Maternal Vitamin C Intake Regulates the Epigenome During Germline Development

The Santos Lab has played a pivotal role in understanding how maternal diet affects the development of embryos. Their findings reveal that maternal vitamin C deficiency dysregulates the epigenetic landscape in embryonic germ cells.

Yayra Gbotsyo, Saloni Modi, Anthea Travas

Dr. Miguel Ramalho-Santos is a Senior Investigator at the Lunenfeld-Tanenbaum Research Institute, specializing in mammalian development. He is also a Professor in the Molecular Genetics Department at the University of Toronto. (Image taken from the Santos Lab website).

Maternal diet is an essential factor that affects the health and development of offspring during pregnancy. Several lines of evidence demonstrate that poor maternal nutrition, such as lack of vitamin C (vitC) during pregnancy, leads to abnormal fetal development. Changes in the in vitro environment due to external factors such as smoking, and drinking pose long-term consequences for the developing embryo. Such consequences are shaped by a process known as epigenetics. Epigenetics is the study of DNA and histone methylation patterns that alter chromatin state and gene expression (Goldberg et al. 2007). At the forefront of developmental research is Dr. Miguel Ramalho-Santos, who uses cutting-edge technology to understand epigenetic regulation during gestation.

Dr. Ramalho-Santos explains that he was drawn to Toronto’s vibrant research community and their collaborative efforts in developmental stem cell biology. He received his Ph.D. at Harvard University in 2002, where he trained as a developmental biologist. He then became a Fellow at the University of California San Francisco (UCSF). In 2007, he became an Assistant Professor at the UCSF and was later promoted to Associate Professor in 2013. In 2018, Dr. Ramalho-Santos was recruited to become the Canada 150 Research Chair in Developmental Epigenetics. This initiative recruits exceptional scholars to enhance Canada’s reputation for research and innovation. Alongside this endeavour, Dr. Ramalho-Santos is a Senior Investigator at the Lunenfeld-Tanenbaum Research Institute and a Professor in the Department of Molecular Genetics, at the University of Toronto. His lab uses mouse models to investigate how environmental inputs such as inadequate diet regulate proper gene transcription. Currently, he and his team aim to understand the underpinnings of gene activation during development at the right place and level.

Tet Enzymes Regulate DNA Methylation Patterns in Embryonic Stem Cells

Developmental epigenetics is the study of how environmental inputs influence gene expression during gestation1–3. Environmental factors such as nutrient availability during pregnancy can positively or negatively affect the way genes are expressed in the fetus1,2,4. According to Dr. Ramalho-Santos, the epigenetic landscape during development facilitates our understanding of how certain disruptions in adulthood can be traced back to insults in early uterine life3.

One of the most important takeaways from the Santos Lab was that “mammalian embryos are acutely aware of their mother’s environment.” In utero, the embryo is remarkably responsive to environmental agents that can alter their development2,5. This became evident when they realised that the epigenetic regulating enzyme, ten-eleven translocation (Tet), is dependent on maternal nutrient availability during development5–7. Tet enzymes play a key role in demethylating cytosine nucleotides, thereby removing methyl modifiers and making DNA more accessible6,8. This process promotes the transcription of many genes and maintains pluripotency, thereby giving rise to several different cell types. Tet enzymes function to demethylate DNA within the germ cells of the developing embryo8. Germ cells develop in the gonads of the growing embryo and will ultimately give rise to gametes9. (Figure 1). During embryonic development, DNA demethylation is important for keeping chromatin in an accessible state so that genes are actively expressed, and cell differentiation is restricted3,7. Therefore, Tet-mediated demethylation is crucial in maintaining embryonic stem cell (ESC) pluripotency10. Previous studies show that the offspring of Tet1 knockout mice have significantly reduced germ cells, which leads to compromised fertility10.  In order to further understand this process, Dr. Ramalho-Santos’ research investigates how environmental conditions, such as adequate access to vitC, modulates Tet activity.

Figure 1. Maternal Vitamin C (VitC) promotes germ-cell development by modulating Tet mediated demethylation. A) VitC taken by maternal F0 activates Tet enzymes, which promotes DNA demethylation in the F1 germ cells. Chromatin remodelling into an open state allows active transcription of genes and keeps embryonic germ cells in a stem cell-like state. B) VitC deficient embryos exhibit impaired Tet demethylation activity. As a result, DNA is kept in a non-permissive state. This dysregulation results in reduced embryonic germ cells in the F1 generation. (Figure is not quantitative) Figure created in Bio Render and adapted from3.

