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,**
*
Center for Environmental and Genetic Medicine, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, TX 77030;
Center for Human Molecular Genetics, University of Nebraska Medical Center, Omaha, NE 68198;
**
National Veterinary Research Institute, 24-100 Pulawy, Poland; and
National Center for Toxicological Research, Division of Biochemical Toxicology, Jefferson, AR 72079
3To whom correspondence should be addressed. E-mail: rfinnell{at}ibt.tamu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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embryos. Complete Folbp1 inactivation is embryolethal but can be reversed by maternal folinic acid (FA) supplementation, and thus we also tested the effect of FA supplementation on DNA methylation in Folbp1 fetuses. Overall, the extent of global DNA methylation seems to be similar across all genotypes in unsupplemented control Folbp1 mice; however, explicit conclusions regarding Folbp1-/- fetuses were not possible because only a single living unsupplemented fetus was viable at GD 15 . FA supplementation induced global DNA hypomethylation across all genotypes. FA-induced hypomethylation is most likely due to its ability to inhibit the enzyme glycine hydroxymethyltransferase, thereby inhibiting the homocysteine remethylation cycle necessary to regenerate S-adenosylmethionine, the methyl donor for DNA methyltransferases. Our hypothesis was that due to defective folate transport in Folbp1-/- embryos and fetuses, DNA would be hypomethylated, thereby altering the temporal expression of critical genes necessary for normal embryonic development. However, these results suggest that an extended examination of changes in DNA methylation prior to GD 15 is required to unequivocally prove or disprove the hypothesis.
KEY WORDS: • Folbp1 • DNA methylation • folinic acid • birth defect • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
). Clinical trials unequivocally demonstrated that periconceptional supplementation of folic acid significantly reduces the risk of neural tube defects and other congenital malformations (4
–6
). Although the mechanism underlying this effect is yet to be elucidated, one of the hypotheses that have been suggested is folate-dependent elevation of serum homocysteine (6
–8
). Hyperhomocysteinemia can result not only from an outright deficiency in folate but also in response to reduced activity on the part of one of various folate pathway enzymes (9
–11
).
The mammalian genome contains two types of information: genetic and epigenetic. The genetic portion comprises the blueprint for the synthesis of all proteins necessary for life, whereas the epigenetic component provides the temporal and spatial framework for the genetic information to be used. The predominant means of epigenetic information is via methylation of DNA cytosine bases, involving the covalent addition of a methyl group at the 5-position of cytosine, predominantly at cytosine guanine dinucleotide (CpG)4
sequences (12
,13
). In most mammals, 5-methylcytosine is present in the majority of CpG dinucleotide sequences, which are a major component of DNA-regulatory sequences (e.g., promoters) located upstream of the majority of human genes (14
,15
). Methylated DNA generally is associated with transcriptional repression via two distinct pathways: enhancement of inactive, condensed and nuclease-resistant heterochromatin; and by sterical hindrance to the binding of gene expression regulatory elements (16
,17
). DNA methylation is carried out by DNA methyltransferases (EC 2.1.1.37), of which several have been cloned, characterized and found to require S-adenosylmethionine (SAM) as the methyl donor (18
). Perturbation of methyltransferase activity results in hypomethylated DNA strands during cell replication, and because unmethylated DNA is a poor substrate for maintenance DNA methyltransferase 1, this newly acquired and altered DNA methylation pattern becomes somatically heritable, thus adversely affecting gene expression (19
–21
).
Mammalian cells harvest folate from circulating blood via folate receptors, also known as folate-binding proteins (Folbp) in the mouse, and via the reduced folate carrier. Although the folate carrier is ubiquitously located in all tissues and cells, folate receptors are highly localized and are expressed in specific tissues and cellular populations (22
,23
). Binding of folates to their receptors is a highly specific process, thereby making these receptors excellent candidate genes for conferring susceptibility to neural tube and other defects. By using homologous recombination in embryonic stem cells, we generated the Folbp1 knockout mouse strain (24
). Matings between Folbp1+/- dams and sires produces both Folbp1+/- and Folbp1+/+ live born fetuses that appear healthy, indistinguishable from one another, and fertile as adults. However, complete inactivation of the Folbp1 allele was embryolethal, given that no Folbp1-/- newborn pups were present in the litters. By examining litters during neurulation, Folbp1-/- embryos were present, although they were lagging developmentally behind their Folbp1+/- and Folbp1+/+ littermates. In addition, their neural tube closure was severely delayed and appeared morphologically abnormal and grossly malformed; eventually, the nullizygotes died by gestational day (GD) 10 (6
,24
). By orally supplementing Folbp1+/- dams with 25 mg/kg · d folinic acid (FA), we were able to substantially minimize prenatal wastage and rescue the majority of Folbp1-/- fetuses. However, in spite of the high-dose FA supplementation, 
20% of live born Folbp1-/- pups had neural tube, conotruncal and craniofacial defects (25
). Using this folate supplementation regimen, we also were successful in bringing several Folbp1- /- fetuses to term, some of which were even able to reproduce.
