Developmental-vitamin D deficiency epigenetically regulates cell cycling genes in the embryonic mesencephalon via DNA methylation

Epidemiological studies have shown developmental vitamin D (DVD)-deficiency increases the risk of later onset of schizophrenia, and animal models reveal that DVD-deficiency impairs dopaminergic neuron maturation. In contrast, vitamin D treatment promotes dopaminergic neuron differentiation in cellular models. Vitamin D also modulates DNA methylation. This study investigates whether maternal vitamin D status influences differentiation of the dopamine-rich ventral mesencephalon via this epigenetic process. Mesencephalon was examined from both DVD-deficient rat dams at gestational day (GD) 14 and dams to which the active form of vitamin D was administered at GD 13. We show that from a panel of DNA methylation or demethylation enzymes, DVD-deficiency increased, whilst vitamin D decreased DNMT3A expression. We then examined the effects of increasing or decreasing DNMT3A on dopaminergic and cell cycle-related genes in mesencephalic neural cultures. DNMT3A overexpression reduced expression of cyclin D1 (CCND1) and CDKN1A (P21), while silencing DNMT3A increased expression of these important cell-cycling genes. Methylation analysis of the promoters of these genes revealed heightened cytosine methylation (5mC) at CCND1 and CDKN1A promoters in DVD-deficient embryos, but vitamin D treatment had no direct impact on these methylation patterns. We conclude that DVD-deficiency's adverse effects on early brain development may be due to heightened methylation of important cell cycle genes via increased DNMT3A. Although the active form of vitamin D decreased DNMT3A expression in utero, the absence of any silencing effect on these same cell cycle genes suggests this hormone may affect early brain differentiation via more direct transcriptional regulatory pathways via its canonical receptor.