Mild to moderate hyperhomocysteinemia has been implicated in neurodevelopmental disorders and neurodegenerative diseases in human studies. Although the molecular mechanisms underlying the effects of homocysteine (Hcy) neurotoxicity on the nervous system are not yet fully understood, inhibition of neural stem cell (NSC) proliferation and alterations in DNA methylation may be involved. The aim of the present study was to characterize the effects of Hcy on DNA methylation in NSCs, and to explore how Hcy-induced changes in DNA methylation patterns affect NSC proliferation. We found that D,L-Hcy (30-1000 lM) but not L-cysteine inhibited cell proliferation and reduced levels of global DNA methylation in NSCs from neonatal rat hippocampus and increased cell injury. High levels of Hcy also induced an increase in S-adenosylhomocysteine (SAH), a decrease in the ratio of S-adenosylmethionine (SAM) to SAH, and a reduction in protein expression of the DNA methyltransferases DNMT1, DNMT3a and DNMT3b and their enzymatic activity. Moreover, the DNMT inhibitor zebularine reduced the global DNA methylation level and inhibited NSC proliferation. Our results suggest that alterations in DNA methylation may be an important mechanism by which high levels of Hcy inhibit NSC viability in vitro. Hcy-induced DNA hypomethylation may be caused by a reduction in the DNMT activity which is regulated by the cellular concentrations of SAM and SAH, or their protein expression levels. Our results also suggest that Hcy may play a role in the pathogenesis of certain nervous system diseases via a molecular mechanism that involves negative regulation of NSC proliferation and alterations in DNA methylation.
Arterial marker genes EphrinB2 and HEY2 are essential for cardiovascular development and postnatal neovascularization. Our previous study confirmed that E2F1 could activate the transcription of EphrinB2 and HEY2 in human mesenchymal stem cells; however, the detailed mechanism has not been resolved yet. In this study, we focused on the interaction between E2F1 and DNMT3A, a de novo DNA methyltransferase, on regulating the expression of EphrinB2 and HEY2, and explored the potential mechanisms. Gain- and loss-of-function experiments implicated the positive effect of E2F1 on the expression of EphrinB2 and HEY2 and tube formation in human umbilical artery endothelial cells. Accumulation of DNMT3A decreased the levels of EphrinB2 and HEY2, and impaired tube formation induced by E2F1, while inhibiting DNMT3A by RNA interference augmented their expression and angiogenesis in E2F1-trasfected cells. We then asked whether the low expressions of EphrinB2 and HEY2 induced by DNMT3A are related to the methylation status of their promoters. Surprisingly, the methylation status of the CpG islands in the promoter region was not significantly affected by overexpression of exogenous DNMT3A. Furthermore, the interaction between E2F1 and DNMT3A was confirmed by co-immunoprecipitation. DNMT3A could inhibit the transcription of EphrinB2 and HEY2 promoters by affecting the binding of E2F1 to its recognition sequences as revealed by luciferase reporter assay and chromatin immunoprecipitation. These results identified a novel mechanism underlying the cooperation of DNMT3A with E2F1 on regulating target gene expression, and revealed their roles in the angiogenic process.
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