Folic acid deficiency affects H3K9 crotonylation and neurodevelopment-related gene expression in embryonic stem cells

Abstract

Objective To analyze the genome-wide profiles of H3K9 crotonylation (H3K9cr) in mouse embryonic stem cells (mESCs) in normal or deficient folic acid (FA) by chromatin immunoprecipitation sequencing (ChIP-seq) in order to observe the genome-wide changes in H3K9cr status and the regulated pathway genes under FA deficiency condition. Methods The mESCs were divided into normal FA group (4 mg/L, FA4) and FA deficient group (0 mg/L, FA0). Western blotting and immunohistochemical (IHC) assay were used to detect the protein level of H3K9cr in the FA deficient mESCs and the embryonic brain tissues of neural tube defects (NTDs) mice, respectively. ChIP-seq technology was carried out for sequence identification of the obtained specific binding DNA fragments. Differentially expressed genes (DEGs) were analyzed and visualized by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways, with the aim of analyzing the whole genome of mESCs with FA deficiency and validating the regulated genes. Results Overall H3K9cr level was significantly lower in the FA deficient mESCs and the embryonic brain tissues of NTDs mice than in the corresponding normal FA mESCs and mice (P < 0.05). Neurodevelopmental-related genes such as Bdnf, Pax6, and App were down-regulated in the FA deficient mESCs (P < 0.05). The KEGG pathway analysis indicated that the DEGs were concentrated in axon-guiding, neuroactive ligand-receptor interaction, glutaminergic synapses and other nervous system-related pathways. Conclusion FA deficiency in embryonic stem cells participates in the regulation of neurodevelopmental genes through modification of H3K9cr, suggesting that the modification of histone crotonylation may be involved in neurodevelopmental diseases induced by FA deficiency.

Keywords

• folic acid deficiency
• histone crotonylation
• chip-seq
• neurogenesis