Kopp M U, Winterhalter K H, Trueb B
MEM-Institute, Division of Biology, University of Bern, Switzerland.
Eur J Biochem. 1997 Oct 15;249(2):489-96. doi: 10.1111/j.1432-1033.1997.00489.x.
The expression of collagen VI, an adhesive glycoprotein of the extracellular matrix, is completely inhibited in virally transformed fibroblasts and in many cell lines derived from spontaneous mesenchymal tumors. Here we present evidence that DNA methylation plays an important role in this inhibition: (a) The mRNA level for DNA methyltransferase is highly increased in simian virus 40 (SV40)-transformed fibroblasts compared with normal cells and this increase correlates with the decrease of the mRNA level for collagen VI. (b) Methylation of the alpha2(VI) collagen promoter in vitro abolishes promoter activity in a transient transfection assay. (c) Genomic sequencing reveals extensive methylation of the promoter region in SV40-transformed cells, but virtually no methylation of the corresponding region in normal cells. Increased methylation is also observed in a rhabdomyosarcoma cell line. (d) Two of the cis-acting elements of the alpha2(VI) collagen promoter lose their affinity for transcription factor AP2 when methylated in vitro as demonstrated by gel retardation experiments. DNA methylation is therefore involved in the silencing of the alpha2(VI) collagen gene. It seems likely that the same mechanism is also responsible for the repression of other transformation-sensitive proteins.
细胞外基质的一种粘附糖蛋白——胶原蛋白VI的表达，在病毒转化的成纤维细胞以及许多源自自发性间充质肿瘤的细胞系中完全受到抑制。在此，我们提供证据表明DNA甲基化在这种抑制中起重要作用：（a）与正常细胞相比，猿猴病毒40（SV40）转化的成纤维细胞中DNA甲基转移酶的mRNA水平显著升高，且这种升高与胶原蛋白VI mRNA水平的降低相关。（b）在瞬时转染实验中，体外α2（VI）胶原蛋白启动子的甲基化消除了启动子活性。（c）基因组测序显示SV40转化细胞中启动子区域存在广泛甲基化，但正常细胞中相应区域几乎没有甲基化。在横纹肌肉瘤细胞系中也观察到甲基化增加。（d）凝胶阻滞实验表明，α2（VI）胶原蛋白启动子的两个顺式作用元件在体外甲基化后失去了对转录因子AP2的亲和力。因此，DNA甲基化参与了α2（VI）胶原蛋白基因的沉默。似乎相同的机制也负责其他转化敏感蛋白的抑制。
