The cell-specific expression of endothelial nitric-oxide synthase - A role for DNA methylation
The cell-specific expression of endothelial nitric-oxide synthase - A role for DNA methylation
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DOI：
10.1074/jbc.m405063200
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发表时间：
2004-08-13
影响因子：
4.8
通讯作者：
Marsden, PA
中科院分区：
文献类型：
--
作者：
Chan, Y;Fish, JE;Marsden, PA
内皮细胞限制性表达内皮一氧化氮合酶（eNOS）的基础尚不清楚。虽然转基因启动子/报告基因小鼠表现出内皮细胞特异性eNOS表达，我们发现在不表达天然eNOS转录本的细胞类型中，附加型eNOS启动子/报告基因构建体有强表达。为了探索基于染色质与基于附加体的eNOS启动子的这种差异活性模式的机制，我们检测了人eNOS基因5 '调控序列的甲基化状态。DNA甲基化在内皮细胞和非内皮细胞类型（包括血管平滑肌细胞）之间存在显著差异。在小鼠主动脉的内皮和血管平滑肌细胞中，在天然小鼠eNOS启动子处观察到体内相同的细胞类型特异性甲基化模式。我们使用体外甲基化启动子/报告基因构建体的瞬时转染来研究甲基化对eNOS转录的功能影响，发现甲基化构建体在Sp1、Sp3和Ets 1对eNOS启动子活性的协同作用中表现出显著降低。甲基CpG结合蛋白2的加入进一步降低了甲基化eNOS构建体的转录活性。重要的是，染色质免疫沉淀证明了在内皮细胞中，而不是在血管平滑肌细胞中的天然eNOS启动子的Sp1，Sp3和Ets 1的存在。最后，强大的eNOS mRNA的表达诱导非内皮细胞类型的DNA甲基转移酶活性与5-氮杂胞苷抑制后，证明了DNA甲基化介导的镇压的重要性。这份报告是第一次表明，启动子DNA甲基化在血管内皮细胞组成型表达基因的细胞特异性表达中起着重要作用。
The basis for the endothelial cell-restricted expression of endothelial nitric-oxide synthase ( eNOS) is not known. While transgenic promoter/reporter mice demonstrated endothelium cell-specific eNOS expression, we found robust expression of episomal eNOS promoter/reporter constructs in cell types that do not express the native eNOS transcript. To explore the mechanism underlying this differential activity pattern of chromatin-versus episome-based eNOS promoters, we examined the methylation status of 5'-regulatory sequences of the human eNOS gene. DNA methylation differed dramatically between endothelial and nonendothelial cell types, including vascular smooth muscle cells. This same cell type-specific methylation pattern was observed in vivo in endothelial and vascular smooth muscle cells of the mouse aorta at the native murine eNOS promoter. We addressed the functional consequences of methylation on eNOS transcription using transient transfection of in vitro methylated promoter/reporter constructs and found that methylated constructs exhibited a marked decrease in the synergistic action of Sp1, Sp3, and Ets1 on eNOS promoter activity. The addition of methyl-CpG-binding protein 2 further reduced the transcriptional activity of methylated eNOS constructs. Importantly, chromatin immunoprecipitation demonstrated the presence of Sp1, Sp3, and Ets1 at the native eNOS promoter in endothelial cells but not in vascular smooth muscle cells. Finally, robust expression of eNOS mRNA was induced in nonendothelial cell types following inhibition of DNA methyltransferase activity with 5-azacytidine, demonstrating the importance of DNA methylation-mediated repression. This report is the first to show that promoter DNA methylation plays an important role in the cell-specific expression of a constitutively expressed gene in the vascular endothelium.