Methyl-CpG binding domain protein 2 represses transcription from hypermethylated π-class glutathione S-transferase gene promoters in hepatocellular carcinoma cells
Methyl-CpG binding domain protein 2 represses transcription from hypermethylated π-class glutathione S-transferase gene promoters in hepatocellular carcinoma cells
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DOI：
10.1074/jbc.m203009200
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发表时间：
2002-06-21
影响因子：
4.8
通讯作者：
Nelson, WG
中科院分区：
文献类型：
--
作者：
Bakker, J;Lin, XH;Nelson, WG
在人肝细胞癌（HCC）的发病过程中，围绕pi类谷胱甘肽S-转移酶基因（GSTP 1）的CpG岛变得高甲基化。甲基CpG结合域（MBD）蛋白介导了CpG岛甲基化导致的转录抑制。我们在这里报告，抑制转录从超甲基化的GSTP 1启动子在Hep 3B肝癌细胞，不能表达GSTP 1 mRNA或GSTP 1多肽，似乎是由MBD 2介导的。用甲基转移酶抑制剂5-氮脱氧胞苷（5-aza-dC）处理Hep 3B细胞，激活GSTP 1表达，而用组蛋白去乙酰化酶抑制剂阿司他丁A处理，效果甚微。为了更精确地评估GST 1 CpG岛甲基化模式对GST 1 mRNA表达的贡献，用5-aza-dC处理Hep 3B细胞72 h，然后进行有限稀释克隆。亚硫酸氢盐测序用于绘制GST 1表达和非表达克隆中GST 1启动子区域的甲基化模式。通过北方印迹分析和定量逆转录酶（RT）-PCR确定表达GST 1 mRNA的克隆中，至少一个GST 1等位基因的广泛去甲基化是明显的。染色质免疫沉淀实验表明，MBD 2的存在下，但不是Sp1，在GST 1启动子在Hep 3B细胞。相反，在表达GST P1的Hep 3B 5-aza-dC亚克隆中，在GST P1启动子处检测到Sp1。为了测试MBD 2是否可能负责抑制来自高甲基化GSTP 1启动子的GSTP 1转录，使用siRNA来降低Hep 3B细胞中的MED 2多肽水平。SssI催化的GST P1启动子序列甲基化导致转染到Hep 3B细胞后荧光素酶报告活性降低。然而，当将高甲基化的GSTP 1启动子序列转染到已经用siRNA靶向MBD 2 mRNA处理的Hep 3B细胞中时，没有明显的荧光素酶报告基因表达的抑制。这些发现暗示MBD 2在HCC细胞中抑制与GST 1 CpG岛高甲基化相关的GST 1表达。
During the pathogenesis of human hepatocellular carcinoma (HCC), the CpG island encompassing the pi-class glutathione S-transferase gene (GSTP1) becomes hypermethylated. Repression of transcription accompanying CpG island hypermethylation has been proposed to be mediated by methyl-CpG binding domain (MBD) proteins. We report here that inhibition of transcription from hypermethylated GSTP1 promoters in Hep3B HCC cells, which fail to express GSTP1 mRNA or GSTP1 polypeptides, appears to be mediated by MBD2. Treatment of Hep3B cells with 5-azadeoxycytidine (5-aza-dC), a methyltransferase inhibitor, activated GSTP1 expression, whereas treatment with trichostatin A, a histone deacetylase inhibitor, had little effect. To more precisely assess the contribution of the pattern of GSTP1 CpG island methylation on GSTP1 mRNA expression, Hep3B cells were treated for 72 h with 5-aza-dC and then subjected to limiting dilution cloning. Bisulfite sequencing was used to map the methylation patterns of the GSTP1 promoter region in GSTP1-expressing and -non-expressing clones. In the clone that expressed GSTP1 mRNA determined by Northern blot analysis and quantitative reverse transcriptase (RT)-PCR, widespread demethylation of at least one GSTP1 allele was evident. Chromatin immunoprecipitation experiments revealed the presence of MBD2, but not Sp1, at the GSTP1 promoter in Hep3B cells. In contrast, Sp1 was detected at the GSTP1 promoter in a GSTP1-expressing Hep3B 5-aza-dC subclone. To test whether MBD2 might be responsible for the inhibition of GSTP1 transcription from hypermethylated GSTP1 promoters, siRNAs were used to reduce MED2 polypeptide levels in Hep3B cells. SssI-catalyzed methylation of GSTP1 promoter sequences resulted in diminished luciferase reporter activity after transfection into Hep3B cells. However, when hypermethylated GSTP1 promoter sequences were transfected into Hep3B cells that had been treated with siRNA-targeting MBD2 mRNA, no repression of luciferase reporter expression was evident. These findings implicate MBD2 in the repression of GSTP1 expression associated with GSTP1 CpG island hypermethylation in HCC cells.