Homocysteine Inhibits Cyclin A Promoter Methylation Via DNMT3 Inactivation in Human Endothelial Cells.

Hyperhomocysteinemia is a significant and independent risk factor for cardiovascular disease. We reported previously that homocysteine (Hcy) inhibits endothelial cell (EC) growth by transcriptional inhibition of the cyclin A gene. This is associated with an increase in S-adenosylhomocysteine, a potent inhibitor of methyltransferase. We hypothesized that Hcy inhibits EC growth and cyclin A transcription via hypomethylation and studied the effect of Hcy on epigenetic regulation of the cyclin A gene in EC. We found that the levels of cyclin A mRNA were significantly suppressed by azacytidne (AZC), a potent DNA methyl transferase (DNMT) inhibitor, in human umbilical vein EC (HUVEC). The cyclin A promoter (−518/256) is rich in GC content (59.1%), and has a CpG island spanning a 477 bp region (−277/200). Bisulphite sequencing followed by PCR amplification of the cyclin A core (−267/37) promoter, which contains 23 CpG sites serving as potential methylation sites, showed that Hcy (50 mM) eliminated methylation at two CpG sites, position 1 and 35, in the cyclin A promoter. Hcy selectively inhibited the activity of DNA methyl transferase 1 (DNMT1) by 40%, and had no effect on DNA methyl transferase 3 (DNMT3) activity in HUVEC. Furthermore, chromatin immunoprecipitation (ChIP) assays demonstrated that Hcy reduced the binding of methyl CpG binding protein 2 (MeCP2) and increased the binding of acetylated histone H3 and H4 in the cyclin A promoter in HUVEC but not in human aortic smooth muscle cells. The binding of MeCP2 to the cyclin A promoter was completely suppressed by AZC and trichostatin A, a histone deacetylase (HDAC) inhibitor, indicating that HDAC and DNA methylation mediate MeCP2 binding to cyclin A promoter. Finally, adenovirus tranduced DNMT1 overexpression, but not DNMT3, reversed Hcy-induced growth inhibition. In conclusion, we found that Hcy inhibits EC growth by suppressing cyclin A transcription, and that Hcy exerts this action by inhibiting DNA methylation through the suppression of DNMT1 and HDAC activity.