Peek R, Niessen R W, Schoenmakers J G, Lubsen N H
Department of Molecular Biology, University of Nijmegen, The Netherlands.
Nucleic Acids Res. 1991 Jan 11;19(1):77-83. doi: 10.1093/nar/19.1.77.
We have investigated the methylation state of the rat gamma-crystallin genes in DNA from lens cells at different developmental stages as well as from kidney and heart cells. A clear correlation between the extent of demethylation of the promoter and 5' gene regions and the expression of these genes was observed. No change in the methylation state of the far upstream or 3' regions of the genes was seen. The demethylation of the promoter region was shown to occur during the differentiation from the lens epithelial to the lens fiber cell. The effect of cytosine methylation on gamma-crystallin promoter activity was tested by measuring gamma-crystallin promoter/chloramphenicol acetyltransferase fusion gene expression after in vitro primed repair synthesis of the promoter region in the presence of either dCTP or 5mdCTP. The hemimethylated promoter was no longer capable of promoting high CAT activity after introduction into lens-like cells. Taken together, our data suggest that DNA demethylation may be the determining step in the developmental stage-specific expression of the rat gamma-crystallin genes.
我们研究了大鼠γ-晶状体蛋白基因在不同发育阶段晶状体细胞以及肾细胞和心脏细胞DNA中的甲基化状态。观察到启动子和5'基因区域的去甲基化程度与这些基因的表达之间存在明显的相关性。基因远上游或3'区域的甲基化状态没有变化。启动子区域的去甲基化发生在晶状体上皮细胞向晶状体纤维细胞分化的过程中。通过在存在dCTP或5mdCTP的情况下对启动子区域进行体外引发修复合成后，测量γ-晶状体蛋白启动子/氯霉素乙酰转移酶融合基因的表达，来测试胞嘧啶甲基化对γ-晶状体蛋白启动子活性的影响。将半甲基化的启动子导入类晶状体细胞后，它不再能够促进高CAT活性。综上所述，我们的数据表明DNA去甲基化可能是大鼠γ-晶状体蛋白基因发育阶段特异性表达的决定性步骤。
