High Folic Acid Intake Could Lead to Changes in Gene Expression

A person's epigenetics can be affected by a number of different factors, including environment, stress, and diet. Folate (FA) is a type of B vitamin that is commonly found in citrus fruits and leafy green vegetables. FA is often recommended to be taken by pregnant women to help prevent potential birth defects.

One issue with FA is that excess amounts do not get excreted from the body without some effort. A buildup of it may have a negative effect on cells, hindering the healthy development of an organism, and can lead to neurological changes.

To help metabolize FA, the body employs an enzyme called methylenetetrahydrofolate reductase (MTHFR). Unfortunately, MTHFR gene mutations are extremely common and can result in the improper functioning of the enzyme, which could lead to a buildup of FA.

Scientists from the University of South Carolina Upstate wanted to determine if a knockdown of the MTHFR gene had any effect on gene expression in the presence of FA. They were particularly interested in how epigenetic modifications like DNA methylation, and histone modifications were affected with or without MTHFR knockdown.

The team, led by Dr. Kimberly Shorter, obtained SHSY5Y cells and split them into four groups: normal untreated (SCR), normal cells treated with FA (SCR+10x FA), and 2 MTHFR knockdown groups: MTHFR untreated cells (KD), and MTHFR treated with 10x FA (KD+10x FA). The cells were harvested after 48 hours and prepped for examination.

To begin, the researchers did a preliminary gene expression array to set a baseline for change in expression., Immediately, they noticed that FA treatment alone had significantly increased expression in some genes. The areas of activity observed included DNA methyltransferases (DNMTs), histone methyltransferases (HMT), and histone acetyltransferases (HAT).

To determine the reason behind these expression changes, the team employed a number of different kits from EpigenTek to measure the different epigenetic modifications being affected by the FA treatments.

They first looked into the DNA methylation patterns of the 4 groups of cells. DNA methylation involves the addition of a methyl group to the 5th cytosine of DNA. This process plays a role in development and disease and can result in a change of gene expression.

They isolated DNA from the four different cell samples, then used the MethylFlash Global DNA Methylation (5-mC) ELISA Easy Kit and Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit to measure the levels of 5-mC and 5-hmC, respectively. FA treated cells alone exhibited no crucial changes to either 5-mC or 5-hmC. In the KD groups, they found that global DNA methylation had decreased significantly, while global hydroxymethylated DNA had increased. This finding is important because a global decrease in 5-hmC has been demonstrated in most cancers; so, FA may have a hand in cancer protection.

To better grasp the effect that FA treatment had on cells, Dr. Shorter and her team isolated nuclear proteins using the EpiQuik Nuclear Extraction Kit to measure the activity of different enzymes.

The ten-eleven translocation (TET) is a family of enzymes responsible for oxidizing 5-mC to create 5-hmC. In this study, the team used the Epigenase 5mC-Hydroxylase TET Activity/Inhibition Assay Kit to measure the activity of TETs 1-3. TET2 expression was found to have substantially increased due to knockdown alone, whereas TET3 and TET1 were unaffected.

As mentioned earlier, 5-hmc levels increased, but 5-mC levels decreased as a result of the MTHFR knockdown. Since TET enzymes have a hand in the reversal of DNA methylation, the team speculates that the increased levels of 5-hmC could have led to the decrease in 5-mC.

DNMTs are responsible for establishing and maintaining DNA methylation patterns, which play a part in determining which genes get expressed or silenced. DNMT activity was measured using the EpiQuik DNMT Activity/Inhibition Assay Ultra Kit (Colorimetric). Of the 4 groups of cells, DNMT activity was substantially increased in both of the KD samples, making the decrease in global 5-mC an interesting discovery. In the FA treated cells, the DNMT levels were not affected, nor were there any significant changes due to the interaction between knockdown and FA treatment.

Histones are proteins that serve as the building blocks for chromatin structure, and modifications can affect how genes are expressed. For histone modification analysis, histones were isolated using the EpiQuik Total Histone Extraction HT Kit. They then measured the Histone H3 modifications using the EpiQuik Histone H3 Modification Multiplex Assay Kit.

They determined that H3 methylation remained largely unchanged across the samples, while the KD+10x FA cells experienced substantial increases in H3K18Ac, H3ser28p and H3ser10p levels. This finding is important because abnormal histone modifications could lead to autoimmune disorders, cancers, and neurological diseases.

To elaborate on their results, the team evaluated the activity of the enzymes involved in histone modification. They used the EpiQuik Histone Methyltransferase Activity/Inhibition Assay Kit (H3K4) to measure the activity of H3K4 HMT and found that both KD and KD+10x FA cells had virtually no effect on HMT levels, which is consistent with the unchanged H3 methylation levels.

Next, they measured HDAC and HAT activity using the Epigenase HDAC Activity/Inhibition Direct Assay Kit (Colorimetric) and EpiQuik HAT Activity/Inhibition Assay Kit, respectively. HDAC activity was shown to have greatly decreased in the KD+10x FA cells, though levels in the KD and SCR+10x FA cells remained stagnant. HAT activity also remained fairly unchanged across all samples.