Mihai D. Niculescu

Dietary choline deficiency alters global and gene-specific DNA methylation in the developing hippocampus of mouse fetal brains

The availability of choline during critical periods of fetal development alters hippocampal development and affects memory function throughout life. Choline deficiency during fetal development reduces proliferation and migration of neuronal precursor cells in the mouse fetal hippocampus and these changes are associated with modifications in the protein levels of some cell cycle regulators and early differentiation markers. We fed C57 BL/6 mouse dams diets deficient or normal in choline content from days 12 to 17 of pregnancy, and then collected fetal brains on embryonic day 17. Using laser‐capture micro‐dissection we harvested cells from the ventricular and subventricular zones of Ammons horn and from the prime germinal zone of the dentate gyrus (hippocampus). In the ventricular and subventricular zones from the cholinedeficient group, we observed increased protein levels for kinase‐associated phosphatase (Kap) and for p15INK4b (two cell cycle inhibitors). In the dentate gyrus, we observed increased levels of calretinin (an early marker of neuronal differentiation). In fetal brain from mothers fed a choline‐deficient diet, DNA global methylation was decreased in the ventricular and sub‐ventricular zones of Ammons horn. We also observed decreased gene‐specific DNA methylation of the gene (Cdkn3) that encodes for Kap, correlating with increased expression of this protein. This was not the case for p15INK4b or calretinin (Cdkn2b and Calb2, respectively). These data suggest that choline deficiency‐induced changes in gene methylation could mediate the expression of a cell cycle regulator and thereby alter brain development. FASEB J. 20, 43–49 (2006)

Phosphatidylethanolamine N-methyltransferase (PEMT) gene expression is induced by estrogen in human and mouse primary hepatocytes

Choline is an essential nutrient for humans, though some of the requirement can be met by endogenous synthesis catalyzed by phosphatidylethanolamine A‐methyltransferase (PEMT). Premenopausal women are relatively resistant to choline deficiency compared with postmenopausal women and men. Studies in animals suggest that estrogen treatment can increase PEMT activity. In this study we investigated whether the PEMT gene is regulated by estrogen. PEMT transcription was increased in a dose‐dependent manner when primary mouse and human hepatocytes were treated with 17‐β‐estradiol for 24 h. This increased message was associated with an increase in protein expression and enzyme activity. In addition, we report a region that contains a perfect estrogen response element (ERE) ~7.5 kb from the transcription start site corresponding to transcript variants NM_007169 and NM‐008819 of the human and murine PEMT genes, respectively, three imperfect EREs in evolutionarily conserved regions and multiple imperfect EREs in nonconserved regions in the putative promoter regions. We predict that both the mouse and human PEMT genes have three unique transcription start sites, which are indicative of either multiple promoters and/or alternative splicing. This study is the first to explore the underlying mechanism of why dietary requirements for choline vary with estrogen status in humans.—Resseguie M., Song, J., Niculescu, M. D., da Costa K., Randall, T. A., Zeisel S. H. Phosphatidylethanolamine A‐methyltrans‐ferase (PEMT) gene expression is induced by estrogen in human and mouse primary hepatocytes. FASEB J. 21, 2622–2632 (2007)

Choline deficiency alters global histone methylation and epigenetic marking at the Re1 site of the calbindin 1 gene

Maternal choline availability is essential for fetal neurogenesis. Choline deprivation (CD) causes hypomethylation of specific CpG islands in genes controlling cell cycling in fetal hippocampus. We now report that, in C57BL/6 mice, CD during gestational days 12–17 also altered methylation of the histone H3 in E17 fetal hippocampi. In the ventricular and subven‐tricular zones, monomethyl‐lysine 9 of H3 (H3K9me1) was decreased by 25% (P<0.01), and in the pyramidal layer, dimethyl‐lysine 9 of H3 (H3K9me2) was decreased by 37% (P<0.05). These changes were region specific and were not observed in whole‐brain preparations. Also, the same effects of CD on H3 methylation were observed in E14 neural progenitor cells (NPCs) in culture. Changes in G9a histone methyltransferase might mediate altered H3K9me2,1. Gene expression of G9a was decreased by 80% in CD NPCs (P<0.001). In CD, H3 was hypomethylated upstream of the RE1 binding site in the calbindin 1 promoter, and 1 CpG site within the calbindin1 promoter was hypermethylated. REST binding to RE1 (recruits G9a) was decreased by 45% (P<0.01) in CD. These changes resulted in increased expression of calbindin 1 in CD (260%; P< 0.05). Thus, CD modulates histone methylation in NPCs, and this could underlie the observed changes in neurogenesis.—Mehedint, M. G., Niculescu, M. D., Craciunescu, C. N., Zeisel, S. H. Choline deficiency alters global histone methylation and epigenetic marking at the Re1 site of the calbindin 1 gene. FASEB J. 24, 184–195 (2010). www.fasebj.org

High fat diet-induced maternal obesity alters fetal hippocampal development.

