Folate-dependent one-carbon metabolism is required for the synthesis of purines and thymidylate and for the remethylation of homocysteine to methionine. Methionine is subsequently adenylated to S-adenosylmethionine (SAM), a cofactor that methylates DNA, RNA, proteins, and many metabolites. Previous experimental and theoretical modeling studies have indicated that folate cofactors are limiting for cytoplasmic folate-dependent reactions and that the synthesis of DNA precursors competes with SAM synthesis. Each of these studies concluded that SAM synthesis has a higher metabolic priority than dTMP synthesis. The influence of cytoplasmic serine hydroxymethyltransferase (cSHMT) on this competition was examined in MCF-7 cells. Increases in cSHMT expression inhibit SAM concentrations by two proposed mechanisms: (1) cSHMT-catalyzed serine synthesis competes with the enzyme methylenetetrahydrofolate reductase for methylenetetrahydrofolate in a glycine-dependent manner, and (2) cSHMT, a high affinity 5-methyltetrahydrofolate-binding protein, sequesters this cofactor and inhibits methionine synthesis in a glycine-independent manner. Stable isotope tracer studies indicate that cSHMT plays an important role in mediating the flux of one-carbon units between dTMP and SAM syntheses. We conclude that cSHMT has three important functions in the cytoplasm: (1) it preferentially supplies one-carbon units for thymidylate biosynthesis, (2) it depletes methylenetetrahydrofolate pools for SAM synthesis by synthesizing serine, and (3) it sequesters 5-methyltetrahydrofolate and inhibits SAM synthesis. These results indicate that cSHMT is a metabolic switch that, when activated, gives dTMP synthesis higher metabolic priority than SAM synthesis.

Thyroid hormone (T3) and retinoic acid (RA) are essential for normal vertebrate development and are known to coregulate several genes. Early development is predominantly retinoic acid sensitive, yet thyroid hormone receptor-alpha (T3R alpha) is expressed along with retinoic acid receptors (RAR)-alpha, -beta, and -gamma. To determine the role of unliganded T3R alpha in early development and on RA-stimulated neural development, we used homologous recombination techniques to inactivate both T3R alpha gene alleles in mouse embryonic stem (ES) cells. Loss of both T3R alpha alleles resulted in an increase in basal and RA-induced expression of the endogenous RA-responsive genes, RAR beta and alkaline phosphatase, which demonstrates that T3R alpha has an inhibitory effect on the RA response. A similar magnitude of T3R inhibition of the RA response was seen in transient transfection assays of RA response elements in both ES and assays of RA response elements in both ES and JEG cells. Cotransfection experiments were used to demonstrate that inhibition of the RA response could be mediated by T3R alpha 1. The addition of T3R alpha 1, but not the T3R alpha variant c-erbA alpha 2, to T3R alpha-null ES cells restored the inhibitory effect on RA-induced gene expression. RA-stimulated neural differentiation was seen in the wild-type, but not in T3R alpha-null ES, cells, consistent with reports of abnormal neural development as a consequence of premature RA stimulation. Our results demonstrate that the early expression of unliganded T3R alpha functions to modulate the RA response and RA-stimulated neural differentiation.