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Hyperhomocysteinemia (HHcy), which is an independent risk factor for atherosclerosis,might cause dysregulation of gene expression, but the characteristics and key links involved in its pathogenic mechanisms are still poorly understood. The objective of the present study was to investigate the effect of HHcy on DNA methylation and the underlying mechanism of homocysteine (Hcy)-induced DNA methylation.HHcy was induced in Sprague-Dawley rats after 4 weeks of a low, medium or high methionine diet. The levels of total homocysteine, S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) were detected by high-performance liquid chromatography. The expression levels of genes and proteins of S-adenosylhomocysteine hydrolase, DNA methyltransferase and methyl-CpG-binding domain 2 were detected by real-time reverse transcription-polymerase chain reaction and Weste blot analysis. A high-throughput quantitative methylation assay using fluorescence-based real-time polymerase chain reaction was employed to determine the levels of DNA methylation. The results indicated that HHcy induced the elevation of AdoHcy concentration, the decline of AdoMet concentration, the ratios of AdoMet/AdoHcy and the RNA and protein expression of S-adenosylhomocysteine hydrolase and methyl-CpG-binding domain 2, as well as an increase of DNA methyltransferase activity. With different methylation-dependent restriction endonucleases, the aberrant demethylation was found to prefer CCGG sequences to CpG islands. Increasing levels of HHcy significantly increased genome hypomethylation in B1 repetitive elements. The impacts of different levels of HHcy showed that the varied detrimental effects of HHcy could be attributed to different concentrations through different mechanisms. In mild and moderate HHcy, the Hcy might primarily influence the epigenetic regulation of gene expression through the interference of transferring methyl-group metabolism. However,at high Hcy concentrations, the impacts might be more injurious through oxidative stress, apoptosis and inflammation.