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. 2022 Feb 24:13:812764.
doi: 10.3389/fgene.2022.812764. eCollection 2022.

Epigenetic Modifier Supplementation Improves Mitochondrial Respiration and Growth Rates and Alters DNA Methylation of Bovine Embryonic Fibroblast Cells Cultured in Divergent Energy Supply

Matthew S Crouse  1 , Joel S Caton  2 , Kate J Claycombe-Larson  3 , Wellison J S Diniz  4 , Amanda K Lindholm-Perry  1 , Lawrence P Reynolds  2 , Carl R Dahlen  2 , Pawel P Borowicz  2 , Alison K Ward  2
Affiliations

Epigenetic Modifier Supplementation Improves Mitochondrial Respiration and Growth Rates and Alters DNA Methylation of Bovine Embryonic Fibroblast Cells Cultured in Divergent Energy Supply

Matthew S Crouse et al. Front Genet. .

Abstract

Epigenetic modifiers (EM; methionine, choline, folate, and vitamin B12) are important for early embryonic development due to their roles as methyl donors or cofactors in methylation reactions. Additionally, they are essential for the synthesis of nucleotides, polyamines, redox equivalents, and energy metabolites. Despite their importance, investigation into the supplementation of EM in ruminants has been limited to one or two epigenetic modifiers. Like all biochemical pathways, one-carbon metabolism needs to be stoichiometrically balanced. Thus, we investigated the effects of supplementing four EM encompassing the methionine-folate cycle on bovine embryonic fibroblast growth, mitochondrial function, and DNA methylation. We hypothesized that EM supplemented to embryonic fibroblasts cultured in divergent glucose media would increase mitochondrial respiration and cell growth rate and alter DNA methylation as reflected by changes in the gene expression of enzymes involved in methylation reactions, thereby improving the growth parameters beyond Control treated cells. Bovine embryonic fibroblast cells were cultured in Eagle's minimum essential medium with 1 g/L glucose (Low) or 4.5 g/L glucose (High). The control medium contained no additional OCM, whereas the treated media contained supplemented EM at 2.5, 5, and 10 times (×2.5, ×5, and ×10, respectively) the control media, except for methionine (limited to ×2). Therefore, the experimental design was a 2 (levels of glucose) × 4 (levels of EM) factorial arrangement of treatments. Cells were passaged three times in their respective treatment media before analysis for growth rate, cell proliferation, mitochondrial respiration, transcript abundance of methionine-folate cycle enzymes, and DNA methylation by reduced-representation bisulfite sequencing. Total cell growth was greatest in High ×10 and mitochondrial maximal respiration, and reserve capacity was greatest (p < 0.01) for High ×2.5 and ×10 compared with all other treatments. In Low cells, the total growth rate, mitochondrial maximal respiration, and reserve capacity increased quadratically to 2.5 and ×5 and decreased to control levels at ×10. The biological processes identified due to differential methylation included the positive regulation of GTPase activity, molecular function, protein modification processes, phosphorylation, and metabolic processes. These data are interpreted to imply that EM increased the growth rate and mitochondrial function beyond Control treated cells in both Low and High cells, which may be due to changes in the methylation of genes involved with growth and energy metabolism.

Keywords: DNA methylation; cell growth; embryonic fibroblasts; mitochondrial respiration; one-carbon metabolism.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Cell growth rate is increased in High cells with increasing epigenetic modifiers (EM) and to ×5 in Low cells. Three replicates per treatment per timepoint were measured. The error bars represent the average standard error of the mean within the glucose level. # indicates a tendency (0.05 < p < 0.10) for an interaction of glucose × EM. * indicates a significant (p ≤ 0.05) interaction of glucose × EM.
FIGURE 2
FIGURE 2
The cell proliferation responses are dependent on the time of measurement and decrease as cells reach confluency. High glucose cells are generally greater than Low cells, and the labeling index in High cells across each treatment is representative of growth rates. Low glucose cells are represented by the solid white bar. High glucose cells are represented by the dashed bar. Means without a common superscript differ in glucose × epigenetic modifiers (p ≤ 0.05). Three replicates per treatment per timepoint were measured. Error bars represent the average standard error of the mean within a glucose level.
FIGURE 3
FIGURE 3
Mitochondrial respiration increases cubically with supplementation of epigenetic modifiers (EM) in High but not in Low cells. Basal respiration and O2-linked respiration increase to ×2.5 in Low, but reserve capacity increases to ×5 in Low. Low glucose cells are represented by the solid white bar. High glucose cells are represented by the dashed bar. Means without a common superscript differ in glucose × EM (p ≤ 0.05). Three replicates per treatment were measured (three technical replicates per biological replicate). Error bars represent the average standard error of the mean within a glucose level at each measurement.
FIGURE 4
FIGURE 4
The expression of MAT2B is greater with epigenetic modifier (EM) supplementation in High cells, and DNMT1 expression is lower in High compared with Low cells. High glucose cells are represented by the dashed bar. MAT2A, methionine adenosyltransferase 2A; MAT2B, methionine adenosyltransferase 2B; DNMT1, DNA methyltransferase 1; DNMT3A, DNA methyltransferase 3A; DNMT3B, DNA methyltransferase 3B; AHCY, S-adenosylhomocysteine hydrolase; MTR, methionine synthase. Low glucose cells are represented by the solid white bar. High glucose cells are represented by the dashed bar. Means without a common superscript differ in glucose × EM (p ≤ 0.05). Only means that are significant for the glucose × EM interaction will have mean separation. Three replicates per treatment per timepoint were measured. Error bars represent the average standard error of the mean within a glucose level.
FIGURE 5
FIGURE 5
Increasing epigenetic modifier (EM) supplementation decreases the number of methylated cytosines (p < 0.01) of embryonic tracheal fibroblast cells across treatments. Data are presented as contrasts between treatments where the number of methylated and demethylated cytosines of the first treatment listed are compared against the second (i.e., the number of differentially methylated and demethylated cytosines in the High ×5 treatment compared with the High Control treatment showing a greater number of demethylated cytosines compared with methylated cytosines in High ×5 compared with High Control). The differentially methylated and demethylated cytosines were defined based on the sign of the log2 fold change. The number of differentially methylated cytosines are represented by the solid black bar. The number of differentially demethylated cytosines are represented by the dashed bar.
FIGURE 6
FIGURE 6
Over-represented biological processes (BPs) of genes with differentially methylated cytosines from embryonic tracheal fibroblast cells treated with different levels of glucose and epigenetic modifiers. The plot is color-coded based on -log10 (false discovery rate) of different BPs.
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