The importance of maternal nutrition for fetal brain development is increasingly recognized. Previous studies have suggested that maternal obesity or maternal exposure to obesogenic diets may permanently alter brain structure and function in the offspring. To test whether maternal exposure to a high‐fat diet, prior and during gestation, alters fetal hippocampal development, we fed 8‐week old C57BL/6 females with a high‐fat diet (60% calories from fat) for 10 weeks prior to matting and 17 days after. Fetal brains at embryonic day E17 were used to determine developmental changes in the hippocampus. We report that maternal exposure to the high‐fat diet induced small for gestational age (SGA) status and fetal resorption. The proliferation of neural progenitors was increased in the neuroepithelium from hippocampus and cortex in fetuses from mothers fed the high‐fat diet when compared to controls, but decreased within the dentate gyrus (DG). Apoptosis in the hippocampus was decreased (Ammons Horn and fimbria). The differentiation of calretinin‐positive neurons within the DG was also decreased.

Choline availability modulates human neuroblastoma cell proliferation and alters the methylation of the promoter region of the cyclin-dependent kinase inhibitor 3 gene

Choline is an important methyl donor and a component of membrane phospholipids. In this study, we tested the hypothesis that choline availability can modulate cell proliferation and the methylation of genes that regulate cell cycling. In several other model systems, hypomethylation of cytosine bases that are followed by a guanosine (CpG) sites in the promoter region of a gene is associated with increased gene expression. We found that in choline‐deficient IMR‐32 neuroblastoma cells, the promoter of the cyclin‐dependent kinase inhibitor 3 gene (CDKN3) was hypomethylated. This change was associated with increased expression of CDKN3 and increased levels of its gene product, kinase‐associated phosphatase (KAP), which inhibits the G1/S transition of the cell cycle by dephosphorylating cyclin‐dependent kinases. Choline deficiency also reduced global DNA methylation. The percentage of cells that accumulated bromodeoxyuridine (proportional to cell proliferation) was 1.8 times lower in the choline‐deficient cells than in the control cells. Phosphorylated retinoblastoma (p110) levels were 3 times lower in the choline‐deficient cells than in control cells. These findings suggest that the mechanism whereby choline deficiency inhibits cell proliferation involves hypomethylation of key genes regulating cell cycling. This may be a mechanism for our previously reported observation that stem cell proliferation in hippocampus neuroepithelium is decreased in choline‐deficient rat and mouse fetuses.

Perinatal manipulation of α-linolenic acid intake induces epigenetic changes in maternal and offspring livers

Previous studies indicated that the intake of α‐linolenic acid (ALA) can alter the concentration of both ω‐6 and ω‐3 fatty acids in both mother and offspring, with consequences on postnatal brain development. This study describes the association between maternal ALA availability during gestation and lactation, and alterations in the Fads2 DNA methylation in both maternal and offspring livers, at the end of lactation period. Both Fads2 promoter and intron 1 DNA methylation were increased in the groups receiving postnatal flaxseed oil containing 50% ALA (mothers or pups), while bivariate analysis indicated a significant association of the Fads2 epigenetic status in the liver between each mother and its offspring. In addition, Fads2 expression was negatively correlated with promoter methylation at the individual level in maternal livers (P<0.05). This study also indicated that the interplay between ALA availability during gestation and lactation can differentially alter the expression of desaturases and elongases involved in ω‐6 and ω‐3 metabolic pathways. In summary, when considering the perinatal dietary ALA requirements in mice, both gestation and lactation periods should be considered as having distinct roles in modulating the metabolism of ω‐6 and ω‐3 fatty acids in maternal mouse livers.—Niculescu, M. D., Lupu, D. S., Craciunescu, C. N. Perinatal manipulation of α‐linolenic acid intake induces epigenetic changes in maternal and offspring livers. FASEB J. 27, 350–358 (2013). www.fasebj.org

The epigenetic effects of a high prenatal folate intake in male mouse fetuses exposed in utero to arsenic.

Inorganic arsenic (iAs) is a complete transplacental carcinogen in mice. Previous studies have demonstrated that in utero exposure to iAs promotes cancer in adult mouse offspring, possibly acting through epigenetic mechanisms. Humans and rodents enzymatically convert iAs to its methylated metabolites. This reaction requires S-adenosylmethionine (SAM) as methyl group donor. SAM is also required for DNA methylation. Supplementation with folate, a major dietary source of methyl groups for SAM synthesis, has been shown to modify iAs metabolism and the adverse effects of iAs exposure. However, effects of gestational folate supplementation on iAs metabolism and fetal DNA methylation have never been thoroughly examined. In the present study, pregnant CD1 mice were fed control (i.e. normal folate, or 2.2 mg/kg) or high folate diet (11 mg/kg) from gestational day (GD) 5 to 18 and drank water with 0 or 85 ppm of As (as arsenite) from GD8 to 18. The exposure to iAs significantly decreased body weight of GD18 fetuses and increased both SAM and S-adenosylhomocysteine (SAH) concentrations in fetal livers. High folate intake lowered the burden of total arsenic in maternal livers but did not prevent the effects of iAs exposure on fetal weight or hepatic SAM and SAH concentrations. In fact, combined folate-iAs exposure caused further significant body weight reduction. Notably, iAs exposure alone had little effect on DNA methylation in fetal livers. In contrast, the combined folate-iAs exposure changed the CpG island methylation in 2,931 genes, including genes known to be imprinted. Most of these genes were associated with neurodevelopment, cancer, cell cycle, and signaling networks. The canonical Wnt-signaling pathway, which regulates fetal development, was among the most affected biological pathways. Taken together, our results suggest that a combined in utero exposure to iAs and a high folate intake may adversely influence DNA methylation profiles and weight of fetuses, compromising fetal development and possibly increasing the risk for early-onset of disease in offspring.