Thus, our working hypothesis is that defective folate transport in the embryo during critical periods of development, as achieved by inactivation of the Folbp1 gene, results in altered intracellular folate homeostasis, which in turn adversely affects DNA methylation. Perturbation of DNA methylation subsequently results in altered expression of critical developmentally regulated genes and may render embryos susceptible to developmental defects. To test this hypothesis directly, we examined the effect of maternal folate supplementation on global DNA methylation using Folbp1 embryos of all possible genotypes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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The mice were housed in clear polycarbonate cages, allowed free access to water and food (Harlan Teklad Rodent Diet #8604, Ralston-Purina, St. Louis, MO, folate content 2.7 mg/kg) and were maintained on a 12-h light/dark cycle in the Swanson Hall Vivarium at the University of Nebraska Medical Center. Virgin Folbp1+ /- females were randomly assigned to receive either a daily oral gavage of FA (Sigma-Aldrich, St. Louis, MO) or water. FA supplementation was provided by a single 25-mg/kg daily oral gavage (10 µL/g body weight) that was fed a minimum of 2 wk prior to the first attempted mating and throughout pregnancy. Females were mated overnight with Folbp1+/- males and examined for the presence of vaginal plugs the following morning. The onset of gestation was set at 22:00 h of the previous night, the midpoint of the dark cycle (26
). On GD 15 dams were killed by cervical dislocation, the abdomens were opened and the gravid uteri were removed and placed in ice-cold phosphate buffer. Each embryo then was cleaned free from surrounding membranes and tissue, and samples from brain and liver were stored at -80°C. The appropriate Institutional Animal Care and Use Committee approved the experiments described herein.
Assessing DNA methylation and quantitation of SAM and S-adenosylhomocysteine
The cytosine extension assay was used to evaluate global DNA methylation as previously described by Pogribny et al. (27
). Briefly, 1 µg of genomic DNA was digested for 16–18 h with 20 U of HpaII. A second DNA aliquot served as background control and similarly was incubated without addition of the restriction enzyme. The single nucleotide extension reaction was performed in a 25-µL reaction mixture containing 0.5 µg of DNA, 1x polymerase chain reaction buffer II, 1.0 mmol/L magnesium chloride, 0.25 U of AmpliTaq DNA polymerase (PerkinElmer, Wellesley, MA) and 0.1 µL of [3H]dCTP (57.4 Ci/mmol; New England Nuclear, Boston, MA), incubated at 56°C for 1 h and then placed on ice. Duplicate 10-µL aliquots from each reaction were applied to a Whatman DE-81 ion exchange filter, washed three times, dried and processed for scintillation counting. Background radioactivity in untreated samples was subtracted from enzyme-treated samples. The results of [3H]dCTP incorporation are expressed as dpm/0.5 µg of DNA. An increase in [3H]dCTP incorporation (higher dpm values) indicates that DNA was hypomethylated. Tissue concentrations of SAM and S-adenosylhomocysteine (SAH) were determined by high pressure liquid chromatography with electrochemical (coulometric) detection as previously described by Melnyk et al. (28
).
Statistical analysis
A pairwise comparison with the t test was used to assess the significance of variance of DNA methylation, SAM and SAH concentration and the SAM/SAH ratio between unsupplemented Folbp1+/+ fetuses and all other unsupplemented and FA-supplemented groups (P < 0.05, two-tailed). No statistical analysis was performed on the single unsupplemented Folbp1-/- fetus.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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and Table 2
depict the extent of DNA methylation and the concentrations of SAM and SAH from liver and brain of Folbp1 fetuses of all genotypes. In the liver, control Folbp1+ /+ and Folbp1+/- samples had comparable DNA methylation, whereas the single sample from a Folbp1-/- fetus appears to be hypomethylated. FA supplementation reduced hepatic DNA methylation in the Folbp1+/- and Folbp1-/- fetuses relative to unsupplemented controls. However, the single control Folbp1-/- fetus surviving to GD 15 precluded statistical comparisons. Similarly, in control brain samples, all three Folbp1 genotypes had comparable extent of DNA methylation. More than a double reduction in DNA methylation was observed across all genotypes in response to FA supplementation. Concomitant to FA-induced DNA hypomethylation in brain (Table 2)
, SAM/SAH ratios as an indicator that the cellular methylation capacity was reduced due to respective changes in SAM and SAH concentrations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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,13
), and the pattern of DNA methylation observed in adult cells is established very early in embryogenesis. During the preimplantation period, a massive wave of global demethylation occurs, in which the gamete-specific methylation patterns are erased and most CpG sites are demethylated by the 8- to 16-cell stage (29
–31
). This genomic reformatting appears to be necessary for the creation of a pluripotent state before specific cell lineages can be established. The process of establishing the adult methylation pattern involves an initial wave of global de novo methylation at the pregastrulation stage, followed by gene-specific demethylation associated with the transcription of tissue-specific genes during organogenesis (31
–33
). It is clear that the DNA methylation patterns that are established during early embryogenesis are nonrandom and form the basis for tissue-specific gene expression (33
). Experimental studies have confirmed that a failure to establish the correct methylation pattern during these early critical stages can lead to apoptosis, embryonic lethality (34
) or multiple developmental malformations (35
–38
).
