Stability of DNA methylation patterns in mouse spermatogonia under conditions of MTHFR deficiency and methionine supplementation.

Little is known about the conditions contributing to the stability of DNA methylation patterns in male germ cells. Altered folate pathway enzyme activity and methyl donor supply are two clinically significant factors that can affect the methylation of DNA. 5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key folate pathway enzyme involved in providing methyl groups from dietary folate for DNA methylation. Mice heterozygous for a targeted mutation in the Mthfr gene (Mthfr(+/-)) are a good model for humans homozygous for the MTHFR 677C>T polymorphism, which is found in 10% of the population and is associated with decreased MTHFR activity and infertility. High-dose folic acid is administered as an empirical treatment for male infertility. Here, we examined MTHFR expression in developing male germ cells and evaluated DNA methylation patterns and effects of a range of methionine concentrations in spermatogonia from Mthfr(+/-) as compared to wild-type, Mthfr(+/+) mice. MTHFR was expressed in prospermatogonia and spermatogonia at times of DNA methylation acquisition in the male germline; its expression was also found in early spermatocytes and Sertoli cells. DNA methylation patterns were similar at imprinted genes and intergenic sites across chromosome 9 in neonatal Mthfr(+/+) and Mthfr(+/-) spermatogonia. Using spermatogonia from Mthfr(+/+) and Mthfr(+/-) mice in the spermatogonial stem cell (SSC) culture system, we examined the stability of DNA methylation patterns and determined effects of low or high methionine concentrations. No differences were detected between early and late passages, suggesting that DNA methylation patterns are generally stable in culture. Twenty-fold normal concentrations of methionine resulted in an overall increase in the levels of DNA methylation across chromosome 9, suggesting that DNA methylation can be perturbed in culture. Mthfr(+/-) cells showed a significantly increased variance of DNA methylation at multiple loci across chromosome 9 compared to Mthfr(+/+) cells when cultured with 0.25- to 2-fold normal methionine concentrations. Taken together, our results indicate that DNA methylation patterns in undifferentiated spermatogonia, including SSCs, are relatively stable in culture over time under conditions of altered methionine and MTHFR levels.

Epigenetic modification of the gene for the vitamin B(12) chaperone MMACHC can result in increased tumorigenicity and methionine dependence.

Methionine dependence, the inability of cells to grow when the amino acid methionine is replaced in culture medium by its metabolic precursor homocysteine, is characteristic of many cancer cell lines and some tumors in situ. Most cell lines proliferate normally under these conditions. The methionine dependent tumorigenic human melanoma cell line MeWo-LC1 was derived from the methionine independent non-tumorigenic line, MeWo. MeWo-LC1 has a cellular phenotype identical to that of cells from patients with the cblC inborn error of cobalamin metabolism, with decreased synthesis of cobalamin coenzymes and decreased activity of the cobalamin-dependent enzymes methionine synthase and methylmalonylCoA mutase. Inability of cblC cells to complement the defect in MeWo-LC1 suggested that it was caused by decreased activity of the MMACHC gene. However, no potentially disease causing mutations were detected in the coding sequence of MMACHC in MeWo-LC1. No MMACHC expression was detected in MeWo-LC1 by quantitative or non-quantitative PCR. There was virtually complete methylation of a CpG island at the 5'-end of the MMACHC gene in MeWo-LC1, consistent with inactivation of the gene by methylation. The CpG island was partially methylated (30-45%) in MeWo and only lightly methylated (2-11%) in control fibroblasts. Infection of MeWo-LC1 with wild type MMACHC resulted in correction of the defect in cobalamin metabolism and restoration of the ability of cells to grow in medium containing homocysteine. We conclude that epigenetic inactivation of the MMACHC gene is responsible for methionine dependence in MeWo-LC1.