Abnormal transcriptome-wide DNA demethylation induced by folate deficiency causes neural tube defects.

Neural tube defect (NTDs) is one of the most common and serious fetal and neonatal birth defects. Neural tube closure (NTC) is an exquisitely coordinated process and this procedure is influenced by both genetic and environmental factor. Folic acid (FA) supplementation is an effective for prevention of a proportion of NTDs, however, the mechanism remains unclear. In this study, our data demonstrated genome-wide enrichment of 5-hydroxymethylcytosine (5hmC) modification on active transcriptional start sites (TSS) and decreased 5-methylcytosine (5mC) binding to TSS under folate deficiency in mESCs (mouse embryonic stem cells). Furthermore, folate deficiency promoted 5hmC enrichment enhancer histone 3 lysine 27 acetylation (H3K27ac) binding to Shh pathway genes in mESCs. Upregulation of Shh target genes was observed in mouse brain tissue under low levels of maternal serum folate, along with increased expression of 5-methylcytosine dioxygenase Tet1 levels. Taken together, we found that folate deficiency promoted DNA demethylation and enriched 5hmC through recruitment of H3K27ac to activate the Shh signaling pathway. These results suggest that the 5hmC modification increases concomitantly with a positive correlation to Shh gene expression in folate deficiency-induced mouse NTDs.

Folate-deficiency induced acyl-CoA synthetase short-chain family member 2 increases lysine crotonylome involved in neural tube defects.

Maternal folate deficiency increases the risk of neural tube defects (NTDs), but the mechanism remains unclear. Here, we established a mouse model of NTDs via low folate diets combined with MTX-induced conditions. We found that a significant increase in butyrate acid was observed in mouse NTDs brains. In addition, aberrant key crotonyl-CoA-producing enzymes acyl-CoA synthetase short-chain family member 2 (ACSS2) levels and lysine crotonylation (Kcr) were elevated high in corresponding low folate content maternal serum samples from mouse NTD model. Next, proteomic analysis revealed that folate deficiency led to global proteomic modulation, especially in key crotonyl-CoA-producing enzymes, and dramatic ultrastructural changes in mouse embryonic stem cells (mESCs). Furthermore, we determined that folate deficiency induced ACSS2 and Kcr in mESCs. Surprisingly, folic acid supplementation restored level of ACSS2 and Kcr. We also investigated overall protein post-translational Kcr under folate deficiency, revealing the key regulation of Kcr in glycolysis/gluconeogenesis, and the citric acid cycle. Our findings suggest folate deficiency leads to the occurrence of NTDs by altering ACSS2. Protein crotonylation may be the molecular basis for NTDs remodeling by folate deficiency.