Mthfd1 Is an Essential Gene in Mice and Alters Biomarkers of Impaired One-carbon Metabolism

Cytoplasmic folate-mediated one carbon (1C) metabolism functions to carry and activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine. C1 tetrahydrofolate (THF) synthase, encoded by Mthfd1, is an entry point of 1Cs into folate metabolism through its formyl-THF synthetase (FTHFS) activity that catalyzes the ATP-dependent conversion of formate and THF to 10-formyl-THF. Disruption of FTHFS activity by the insertion of a gene trap vector into the Mthfd1 gene results in embryonic lethality in mice. Mthfd1^gt/+ mice demonstrated lower hepatic adenosylmethionine levels, which is consistent with formate serving as a source of 1Cs for cellular methylation reactions. Surprisingly, Mthfd1^gt/+ mice exhibited decreased levels of uracil in nuclear DNA, indicating enhanced de novo thymidylate synthesis, and suggesting that serine hydroxymethyltransferase and FTHFS compete for a limiting pool of unsubstituted THF. This study demonstrates the essentiality of the Mthfd1 gene and indicates that formate-derived 1Cs are utilized for de novo purine synthesis and the remethylation of homocysteine in liver. Further, the depletion of cytoplasmic FTHFS activity enhances thymidylate synthesis, affirming the competition between thymidylate synthesis and homocysteine remethylation for THF cofactors.Folate-mediated one-carbon (1C)³ metabolism is compartmentalized in the cytoplasm, mitochondria, and nucleus of mammalian cells (1). In the cytoplasm, 1C metabolism functions to carry and chemically activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine (2) (see Fig. 1). Methionine can be adenosylated to form S-adenosylmethionine (AdoMet), the major cellular methyl group donor required for the methylation of DNA, RNA, histones, small molecules, and lipids. Nuclear 1C metabolism functions to synthesize thymidylate from dUMP and serine during S phase through the small ubiquitin-like modifier-dependent translocation of cytoplasmic serine hydroxymethyltransferase (cSHMT), dihydrofolate reductase, and thymidylate synthase into the nucleus (3).Open in a separate windowFIGURE 1.Folate-mediated one-carbon metabolism occurs in the mitochondria, nucleus, and cytoplasm. Mitochondrial-derived formate traverses to the cytoplasm where it is incorporated into the folate-activated one-carbon pool through the activity of FTHFS and utilized in the synthesis of purines, thymidylate, and the methylation of homocysteine to methionine. Methionine can be converted to a methyl donor through its adenosylation to AdoMet. Thymidylate biosynthesis occurs in the cytoplasm and nucleus. The one-carbon unit is labeled in bold. GCS, glycine cleavage system; mSHMT, mitochondrial serine hydroxymethyltransferase; mMTHFD, mitochondrial methylenetetrahydrofolate dehydrogenase; mMTHFC, mitochondrial methenyltetrahydrofolate cyclohydrolase; mFTHFS, mitochondrial formyltetrahydrofolate synthetase; MTHFD, methylenetetrahydrofolate dehydrogenase; MTHFC, methenyltetrahydrofolate cyclohydrolase; FTHFS, formyltetrahydrofolate synthetase; MTHFR, methylenetetrahydrofolate reductase; TS, thymidylate synthase; DHFR, dihydrofolate reductase; and cSHMT, cytoplasmic serine hydroxymethyltransferase.Serine, through its conversion to glycine by SHMT, is a primary source of 1Cs for nucleotide and methionine synthesis (4). SHMT generates 1Cs in the cytoplasm, mitochondria, and nucleus, although the generation of 1Cs through SHMT activity in the cytoplasm is not essential in mice, indicating the essentiality of mitochondria-derived 1Cs for cytoplasmic 1C metabolism (5). In mitochondria, the hydroxymethyl group of serine and the C2 carbon of glycine are transferred to tetrahydrofolate (THF) to generate 5,10-methylene-THF by the mitochondrial isozyme of SHMT and the glycine cleavage system, respectively (6). The 1C carried by methylene-THF is oxidized and hydrolyzed to generate formate by the NAD-dependent methylene-THF dehydrogenase (MTHFD) and methenyl-THF cyclohydrolase (MTHFC) activities encoded by a single gene, Mthfd2 (7), and 10-formyl-THF synthetase (FTHFS) activity, encoded by Mthfd1L (8) (see Fig. 1).In the cytoplasm, the product of the Mthfd1 gene, C1THF synthase, is a trifunctional enzyme that contains NADP-dependent MTHFD and MTHFC activities on the N-terminal domain of the protein, and FTHFS activity on the C-terminal domain (9). These three activities collectively catalyze the interconversion of THF, 10-formyl-THF, 5,10-methenyl-THF, and 5,10-methylene-THF (10) (Fig. 1). The ATP-dependent FTHFS activity of C1THF synthase condenses mitochondria-derived formate with THF to form 10-formyl-THF, which is required for the de novo synthesis of purines (9). The MTHFC and MTHFD activities convert 10-formyl-THF to methylene-THF (11). Methylene-THF is utilized in the de novo synthesis of thymidylate or, alternatively, can be irreversibly reduced by methylene-THF reductase to 5-methyl-THF, which is used in the remethylation of homocysteine to methionine (12).Impairments in 1C metabolism, due to insufficient folate cofactors and/or single nucleotide polymorphisms in genes that encode folate-dependent enzymes, are associated with numerous pathologies and developmental anomalies, including cancers, cardiovascular disease, and neural tube defects. The causal mechanisms underlying the folate-pathology relationship(s) remains to be established. However, a number of hypotheses have been proposed related to the role of 1C metabolism in genome stability and gene expression. Decreased thymidylate synthesis results in increased uracil misincorporation into DNA and decreased rates of cell division, causing double strand breaks in DNA and genomic instability (13). Decreased AdoMet synthesis alters methylation patterns in CpG islands in DNA and can result in histone hypomethylation, which can alter gene expression (2). Proliferating cells also require the de novo synthesis of purines to maintain rates of DNA synthesis (14).It has been shown that the gene product of Mthfd2, mitochondrial MTHFC/MTHFD is essential in mice, and Mthfd2 deficiency results in embryonic lethality (15). This protein is required for the generation of formate from serine in the mitochondria of embryonic cells. Here, we have investigated the essentiality of the Mthfd1 gene in mice and the effect of altered Mthfd1 gene expression on biomarkers of cytoplasmic 1C metabolism. Our data demonstrate that Mthfd1 is an essential gene in mice and that Mthfd1-deficient mice are a model for the study of folate-associated pathologies.