Methylation deficiency disrupts biological rhythms from bacteria to humans

The methyl cycle is a universal metabolic pathway providing methyl groups for the methylation of nuclei acids and proteins, regulating all aspects of cellular physiology. We have previously shown that methyl cycle inhibition in mammals strongly affects circadian rhythms. Since the methyl cycle and circadian clocks have evolved early during evolution and operate in organisms across the tree of life, we sought to determine whether the link between the two is also conserved. Here, we show that methyl cycle inhibition affects biological rhythms in species ranging from unicellular algae to humans, separated by more than 1 billion years of evolution. In contrast, the cyanobacterial clock is resistant to methyl cycle inhibition, although we demonstrate that methylations themselves regulate circadian rhythms in this organism. Mammalian cells with a rewired bacteria-like methyl cycle are protected, like cyanobacteria, from methyl cycle inhibition, providing interesting new possibilities for the treatment of methylation deficiencies.

The methyl cycle is a conserved regulator of biological clocks

The methyl cycle is a universally conserved metabolic pathway operating in prokaryotes and eukaryotes. In this pathway, the amino acid methionine is used to synthesize S-adenosylmethionine, the methyl donor co-substrate in the methylation of nucleic acids, histone and non-histone proteins and many other molecules within the cell. The methylation of nucleic acids and proteins is the foundation of epigenetic and epitranscriptomic regulations of gene expression, but whether the methyl cycle centrally regulates gene expression and function by controlling the availability of methyl moieties is poorly understood.From cyanobacteria to humans, a circadian clock that involves an exquisitely regulated transcription-translation-feedback loop driving oscillations in gene expression and orchestrating physiology and behavior has been described. We reported previously that inhibition of the methyl cycle in mammalian cells caused the lengthening of the period of these oscillations, suggesting the methyl cycle may indeed act as a central regulator of gene expression, at least in mammals. Here, we investigated whether the methyl cycle, given its universal presence among living beings, regulates the circadian clock in species across the phylogenetic tree of life.We reveal a remarkable evolutionary conservation of the link between the methyl cycle and the circadian clock. Moreover, we show that the methyl cycle also regulates the somite segmentation clock, another transcription-translation negative feedback loop-based timing mechanism that orchestrate embryonic development in vertebrates, highlighting the methyl cycle as a master regulator of biological clocks.SIGNIFICANCE STATEMENTHere we reveal that the methyl cycle, a universal metabolic pathway leading to the synthesis of S-adenosylmethionine, the methyl donor co-substrate in virtually all transmethylation reactions within the cell, is a conserved regulator of biological clocks. These discoveries highlight the methyl cycle as a metabolic hub that regulates gene expression via the availability of methyl moieties for the methylation of nucleic acids, proteins and many other molecules with the cell.

Maternal Methyl-Cycle Amino Acid Profile and Kinetics: Relation with Placental Growth

Maternal intake of quality protein regulates placental development and function thereby affecting fetal growth. Considering the prevalence of inadequate intakes of quality protein in Indian pregnant women, understanding the interplay between maternal supply of protein, its metabolism and fetoplacental growth becomes important. A secondary analysis of data from an open labelled-randomized intervention trial with 500 ml milk/day on south Indian pregnant women with marginally low vitamin B12 status, was performed to assess the relations between placental parameters and maternal trimester 3 methyl-cycle amino acid status as well as kinetics. This analysis was performed for 42 pregnancies from the trial where placentae had been collected and placental parameters had been measured. For these pregnancies, data on trimester 3 methionine, serine and glycine kinetics as well as plasma free amino acid concentrations were available. Protein intake and plasma citrulline concentrations were positively correlated at trimester 3 (ρ = 0.34, P = 0.027). Placental weight correlated positively with methyl-cycle specific amino acid concentrations [methionine (ρ = 0.32, P = 0.0388), serine (ρ = 0.49, P = 0.0009)], methionine kinetics [total methionine flux rates (ρ = 0.42, P = 0.006), RM (ρ = 0.45, P = 0.003), TS (ρ = 0.32, P = 0.046), TM (ρ = 0.45, P = 0.004)] and with birth weight (ρ = 0.57, P < 0.001). Findings from the current study indicate that maternal amino acid availability and more importantly, maternal methionine kinetics, positively influenced placental growth, likely mediated by key amino acids such as citrulline, which is known to regulate placental blood flow and function. As an appropriately functioning placenta is indispensable for fetal growth, these findings will form the basis for detailed mechanistic explorations into the placental regulation of maternal supply of amino acid to the fetus for designing effective intervention strategies towards optimizing fetomaternal health during and after pregnancy.

Effect of Increased Dietary Protein on the Plasma Methyl-Cycle Amino Acid Profile and Kinetics during Pregnancy

Low intakes of quality dietary protein could affect the methionine cycle during pregnancy, which is important for fetal growth and epigenetic regulations. Since low quality protein intake is prevalent in India, it is important to define biomarkers of the low protein intake, specifically of methyl cycle homeostasis. A secondary analysis of data was performed, from a randomized intervention trial with 500 ml milk/d on south Indian pregnant women, to examine the association of dietary protein intake with concentrations of specific amino acids (methionine, glycine and serine). The subjects also underwent isotopic infusions (n = 52) for the measurement of amino acid kinetics. Dietary intakes were measured each week by multiple 24 h recall until delivery. The plasma concentrations of amino acids (methionine, serine and glycine) were compared with kinetics of methionine i.e. transmethylation, remethylation and transulfuration (TM, RM, TS) and serine to glycine conversion rates, as measured by stable isotope labeled amino acid infusion. Dietary protein intake in the 3^rd trimester correlated positively with intakes of milk and milk based food products (ρ=0.52, p<0.001) and methionine (ρ=0.97, p<0.001) and with gestational weight gain (GWG, ρ=0.32, p=0.044). While the methionine concentration did not correlate with methyl cycle flux parameters (TM, RM and TS), the plasma concentrations of conditionally essential serine and glycine were positively correlated with their respective flux rate and with RM, TM and TS rates. Further, glycine concentrations specifically correlated positively with serine to glycine conversion rates (ρ=0.32, p=0.027). Dietary protein and methionine supply are important for the conservation of methionine during pregnancy. This had an effect on GWG, but not on birth weight, though this may have been due to the relatively small sample size. The plasma concentration of glycine was correlated with the serine-glycine conversion, which affords methyl groups for the body and supplies these when dietary protein/methionine is in poor supply. This indicates that itcan act as a biomarker of the serine-glycine conversion flux rate, which increases in the presence of a poor protein supply. In general, the plasma concentrations of these conditionally essential amino acids may be biomarkers of the methyl cycle during pregnancy, but this needs to be tested in a larger sample.