Nutritional influence on epigenetics and effects on longevity.

Purpose of reviewThis review synthesizes recently published information regarding nutrition and its impact upon epigenetically mediated mechanisms involved in longevity and aging. Recent findingsRecent studies enriched considerably our understanding of the relationship between aging and gene–nutrient interactions that continuously shape our phenotype. Epigenetic mechanisms play an important role in mediating between the nutrient inputs and the ensuing phenotypic changes throughout our entire life and seem to be responsible, in part, for the biological changes that occur during aging. Less is known about the epigenetic role that nutrients have in directly influencing longevity and aging. However, recent studies clearly indicated that because nutrition modulates epigenetic events associated with various diseases (e.g., cancer, obesity, and diabetes), there is at least an indirect epigenetic link between nutrition and longevity and, therefore, biologic plausibility to hypothesize the epigenetic role of nutrition in altering longevity. Apart from limited human studies, promising animal studies brought us much closer to understanding how nutrition could have such an impact upon longevity and aging. SummaryComplex epigenetic mechanisms are involved in aging and longevity, directly or indirectly via disease mechanisms. Nutrition has a strong impact upon epigenetic processes and, therefore, holds promise in having important roles in regulating longevity and aging.

Maternal α-linolenic acid availability during gestation and lactation alters the postnatal hippocampal development in the mouse offspring

The availability of ω‐3 polyunsaturated fatty acids is essential for perinatal brain development. While the roles of docosahexaenoic acid (the most abundant ω‐3 species) were extensively described, less is known about the role of α‐linolenic acid (ALA), which is the initial molecular species undergoing elongation and desaturation within the ω‐3 pathways. This study describes the association between maternal ALA availability during gestation and lactation, and alterations in hippocampal development (dentate gyrus) in the mouse male offspring, at the end of lactation (postnatal day 19, P19). Postnatal ALA supplementation increased cell proliferation (36% more proliferating cells compared to a control group) and early neuronal differentiation, while postnatal ALA deficiency increased cellular apoptosis within the dentate gyrus of suckling pups (61% more apoptotic cells compared to a control group). However, maternal ALA deficiency during gestation prevented the increased neurogenesis induced by postnatal supplementation. Fatty acid analysis revealed that ALA supplementation increased the concentration of the ω‐3 species in the maternal liver and serum, but not in the brain of the offspring, excepting for ALA itself. Interestingly, ALA supplementation also increased the concentration of dihomo γ‐linolenic acid (a ω‐6 species) in the P19 brains, but not in maternal livers or serum. In conclusion, postnatal ALA supplementation enhances neurogenesis in the dentate gyrus of the offspring at postnatal day 19, but its beneficial effects are offset by maternal ALA deficiency during gestation. These results suggest that ALA is required in both fetal and postnatal stages of brain development.

Identification of new genetic polymorphisms that alter the dietary requirement for choline and vary in their distribution across ethnic and racial groups

Effect alleles (alleles with a polymorphism that is associated with the effect being measured) in a small number of single‐nucleotide polymorphisms (SNPs) are known to influence the dietary requirement for choline. In this study, we examined a much larger number of SNPs (n=200) in 10 genes related to choline metabolism for associations with development of organ dysfunction (liver or muscle) when 79 humans were fed a low‐choline diet. We confirmed that effect alleles in SNPs such as the C allele of PEMT rs12325817 increase the risk of developing organ dysfunction in women when they consume a diet low in choline, and we identified novel effect alleles, such as the C allele of CHKA SNP rs7928739, that alter dietary choline requirements. When fed a low‐choline diet, some people presented with muscle damage rather than liver damage; several effect alleles in SLC44A1 (rs7873937, G allele; rs2771040, G; rs6479313, G; rs16924529, A; and rs3199966, C) and one in CHKB (rs1557502, A) were more common in these individuals. This suggests that pathways related to choline metabolism are more important for normal muscle function than previously thought. In European, Mexican, and Asian Americans, and in individuals of African descent, we examined the prevalence of the effect alleles in SNPs that alter choline requirement and found that they are differentially distributed among people of different ethnic and racial backgrounds. Overall, our study has identified novel genetic variants that modulate choline requirements and suggests that the dietary requirement for choline may be different across racial and ethnic groups.—Da Costa, K.‐A., Corbin, K. D., Niculescu, M. D., Galanko, J. A., Zeisel, S. H. Identification of new genetic polymorphisms that alter the dietary requirement for choline and vary in their distribution across ethnic and racial groups. FASEB J. 28, 2970–2978 (2014). www.fasebj.org