Jamaluddin M D S, Chen Irene, Yang Fan, Jiang Xiaohua, Jan Michael, Liu Xiaoming, Schafer Andrew I, Durante William, Yang Xiaofeng, Wang Hong
Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Blood. 2007 Nov 15;110(10):3648-55. doi: 10.1182/blood-2007-06-096701. Epub 2007 Aug 13.
We reported previously that homocysteine (Hcy) inhibits endothelial cell (EC) growth by transcriptional inhibition of the cyclin A gene via a hypomethylation-related mechanism. In this study, we examined the effect of Hcy on epigenetic modification of the cyclin A gene and its biologic role in human ECs. Cyclin A mRNA levels were significantly suppressed by Hcy and a DNA methyltransferase inhibitor. The cyclin A promoter contains a CpG island spanning a 477-bp region (-277/200). Bisulfite sequencing followed by polymerase chain reaction (PCR) amplification of the cyclin A promoter (-267/37) showed that Hcy eliminated methylation at 2 CpG sites in the cyclin A promoter, one of which is located on the cycle-dependent element (CDE). Mutation of CG sequence on the CDE leads to a 6-fold increase in promoter activity. Hcy inhibited DNA methyltransferase 1 (DNMT1) activity by 30%, and reduced the binding of methyl CpG binding protein 2 (MeCP2) and increased the bindings of acetylated histone H3 and H4 in the cyclin A promoter. Finally, adenovirus-transduced DNMT1 gene expression reversed the inhibitory effect of Hcy on cyclin A expression and EC growth inhibition. In conclusion, Hcy inhibits cyclin A transcription and cell growth by inhibiting DNA methylation through suppression of DNMT1 in ECs.
我们之前报道过，同型半胱氨酸（Hcy）通过一种与低甲基化相关的机制，对细胞周期蛋白A基因进行转录抑制，从而抑制内皮细胞（EC）生长。在本研究中，我们检测了Hcy对细胞周期蛋白A基因表观遗传修饰的影响及其在人内皮细胞中的生物学作用。细胞周期蛋白A的mRNA水平被Hcy和一种DNA甲基转移酶抑制剂显著抑制。细胞周期蛋白A启动子包含一个跨越477bp区域（-277/200）的CpG岛。对细胞周期蛋白A启动子（-267/37）进行亚硫酸氢盐测序，随后进行聚合酶链反应（PCR）扩增，结果显示Hcy消除了细胞周期蛋白A启动子中2个CpG位点的甲基化，其中一个位于细胞周期依赖性元件（CDE）上。CDE上CG序列的突变导致启动子活性增加6倍。Hcy使DNA甲基转移酶1（DNMT1）的活性降低了30%，减少了甲基化CpG结合蛋白2（MeCP2）的结合，并增加了细胞周期蛋白A启动子中乙酰化组蛋白H3和H4的结合。最后，腺病毒转导的DNMT1基因表达逆转了Hcy对细胞周期蛋白A表达的抑制作用以及对内皮细胞生长的抑制作用。总之，Hcy通过抑制内皮细胞中DNMT1来抑制DNA甲基化，从而抑制细胞周期蛋白A的转录和细胞生长。