From our data we see that both control Folbp1+/+ and Folbp1+/- samples from liver and brain have the same extent of global DNA methylation. Due to the embryolethal effect of complete Folbp1 inactivation, Folbp1-/- embryos die in utero by GD 101/2 (24
,25
); thus it was a very rare event to find a single living unsupplemented Folbp1-/- fetus at GD 151/2. This grossly abnormal and severely malformed Folbp1-/- fetus had craniorrhachischisis, gastroschisis and a conotruncal heart defect. Analysis of its DNA methylation status revealed that the liver was hypomethylated compared with Folbp1+/- and Folbp1+/+ embryos and that its brain was as methylated as those of Folbp1+/- and Folbp1+/+ embryos. Because we had only one Folbp1-/- embryo, explicit conclusions regarding the methylation status of Folbp1-/- embryos cannot be made from this study. An extended examination of the temporal changes in DNA methylation prior to GD 151/2, when the majority of nonsupplemented Folbp1-/- embryos are still alive, is required. Recently, a methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) mutant mouse strain was generated, and analysis of its global DNA methylation status demonstrated a proportional increase in hypomethylation in the brain of both MTHFR+/- and MTHFR-/- mutants compared with the wild-type MTHFR+/+ mice (39
). Because we expected a similar proportional response in brain samples of our Folbp1 fetuses, it was a rather unexpected finding that control mice of all genotypes had the same degree of DNA methylation, that was comparable with values obtained by Chen et al. (39
). The present study was designed to test the hypothesis that embryonic lethality and developmental defects in Folbp1-/- embryos are due to severe DNA hypomethylation secondary to defective intracellular folate transport. We postulated that FA supplementation would promote survival by increasing DNA methylation potential to provide for normal epigenetic programming, cell differentiation and development. However, it is evident that overall DNA from FA-supplemented embryos was hypomethylated compared with unsupplemented control embryos. FA has been shown to inhibit glycine hydroxymethyltransferase (EC 2.1.2.1), most likely due to its role as a cellular regulator of 5-methyltetrahydrofolate concentrations as part of the homeostasis of folate metabolism and methyl pool by the cell (40
,41
). Thus high dose FA administration to pregnant mice is likely to inhibit glycine hydroxymethyltransferase, thereby reducing levels of 5,10-methylenetetrahydrofolate, the precursor of 5-methyltetrahydrofolate, and altering SAM and SAH concentration. This in turn would reduce DNA methylation both by reducing the methylating potential and inhibiting DNA methyltransferase, thus providing a plausible explanation for the hypomethylating effect of FA across all genotypes of the Folbp1 embryos and fetuses.
The significant degree of DNA hypomethylation observed in the FA-supplemented embryos suggests that the observed level of hypomethylation was compatible with survival and normal development. The minimal level of DNA hypomethylation that can be tolerated without developmental defects has not been definitively established, but several recent studies have suggested that adequate methylation potential during the very early stages of development is more critical for normal development than at later stages (42
–44
). Based on this evidence, a plausible explanation for observed survival in the FA-supplemented Folbp1 nullizygous embryos can be proposed despite the considerable DNA hypomethylation observed at GD 151/2. It is possible that FA supplementation provided sufficient methyl groups to support the minimal methylation requirements for Folbp1-/- survival during the crucial period of de novo methylation following the preimplantation wave of global demethylation. Once the tissue-specific methylation patterns and cell lineage programming have been established, the embryo’s genome may be able to withstand considerable hypomethylation without phenotypic consequences (43
). Future experiments will determine whether unsupplemented Folbp1- /- embryos prior to GD 151/2 exhibit increased apoptosis and lethal levels of DNA hypomethylation relative to the surviving FA-supplemented embryos.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 This research was supported in part by grants DE13613, HL66398, DE12898 and P30-ES09106 from the National Institutes of Health. The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.
4 Abbreviations used: CpG, cytosine guanine dinucleotides; FA, folinic acid; Folbp, folate-binding proteins; Folbp1, murine folate-binding protein 1; Folbp1+/-, heterozygotes for folate-binding protein 1; Folbp+/+, homozygotes for folate-binding protein 1; Folbp- /-, nullizygotes for folate-binding protein 1; GD, gestational day; MTHFR, methylenetetrahydrofolate reductase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine.
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