Maternal Vitamin C Deficiency Hinders Fecundity in Offspring

In 2013, Dr. Ramalho-Santos and his group discovered that Tet enzymes induced ESC to stay pluripotent when cultured in media containing vitC5. VitC is a potential cofactor of Tet enzymes and helps mediate demethylation5. More recently, these findings were implemented in pregnant mice models to understand how maternal vitC intake regulates Tet demethylation and thus embryonic germline development. 3,11 Dr. Ramalho-Santos explains that vitC supplemented from the diet can modulate Tet-mediated demethylation activity in the embryonic germ line cells (Fig. 1A)3,5,11. This keeps gene promoters accessible for transcription. While VitC deficient F1 embryos are viable, there is a reduction in Tet Demethylation activity in the F1 germ cells, which hinders their ability to give rise to the next generation (Figure 1B, 2)3,11. Interestingly, embryos deficient in vitC have transcriptomes and phenotypes that are remarkably similar to embryos with Tet1 deficiency3. These phenotypes include a reduced number of germ cells in the ovary, reduced fertility, and defects in meiosis3,10,11. In Tet1-deficient mice, defects in meiosis are proposed to be due to insufficient demethylation that fails to activate meiotic genes. These novel findings exemplify the effects of intergenerational epigenetics. This highlights that the F0 maternal environment propagates long-term impacts in the F1 generation.

Figure 2. Maternal Vitamin C deficiency causes defects in embryonic germ cells. VitC deficiency in F0 females leads to intergenerational effects, where the F1 embryo has reduced germ cell count and reduced fecundity in adulthood. Figure created in Bio Render and adapted from4.

When asked whether the effects of vitC deficiency were reversible in mice, Dr. Ramalho-Santos explained that this is only possible if vitC is reintroduced before mid-gestation11. After this point, the adverse effect on germ cells was irreversible. This became an important discovery for demonstrating that vitC is essential during mid-gestation11. The irreversible effects of vitC dysregulates demethylation, thereby hindering germline development11.

Environmental Factors Influencing the Epigenetic Role of Vitamin C

Dr. Ramalho-Santos’ work reveals exciting insights into how maternal vitC intake regulates the germ cells of mice embryos. To this end, one may wonder how these findings relate to the world outside of a lab setting. Inadequate vitC intake may be a reality for individuals with lower incomes and inadequate access to fresh produce12,13. Additionally, studies demonstrate that exposure to pollutants such as cigarette smoke and heavy metals can inhibit or inactivate vitC through oxidation reactions 14,15. This ultimately hamper’s vitC’s role as a cofactor of Tet, thereby dysregulating the epigenetic landscape of developing embryonic germ cells16,17. In the current day and age, regardless of geographical location, exposure to the aforementioned toxic substances has become common and further compounds the effects of vitC deficiency. This reality poses a concern for pregnant mothers as these adverse environmental contributors can lead to dysregulation of Tet.  This imposes serious epigenetic impacts that may not be noticed until after the fetus becomes an adult (i.e. reduced fecundity).

Translating the Effects of Maternal Vitamin C Deficiency from Mice to Humans

Today, researchers strive to accumulate insights into factors that hinder molecular pathways and lead to downstream effects on fetal development. However, it is also important to understand how these scientific findings in animal models translate to improving the health of human populations. Previous studies have demonstrated that vitC modulates Tet enzymes and maintains pluripotency in human ESCs (cells extracted from early human embryos)18. While many of the experiments done in the Santos lab are modelled in mice, they are difficult to replicate in humans and hold ethical barriers. Translation into human subjects would require studying vitC deficiency during pregnancy and then tracing the fecundity of the human offspring throughout adulthood. While this data does not currently exist; Mount Sinai Hospital’s ‘Ontario Birth Study’ program holds promise for recruiting relevant cohorts. This program collects clinical data for pregnant women to understand factors that contribute to maternal and child health. This data is based on multi-generational cohorts, making it immediately accessible for researchers to study trends in the role of epigenetics across many generations.

It is well-known that only a fraction of a disease is attributed to specific genetic defects. Research in the Santos lab addresses some of the missing gaps towards  explaining the epigenetic context of diseases. By studying the role of vitC in fetal development, many further opportunities have opened up for exploring the epigenetic effect of other environmental factors. Some of these factors include the availability of food, temperature changes, stress, and exposure to pathogens. Studies based on these factors will provide insights into how environmental inputs shape the genome over time.

Future Directions

For the Santos Lab, the effects of vitC on mammalian development has opened doors to new questions and research directions. If embryos can respond to their surroundings, “we wonder what else the offspring can sense”. He aims to further explore how environmental inputs, both good and bad, ensure that gene expression happens at the appropriate pace and level. Alongside this environment-epigenome project, Dr. Ramalho-Santos is interested in understanding the biological significance of hyper-transcription, a state of accelerated gene expression in ESCs and other stem cells19. Recent work has shed light on the importance of hyper-transcription in processes such as embryogenesis, neurogenesis, and development19. Dr. Ramalho-Santos explains that there is a link between nutrient availability and hyper-transcription. Instead of entering a hyper-transcription state, the lack of nutrition leads ESCs to become dormant. Ultimately resulting in serious consequences such as missed developmental milestones.

