Abstract

Methionine (Met) is an essential precursor of S-adenosylmethionine (SAM), which is the primary methyl donor required for biological processes such as DNA and histone methylation, which alter gene expression. In dairy cows, dietary Met has been observed to exert transcriptional alterations with beneficial effects on milk biosynthesis; however, the extent of these effects via SAM remains unknown. Therefore, we evaluated the effect of Met supply on histone methylation in lysine residues K9 and K27 in the histone tail H3 via a fluorescence resonance energy transfer (FRET) system in immortalized bovine mammary alveolar epithelial cells (MACT) incubated varying concentration of Met. The histone methylation data was complemented with global DNA methylation, cellular protein synthesis, and RT-qPCR analysis of genes related to Met cycle, DNA and histone methylation, AA transporters, and protein synthesis. The histone methylation data was performed on MACT cells seeded at 30,000 cells/well in 96-well plates 24 h prior to transfection. The transfections of FRET gene reporter plasmids H3K9 and H3K27 was performed with 0.3 μL/well of Lipofectamine® 3000 and 50 ng of plasmid DNA per well. At 24 h post-transfection, cells were treated with 0, 125, 250, and 500 μM of Met, and quantification of histone methylation was performed at 0, 12, and 24 h post-treatment as well as cell viability at 24 h using CellProfiler software. An inverted microscope for live imagining (EVOS® FL Auto) equipped with a motorized scanning stage, and an environment-controlled chamber at 37˚C and 5.0% of CO2 was used to take 4 pictures/well at 4x magnification. A more defined response on histone methylation was observed in H3K9 than H3K27 to Met supply, where maximal histone methylation in H3K9 was observed with 125 μM of Met. This greater histone methylation in H3K9 at 125 μM was accompanied by greater cellular protein concentration. The linear increase in Met supply causes a linear decrease in global DNA methylation, while linearly upregulating genes related to the Met cycle (i.e., MAT1A, PEMT, SAHH, and MTR). The histone methylation data suggest that, to some extent, methyl-donors such as Met may affect the methylation sites, H3K9 and H3K27, and consequently causing a different epigenetic alteration. In the context of the dairy cow, further refinement to this FRET assay to study histone methylation could lead to establishing novel potential mechanisms of how dietary methyl donors may control the structural conformation of the bovine genome and, by extension, gene expression.

Highlights

  • Methionine (Met) and lysine [1] are the most limiting amino acids (AA) in a wide range of diets for dairy cows [2]

  • In the context of the dairy cow, further refinement to this fluorescence resonance energy transfer (FRET) assay to study histone methylation could lead to establishing novel potential mechanisms of how dietary methyl donors may control the structural conformation of the bovine genome and, by extension, gene expression

  • Among the various biological functions associated with Met, besides milk protein synthesis, gene expression regulation in dairy cows has become more evident in recent years [3, 4]

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Summary

Introduction

Methionine (Met) and lysine [1] are the most limiting amino acids (AA) in a wide range of diets for dairy cows [2]. The extent of Met effects through these mechanisms in transition dairy cows remains to be elucidated. Histone modifications such as methylation can considerably alter the available regions or information contained in the DNA that can be transcribed into mRNA and subsequent translation into proteins. This can potentially change the metabolism, health, and performance of dairy cows. The limited amount of data on bovine histone methylation has focused on mastitis [8, 9], but based on the substantial effects of Met supplementation on DNA methylation in transition dairy cows [10], it is conceivable that this nutrient can have a significant impact on histone modifications through methylation

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