Schmitz A, Short M, Ammerpohl O, Asbrand C, Nickel J, Renkawitz R
Genetisches Institut, Justus-Liebig-Universität, Heinrich-Buff-Ring 58-62, D35392 Giessen, Germany.
J Biol Chem. 1997 Aug 15;272(33):20850-6. doi: 10.1074/jbc.272.33.20850.
The mouse lysozyme downstream enhancer was previously colocalized with the DNase I-hypersensitive site in the chromatin of mature macrophages. This hypersensitive site was shown to be macrophage differentiation-dependent. Demethylation of CpG sequences within the enhancer is correlated with lysozyme expression in mature macrophages. Binding of the GABP heterotetrameric transcription factor to the enhancer core element (MLDE), only seen in vivo on the demethylated MLDE element in macrophages, is inhibited by DNA methylation. Here, we analyzed the DNA sequences required for demethylation. In electrophoretic mobility shift experiments we found that in addition to the complete methylated MLDE the hemimethylated form of the lower strand inhibits GABP binding as well. Therefore, GABP is unlikely to be the mediator of demethylation. In addition, we show by stable DNA transfections of methylated mouse lysozyme enhancer sequences that MLDE-flanking sequences are required for demethylation. We narrowed down these DNA elements to two short regions of 163 and 79 base pairs on either side of the MLDE, each of which is sufficient to mediate demethylation of the GABP site.
小鼠溶菌酶下游增强子先前与成熟巨噬细胞染色质中的DNase I超敏位点共定位。该超敏位点显示为巨噬细胞分化依赖性。增强子内CpG序列的去甲基化与成熟巨噬细胞中的溶菌酶表达相关。GABP异源四聚体转录因子与增强子核心元件（MLDE）的结合，仅在体内巨噬细胞中去甲基化的MLDE元件上可见，被DNA甲基化所抑制。在此，我们分析了去甲基化所需的DNA序列。在电泳迁移率变动实验中，我们发现除了完全甲基化的MLDE外，较低链的半甲基化形式也抑制GABP结合。因此，GABP不太可能是去甲基化的介质。此外，我们通过对甲基化小鼠溶菌酶增强子序列进行稳定的DNA转染表明，MLDE侧翼序列是去甲基化所必需的。我们将这些DNA元件缩小到MLDE两侧的两个短区域，分别为163和79个碱基对，每个区域都足以介导GABP位点的去甲基化。
