Shin T H, Paterson A J, Grant J H, Meluch A A, Kudlow J E
Department of Cell Biology, University of Alabama, Birmingham 35294.
Mol Cell Biol. 1992 Sep;12(9):3998-4006. doi: 10.1128/mcb.12.9.3998-4006.1992.
Evidence indicates DNA methylation as a part of the regulatory machinery controlling mammalian gene expression. The human melanoma cell line HA-A expresses low levels of transforming growth factor alpha (TGF-alpha). TGF-alpha mRNA accumulated, however, in response to DNA demethylation induced by a nucleoside analog, 5-azacytidine (5-azaC). The importance of DNA methylation in the TGF-alpha promoter region was examined by a transient transfection assay with luciferase reporter plasmids containing a portion of the TGF-alpha promoter. 5-azaC treatment of HA-A cells before the transfection caused a significant increase in the luciferase activity. Since input plasmids were confirmed to remain unmethylated, DNA demethylation of the TGF-alpha promoter itself does not account for the observed increase in TGF-alpha mRNA. Using an electrophoretic mobility shift assay, enhanced formation of protein-TGF-alpha promoter complex was detected in response to 5-azaC treatment. This 5-azaC-induced complex was shown to contain the transcription factor Sp1 by the following criteria: the protein-DNA complex formed on the TGF-alpha promoter contained immunoreactive Sp1; the mobility of the complex in an electrophoretic mobility shift assay was similar to that formed by recombinant Sp1; and DNase I footprinting analysis demonstrated that the 5-azaC-induced complex produced a footprint on the TGF-alpha promoter identical to that of authentic Sp1. These observations suggest that 5-azaC induces TGF-alpha expression by augmenting the Sp1 activity. However, neither the Sp1 mRNA nor its protein was induced by 5-azaC. These results suggest that in HA-A cells, TGF-alpha expression is down-modulated by DNA methylation. In addition, this process may involve the specific regulation of Sp1 activity without altering the amount of the transcription factor.
有证据表明DNA甲基化是控制哺乳动物基因表达的调控机制的一部分。人黑色素瘤细胞系HA-A表达低水平的转化生长因子α（TGF-α）。然而，TGF-α mRNA在核苷类似物5-氮杂胞苷（5-azaC）诱导的DNA去甲基化反应中积累。通过使用含有部分TGF-α启动子的荧光素酶报告质粒进行瞬时转染试验，研究了TGF-α启动子区域中DNA甲基化的重要性。在转染前用5-azaC处理HA-A细胞导致荧光素酶活性显著增加。由于证实输入质粒保持未甲基化状态，因此TGF-α启动子本身的DNA去甲基化并不能解释所观察到的TGF-α mRNA的增加。使用电泳迁移率变动分析，检测到响应5-azaC处理后蛋白质-TGF-α启动子复合物的形成增强。通过以下标准表明这种5-azaC诱导的复合物含有转录因子Sp1：在TGF-α启动子上形成的蛋白质-DNA复合物含有免疫反应性Sp1；在电泳迁移率变动分析中复合物的迁移率与重组Sp1形成的复合物相似；并且DNase I足迹分析表明5-azaC诱导的复合物在TGF-α启动子上产生的足迹与真实Sp1的足迹相同。这些观察结果表明5-azaC通过增强Sp1活性诱导TGF-α表达。然而，5-azaC既不诱导Sp1 mRNA也不诱导其蛋白质。这些结果表明在HA-A细胞中，TGF-α表达受到DNA甲基化的下调调节。此外，这个过程可能涉及Sp1活性的特异性调节而不改变转录因子的量。
