using both Oct4–eGFP and SSEA1 positivity. n = 6 matched biological replicates per condition. e, Gestational vitamin C deficiency does not affect average somatic cell number. n = 13 control and n = 10 vitamin-C-depleted biological replicates. f, Vitamin-C-deficient E13.5 female germ cells express lower levels of key germline genes, as measured by qRT–PCR. n = 3 control and n = 4 vitamin-C-depleted biological replicates. The variability in Stra8 expression in controls as assessed by qRT–PCR at E13.5 is because it is just being induced at this stage; RNA-seq and immunofluorescence data further document the significant reduction in Stra8 RNA and protein levels in vitamin-C-deficient PGCs (Extended Data Fig. 3b, c). g, Expression of select germline genes are induced in a TET1/2-dependent manner after the addition of vitamin to cultured mouse ES cells11. Gene expression was measured by qRT–PCR, plotted as mean of technical triplicates. Data are from Blaschke et al.11. h, Decreased expression of select germline genes in Tet1−/− E13.5 germ cells as measured by RNA-seq. Data are from Yamaguchi et al.8, and denote averages of three controls and two Tet1−/− samples. i, Select germline genes induced by vitamin C in cultured ES cells (c) and downregulated in Tet1−/− germ cells (d) are also downregulated in vitamin-C-deficient E13.5 germ cells. Gene expression was measured by RNA-seq. n = 6 biological replicates. j, Gestational vitamin C deficiency does not affect transcription in the somatic cells of E13.5 female gonads as shown by unsupervised clustering of RNA-seq analysis. Clustering performed on n = 6 biological replicates per condition. Data are single values per stage (a), mean values (b, h), mean ± s.e.m. (c, i) or mean ± s.d. (dg). P values determined by two-tailed t-test with Welch’s correction (c, d, e), two-tailed Student’s t-test (f). Source Data

Extended Data Fig. 2 Analyses of gonad development upon vitamin C deficiency.

a, There is an overall normal induction of key markers of developmental progression of somatic cells of the ovaries between E11.5 and E13.5 in vitamin-C-deficient embryos, as measured by qRT–PCR. E13.5 somatic ovary cells are matched with PGCs in Extended Data Fig. 1f. In this analysis, Foxl2 expression is strongly induced after vitamin C deficiency, but to a slightly lower extent than in control samples. However, no significant changes in Foxl2 were detected by RNA-seq (Fig. 1f) or immunofluorescence (b). Error bars of E13.5 samples depict mean ± s.d. of four biological replicates; E11.5 data are from two biological replicates. b, Sexual differentiation is controlled by a balance between SOX9 (leading to expression of anti-Müllerian hormone (AMH)) in male and FOXL2 in female mice. Expression of FOXL2 in E14 and E18 ovaries (O14 and O18, respectively) does not change after vitamin C depletion as measured by immunohistochemistry. As expected, FOXL2 was not detected in E14 testes (T14). Images are representative of n = 5 control and n = 4 vitamin-C-deficient ovaries, and n = 2 control and n = 3 vitamin-C-deficient testes. c, Expression of AMH in E14 testis does not change after vitamin C depletion as measured by immunohistochemistry. As expected, AMH was not detected in E14 or E18 ovaries. Images are representative of n = 5 control and n = 4 vitamin-C-deficient ovaries, and n = 2 control and n = 3 vitamin-C-deficient testes. d, Gestational vitamin C deficiency does not affect the presence of Leydig cells (3BHSD) in developing testes or the absence of Leydig cells in developing ovaries. Images are representative of n = 5 control and n = 4 vitamin-C-deficient ovaries, and n = 2 control and n = 3 vitamin-C-deficient testes. Source Data

Extended Data Fig. 3 Detailed analyses of meiotic staging in germ cells of E14.5 vitamin-C-deficient ovaries.

a, Representative images of STRA8 and SYCP3 abundance in E14.5 control or vitamin-C-deficient female germ cells. Images are representative of n = 7 STRA8 and n = 8 SYCP3 biologically independent experiments, as indicated in b and d. b, Significant reduction in the percentage of DDX4+ germ cells that are STRA8+ after vitamin C deficiency. c, Stra8 mRNA abundance is significantly reduced at E13.5 as measured by RNA-seq. n = 6 biological replicates. d, There was a non-significant reduction in the average percentage of DDX4+ germ cells that are SYCP3+ in vitamin-C-deficient E14.5 female germ cells compared with controls. e, Abundance of Sycp3 mRNA is significantly reduced at E13.5 as measured by RNA-seq. n = 6 biological replicates. f, Representative haematoxylin and eosin staining of E14.5 embryonic ovaries for data quantified in Fig. 1h. Black arrowheads indicate germ cells at the indicated stage of meiosis. Images are representative of n = 8 biologically independent experiments. g, The percentage of germ cells in meiotic S phase versus post-S phase is significantly higher in vitamin-C-deficient E14.5 female mice, relative to controls. Additional analysis of data from meiosis staging is shown in Fig. 1h. n = 8 biologically independent experiments. h, Representative images of CREST and SYCP1 staining in E18.5 germ cells, as in Fig. 1h. i, Ovarian follicle volume and frequency are not significantly different in P7 female mice deprived of vitamin C in utero (P = 0.206). Ovarian follicles were stained by NOBOX and quantified using whole-mount imaging. n = 6 control and n = 9 vitamin-C-depleted ovaries per condition. Data are mean (c, e), mean ± s.e.m. (b, d) or mean ± s.d. (i). P values determined by two-sided Mann–Whitney U-test (b, d), Wald χ2 test (c, e, g) or paired two-sided homoscedastic Student’s t-test (i). Source Data

