Folic Acid Attenuates Fragile x Mental Retardation Protein Expression in Lymphoblastoid Cells By Activating DNA Methyltransferases
Objectives: We examined whether environmental epigenetic dysregulation during gestational development plays any contributing role in ASD. Specifically, we studied the effects of excessive folic acid (FA) supplementation on gene expression, which may play a role in fetal brain development, culminating in the onset of psychiatric conditions later in life. FA is an essential vitamin recommended during pregnancy for the prevention of neural tube defects in infants, which also induces epigenetic changes in gene-regulatory DNA sequences. Prescriptions for daily doses of FA, along with the presence of FA in over-the-counter medicines and energy drinks, together with enriched flour, are providing an excessive supply of FA, the physiological significance of which to the developing fetus is unknown. We have previously shown widespread dysregulation of gene expression in lymphoblastoid cells and in the C57BL mouse model after excessive FA supplementation.
Methods: In the present study, we used a combination of Western blot analyses, DNA sequencing following bisulfite treatment of genomic DNA, and enzyme assays in lymphoblastoid cells to identify the effect of excessive FA supplementation on the expression of the FMR gene.
Results: We found a time- and concentration-dependent decrease in the expression of the fragile X protein (FMRP) in response to FA supplementation. The decreased FMRP expression persisted for more than 48 hours after the withdrawal of FA, suggesting a possible lasting epigenome modification. Bisulfite sequencing of DNA from cells treated with low- and high-FA-supplemented cell cultures revealed the methylation of specific cytosine residues around the promoter region and transcription initiation site, indicating possible enzyme-mediated epigenetic modifications. Furthermore, we found that FA supplementation stimulated the activities of the DNA methyltransferases (DNMTs) 1, 3A, and 3B in lymphoblastoid cell extracts. These enzymes are involved in genomic DNA methylation.
Conclusions: These results demonstrate that FA supplementation of lymphoblastoid cells can cause stimulation of DNMTs that contain FA as coenzymes, leading to the increased methylation of specific cytosine residues in the gene promoter, thereby affecting protein expression.