Dr. Ramalho-Santos’ overarching goal is to provide scientific evidence that “development doesn’t happen in a vacuum.” Instead, embryonic development is remarkably reflective of its surrounding maternal environment. Overall, the Santos lab has highlighted that vitC consumption during pregnancy is important for DNA demethylation and plays a key role in establishing the epigenetic landscape of embryonic germ cells. It is important to understand that vitC influences epigenetic changes as they leave a long-lasting effect on many characteristics in offspring.

References

1.   John, R. M. & Rougeulle, C. Developmental Epigenetics: Phenotype and the Flexible Epigenome. Front. Cell Dev. Biol. 6, 130 (2018).

2.   Legoff, L., D’Cruz, S. C., Tevosian, S., Primig, M. & Smagulova, F. Transgenerational Inheritance of Environmentally Induced Epigenetic Alterations during Mammalian Development. Cells 8, 1559 (2019).

3.   DiTroia, S. P. et al. Maternal vitamin C regulates reprogramming of DNA methylation and germline development. Nature 573, 271–275 (2019).

4.   Coker, S. J., Smith-Díaz, C. C., Dyson, R. M., Vissers, M. C. M. & Berry, M. J. The Epigenetic Role of Vitamin C in Neurodevelopment. Int. J. Mol. Sci. 23, 1208 (2022).

5.   Blaschke, K. et al. Vitamin C induces Tet-dependent DNA demethylation in ESCs to promote a blastocyst-like state. Nature 500, 222–226 (2013).

6.   Dawlaty, M. M. et al. Loss of Tet Enzymes Compromises Proper Differentiation of Embryonic Stem Cells. Dev. Cell29, 102–111 (2014).

7.   Jenkins, T. G. & Carrell, D. T. Dynamic alterations in the paternal epigenetic landscape following fertilization. Front. Genet. 3, 143 (2012).

8.   Kohli, R. M. & Zhang, Y. TET enzymes, TDG and the dynamics of DNA demethylation. Nature 502, 472–479 (2013).

9.   Cinalli, R. M., Rangan, P. & Lehmann, R. Germ Cells Are Forever. Cell 132, 559–562 (2008).

10. Caldwell, B. A. et al. Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency. Mol. Cell 81, 859-869.e8 (2021).

11. Ebata, K. T. et al. Vitamin C induces specific demethylation of H3K9me2 in mouse embryonic stem cells via Kdm3a/b. Epigenetics Chromatin 10, 36 (2017).

12. Mosdol, A., Erens, B. & Brunner, E. J. Estimated prevalence and predictors of vitamin C deficiency within UK’s low-income population. J. Public Health 30, 456–460 (2008).

13. Shohaimi, S. et al. Occupational social class, educational level and area deprivation independently predict plasma ascorbic acid concentration: a cross-sectional population based study in the Norfolk cohort of the European Prospective Investigation into Cancer (EPIC-Norfolk). Eur. J. Clin. Nutr. 58, 1432–1435 (2004).

14. Rider, C. F. & Carlsten, C. Air pollution and DNA methylation: effects of exposure in humans. Clin. Epigenetics 11, 131 (2019).

15. Liu, S. et al. Arsenite Targets the Zinc Finger Domains of Tet Proteins and Inhibits Tet-Mediated Oxidation of 5-Methylcytosine. Environ. Sci. Technol. 49, 11923–11931 (2015).

16. Efimova, O. A., Koltsova, A. S., Krapivin, M. I., Tikhonov, A. V. & Pendina, A. A. Environmental Epigenetics and Genome Flexibility: Focus on 5-Hydroxymethylcytosine. Int. J. Mol. Sci. 21, 3223 (2020).

17. Yin, R. et al. Ascorbic Acid Enhances Tet-Mediated 5-Methylcytosine Oxidation and Promotes DNA Demethylation in Mammals. J. Am. Chem. Soc. 135, 10396–10403 (2013).

18. Chung, T.-L. et al. Vitamin C Promotes Widespread Yet Specific DNA Demethylation of the Epigenome in Human Embryonic Stem Cells. Stem Cells 28, 1848–1855 (2010).

19. Bulut-Karslioglu, A. et al. Chd1 protects genome integrity at promoters to sustain hypertranscription in embryonic stem cells. Nat. Commun. 12, 4859 (2021).

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