Extended Data Fig. 4 Analyses of the effects of vitamin C deficiency on the development of male germ cells.

a, Unlike the consistent reduction of female germ cells (top), vitamin C (VC) deficiency does not decrease the number of germ cells in E13.5 male gonads (bottom). ns, not significant. The number of biological replicates is indicated. b, The meiotic germ-cell marker SYCP3 was not identified in n = 2 or n = 3 male gonads with or without vitamin C depletion, respectively. n = 6 control and n = 5 vitamin-C-deficient biological replicates. c, The meiotic germ-cell marker STRA8 was not identified in n = 2 or n = 3 male gonads with or without vitamin C depletion, respectively. Female graph includes n = 6 biological replicates. d, Most germ cells in developing testis are proliferative (Ki67+), with a few quiescent (Ki67) germ cells, and no deviations from this pattern are detected with vitamin C deficiency. Images are representative of n = 2 control and n = 3 vitamin-C-deficient biological replicates. Data are mean ± s.d. (a) or mean ± s.e.m. (b, c). P values determined by two-sided Welch’s t-test (a) or two-tailed Mann–Whitney U-test (bd). Source Data

Extended Data Fig. 5 Further analyses of RNA-seq data from vitamin-C-deficient E13.5 female germ cells.

a, Table of RNA-seq samples and number of germ cells per sample. Each sample represents germ cells from a single E13.5 female. b, Unsupervised hierarchical clustering of n = 6 biological replicates in each condition documents the overall separation between control and vitamin-C-deficient samples (columns) and relative gene expression (rows). 1CF1 denotes control litter 1, female embryo 1; 4VF2 denotes vitamin-C-deficient litter 4, female embryo 2. c, Scatterplot of differential gene expression in vitamin-C-deficient E13.5 female PGCs of genes differentially expressed in E13.5 Tet1−/− female PGCs8. Spearman’s rho statistic is used to estimate a rank-based measure of association. d, Expression of Dnmt and Tet genes in vitamin-C-deficient samples is similar to control samples. n = 6 biological replicates per condition. e, Heat map documenting the consistent expression of Tet and Dnmt genes across the six control and six vitamin-C-deficient samples. f, Expression of other genes belonging to families of enzymes with the potential to be vitamin C-sensitive (such as Kdm genes, collagen hydroxylases (P4h genes), prolyl hydroxylases (Lepre genes), HIF hydroxylases (Egln genes)). Lepre1 is also known as P3h1; Leprel1 is also known as P3h2; Leprel2 is also known as P3h3; and Leprel4 is also known as P3h4. None of these displays differential expression in vitamin-C-deficient female germ cells. n = 6 biological replicates per condition. Data are mean ± s.d. (d, f). Statistical significance assessed by two-tailed Student’s t-test (d, f). n.d., not determined. Source Data

Extended Data Fig. 6 Identification of the window of susceptibility to vitamin C deficiency between E3.5 and E13.5.

a, Pregnant female mice mated in vitamin-C-deficient conditions were either maintained without vitamin C (−VitC) or returned to vitamin-C-containing water at E3.5 (3.5 return). b, Adding back vitamin C from E3.5 to E13.5 tends to rescue the defects in the expression of key germline regulators induced with full vitamin C deficiency. Gene expression was measured by qRT–PCR in E13.5 female germ cells. n = 4 biological replicates. c, The numbers of E13.5 Oct4–eGFP+ germ cells are mostly recovered after the return of vitamin C at E3.5. Data are normalized to the germ-cell count of the control embryos. n = 11–22 biological replicates of each condition as indicated. Data are mean ± s.e.m. (b, c). *P < 0.05; **P < 0.01; two-tailed Student’s t-test (b, c). Source Data

Extended Data Fig. 7 DNA methylation defects in E13.5 vitamin-C-deficient female germ cells.

