Zhang Ming-Xiang, Zhang Cheng, Shen Ying H, Wang Jian, Li Xiao-Nan, Chen Liang, Zhang Yun, Coselli Joseph S, Wang Xing Li
Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Shandong University, Qilu Hospital, Jinan, Shandong 250012, China.
Mol Biol Cell. 2008 Sep;19(9):3997-4005. doi: 10.1091/mbc.e07-11-1186. Epub 2008 Jul 9.
We have reported previously that the 27nt repeat polymorphism in endothelial nitric-oxide synthase (eNOS) intron 4--a source of 27nt small RNA--inhibits eNOS expression. In the current study, we have investigated how 27nt small RNA suppresses eNOS expression. Using a chromatin immunoprecipitation assay, we examined histone acetylation in the 27nt repeat element of eNOS intron 4, the promoter region up to -1486 bp, and the 5' enhancer region (-4583/-4223bp) in human aortic endothelial cells (HAECs) treated with 27nt RNA duplex. 27nt RNA duplex induced hyperacetylation in H3 (lysine8, 12, and 23) and H4 (lysine 9 and 12) at the 27nt repeat element, which then interacted with nuclear actin, histone deacetylase 3 (HDAC3), and NonO proteins. In contrast, the histone H3 and H4 became hypoacetylated at the eNOS core promoter. HAECs treated with 27nt RNA duplex had reduced eNOS expression, but treatment with either HDAC3 small interfering RNA or NonO siRNA significantly attenuated the 27nt small RNA-induced suppression. We further found that 27nt small RNA induced DNA methylation in a region approximately 750nt upstream of the intron 4 repeats, and a methyltransferase inhibitor reversed the effect on methylation and eNOS expression. Our study demonstrates that 27nt small RNA may suppress eNOS expression by altering histone acetylation and DNA methylation in regions adjacent to the 27nt repeat element and core promoter.
我们之前报道过，内皮型一氧化氮合酶（eNOS）内含子4中的27nt重复多态性——一种27nt小RNA的来源——会抑制eNOS表达。在当前研究中，我们探究了27nt小RNA如何抑制eNOS表达。使用染色质免疫沉淀测定法，我们检测了在用27nt RNA双链体处理的人主动脉内皮细胞（HAECs）中，eNOS内含子4的27nt重复元件、直至-1486 bp的启动子区域以及5'增强子区域（-4583/-4223bp）中的组蛋白乙酰化情况。27nt RNA双链体在27nt重复元件处诱导H3（赖氨酸8、12和23）和H4（赖氨酸9和12）发生高乙酰化，随后这些组蛋白与核肌动蛋白、组蛋白去乙酰化酶3（HDAC3）和NonO蛋白相互作用。相反，在eNOS核心启动子处，组蛋白H3和H4发生低乙酰化。用27nt RNA双链体处理的HAECs中eNOS表达降低，但用HDAC3小干扰RNA或NonO siRNA处理可显著减弱27nt小RNA诱导的抑制作用。我们进一步发现，27nt小RNA在内含子4重复序列上游约750nt的区域诱导DNA甲基化，而一种甲基转移酶抑制剂可逆转对甲基化和eNOS表达的影响。我们的研究表明，27nt小RNA可能通过改变27nt重复元件和核心启动子附近区域的组蛋白乙酰化和DNA甲基化来抑制eNOS表达。
