Activation of the methylation cycle in cells reprogrammed into a stem cell-like state

Abstract

Generation of induced pluripotent stem (iPS) cells and cancer biogenesis share similar metabolic switches. Most studies have focused on how the establishment of a cancer-like glycolytic phenotype is necessary for the optimal routing of somatic cells for achieving stemness. However, relatively little effort has been dedicated towards elucidating how one-carbon (1C) metabolism is retuned during acquisition of stem cell identity. Here we used ultra-high pressure liquid chromatography coupled to an electrospray ionization source and a triple-quadrupole mass spectrometer [UHPLC-ESI-QqQ-MS/MS] to quantitatively examine the methionine/folate bi-cyclic 1C metabolome during nuclear reprogramming of somatic cells into iPS cells. iPS cells optimize the synthesis of the universal methyl donor S-adenosylmethionine (SAM), apparently augment the ability of the redox balance regulator NADPH in SAM biosynthesis, and greatly increase their methylation potential by triggering a high SAM:S-adenosylhomocysteine (SAH) ratio. Activation of the methylation cycle in iPS cells efficiently prevents the elevation of homocysteine (Hcy), which could alter global DNA methylation and induce mitochondrial toxicity, oxidative stress and inflammation. In this regard, the methyl donor choline is also strikingly accumulated in iPS cells, suggesting perhaps an overactive intersection of the de novo synthesis of choline with the methionine-Hcy cycle. Activation of methylogenesis and maintenance of an optimal SAM:Hcy ratio might represent an essential function of 1C metabolism to provide a labile pool of methyl groups and NADPH-dependent redox products required for successfully establishing and maintaining an embryonic-like DNA methylation imprint in stem cell states.

Keywords: Homocysteine; S-adenosylhomocysteine; iPS cells; one-carbon metabolism; stem cells.

Figure 1. 1C metabolomic profiling of iPS cells

2D (A) and 3D (B) PLS-DA models for the parental MEFs and iPS cells groups. C. Heatmap visualization and hierarchical clustering analysis with MetaboAnalyst's data annotation tool. Rows: metabolites; Columns: samples; color key indicates metabolite expression value (i.e., blue: lowest; red: highest).

Figure 2. Metabolomic profile of the methionine/folate bi-cyclic 1C metabolism in iPS cells

SAM serves as the methyl donor for numerous methylation reactions, including DNA methylation and synthesis of phosphatidylcholine. During methyl group donation, SAM is converted into SAH, a potent competitive inhibitor of many methyltransferases including DNMTs. Demethylated SAM then is further processed to Hcy, which in turn is converted back to methionine either through a folate-dependent mechanism (the folate cycle on the right side of the schematic, in which 5-mTHF is the methyl donor in the MS pathway) or a folate-independent mechanism (in the left side of the schematic, in which betaine is the methyl donor in the BHMT pathway). MAT: Methionine adenosyltransferase; PEMT: Phosphatidylethanolamine-N-methyl transferase; MTs: Methyl transferases; DNMTs: DNA methyl transferases; SAHH: SAH hydrolase; Hcy: Homocysteine; BHMT: Betaine homocysteine methyltransferases; MS: Methionine synthase; SHMT: serine hydroxymethyltransferase; MTHFR: methylenetetrahydrofolate reductase; 5-mTHF: 5-methyltetrahydrofolate; 5,10-mTHF: 5,10-methylenetetrahydrofolate; Cob: Cobalamine; Zn: Zinc; CBS: Cystathione-β-synthase; DMG: Dimethylglycine; FAD: Flavin adenine dinucleotide; PE: Phosphatidylethanolamine; PC: Phosphatidylcholine; TS: Thymidylate synthase.

Figure 3. 1C metabolism in iPS cells: Balancing methylogenesis, redox, and inflammation

Activation of 1C metabolism and the process of methylogenesis during nuclear reprogramming of somatic cells into iPS cells might ensure the correct correlation between the SAM/Hcy cycle and the DNA methylation imprint of stem cell states by providing a labile pool of methyl groups and NADPH-dependent redox products while avoiding the pro-oxidant and pro-inflammatory effects of Hcy.