a, Average methylation of cytosine according to sequence context. Genome-wide CpG methylation is 3–6% regardless of vitamin C supplementation. Methylation of cytosine in a CHG or CHH context (in which H correspond to A, T or C) is below 1%. b, Average methylation according to genomic context in control and vitamin-C-deficient samples. Box plots indicate minimum to maximum measurements of n = 6 biological replicates, with centre line denoting the mean. c, Top, density plot of 460 DMRs with a >5% methylation change reveals an overall increase in the number and magnitude of methylation gains over losses after vitamin C deficiency. Bottom, GREAT analysis of 175 hypomethylated DMRs (hypermethylated DMRs are in Fig. 4b). d, Transposable elements of the LINE1, LTR, ERVK and IAP families that are associated with DMRs show a consistent pattern of hypermethylation after vitamin C deficiency. n = 6 biological replicates. e, Average methylation in control or vitamin-C-deficient samples in different transposable element families across the genome. Data are from uniquely mapped transposable elements annotated by RepeatMasker and captured by RRBS, regardless of the DMR calls, based on 558 elements and 3,180 CpGs (LINE1), 185 elements and 1,065 CpGs (full-length LINE1; >6 kb), 384 elements and 1,724 CpGs (SINE), 568 elements and 2,691 CpGs (ERVK), 226 elements and 1,057 CpGs (ERVK-IAP), 10 elements and 73 CpGs (satellite). The box extends to 25th and 75th percentiles, with centre line indicating the median transposable element methylation across n = 6 biological replicates. Error bars extend to 5th and 95th percentiles. f, hmC quantification by dot blot in E13.5 brain and liver indicate that hmC abundance is higher in brain and reduced with vitamin C depletion. Each group represents three biological replicates (across) and two technical replicates (vertical pairs). g, hmC quantification by ELISA (Active Motif) confirmed a significant decrease of hmC in both E13.5 brain and liver. Quantification was performed using 50 ng of DNA. n = 5 biological replicates. Data are mean ± s.e.m. (d, g). P values were determined by two-tailed Student’s t-tests (d), two-tailed Wilcoxon matched-pairs signed-rank test (e) or two-sided Welch’s t-test (g). Source Data

Extended Data Fig. 8 CUT&RUN analysis of H3K9me2 abundance in E13.5 female gonads.

a, Diagram of CUT&RUN experiments. E13.5 control or vitamin-C-deficient gonads were dissociated and the PGCs and soma were separated by FACS for cryopreservation and CUT&RUN analysis. PGCs and soma from 3–5 independent embryos per condition were subjected to H3K9me2 (K9me2) or control (IgG) CUT&RUN. b, K9me2 enrichment is highest at non- or low-expressed genes. Box plots of average K9me2 coverage at genes in control soma or PGC samples, separated into quartiles from lowest (quartile 1) to highest (quartile 4) expression level in control E13.5 soma. Coverage is calculated from average (K9me2/IgG + 0.001) of n = 4 control and n = 3 vitamin-C-depleted soma replicates, and n = 5 PGC replicates per condition. c, Scatter plots of average K9me2 coverage at genes or transposable element families in control or vitamin-C-deficient E13.5 female soma and PGCs. n = 4 control and n = 3 vitamin-C-depleted soma replicates, and n = 5 PGC replicates per condition. Significantly upregulated (yellow) or downregulated (blue) genes or transposable element families are indicated (log2-transformed fold change > |1|, FDR < 0.05). d, Increases in soma K9me2 levels are highest at already K9me2-marked genes. Box plot showing K9me2 log2-transformed fold change after vitamin C depletion in soma in groups of genes ranked according to K9me2 enrichment in control (lowest and highest quartile of K9me2 enrichment, and all genes). n = 4 control and n = 3 vitamin-C-depleted soma replicates, and n = 5 PGC replicates per condition. e, f, Histograms of K9me2 or IgG coverage at selected transposable element families in E13.5 female soma (e) or PGCs (f). n = 4 control and n = 3 vitamin-C-depleted soma replicates, and n = 5 PGC replicates per condition. Box plots in b and d indicate 25th and 75th percentiles, with centre lines marking the median values. Data are mean ± s.e.m. (e, f). P values were determined by two-sided Wilcoxon rank-sum test (b, d) or are the FDR from Limma analysis (c, e, f). See Supplementary Data 5 and Methods for details.

Extended Data Fig. 9 Model for the role of vitamin C in DNA methylation reprogramming and development of the embryonic germline.

Embryonic germline cells require vitamin C for proper DNA demethylation of key regulators of meiosis and transposable elements (TEs). Gestational vitamin C deficiency is compatible with development to term and adulthood, but induces a phenotype akin to a TET1 hypomorph, with incomplete DNA demethylation and downregulation of germline genes, reduced numbers of germ cell, meiosis defects and decreased fecundity. Vitamin C deficiency may also affect other enzymatic reactions in the germline.

Supplementary information

Reporting Summary

Supplementary Data 1

E13.5 germ cell RNA-seq from n = 6 control and Vitamin C-deficient female embryos. Toptable output from DESeq2.

Supplementary Data 2

qRT-PCR primer list

Supplementary data 3

RNA-seq significant genes’ gene ontology. Significant genes defined as padj < 0.05 from Supplementary Data 1.

Supplementary Data 4

Days of Vitamin C depletion per experiment.

Supplementary Data 5

Limma CUT&RUN toptable

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DiTroia, S.P., Percharde, M., Guerquin, M. et al. Maternal vitamin C regulates reprogramming of DNA methylation and germline development. Nature 573, 271–275 (2019) doi:10.1038/s41586-019-1536-1

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