Thompson J P, Simkevich C P, Holness M A, Kang A H, Raghow R
Departent of Medicine, University of Tennessee, Memphis 38163.
J Biol Chem. 1991 Feb 5;266(4):2549-56.
We created pCOL-KT, a plasmid construct in which the promoter/enhancer of human Pro alpha 1(I) gene is linked to the chloramphenicol acetyl transferase reporter gene. The Pro alpha 1(I) promoter/enhancer in pCOL-KT was methylated in vitro and tested for transcriptional activity by transient expression analysis. Methylation of the construct with bacterial methylases reduced transcriptional activity about 25-fold. Site-specific methylation of eight potential canonical sites of eukaryotic methylation within the promoter greatly reduced transcriptional activity. Chromatin conformation of the transfected pCOL-KT DNA was analyzed by nuclease sensitivity. Although both methylated and unmethylated transfected DNA had increased susceptibility to DNase I compared with the endogenous gene, the methylated transfected DNA showed increased resistance to nuclease when compared with unmethylated transfected DNA, indicating that the methylation of the DNA alters the chromatin conformation. We also tested the ability of a human rhabdomyosarcoma cell line that does not express type I collagen to support transcription from an exogenously added Pro alpha 1(I) promoter/enhancer. The transformed cell line is able to support transcription from the Pro alpha 1(I) promoter/enhancer. Treatment of the transformed cell line with 5-azacytidine, a potent inhibitor of DNA methylation, resulted in transcriptional activation of the Pro alpha 1(I) gene. These findings, along with the extreme methylation sensitivity of the Pro alpha 1(I) promoter and enhancer, suggest that DNA methylation may be an important mechanism of transcriptional inactivation of interstitial collagen genes.
我们构建了pCOL-KT，这是一种质粒构建体，其中人原α1（I）基因的启动子/增强子与氯霉素乙酰转移酶报告基因相连。pCOL-KT中的原α1（I）启动子/增强子在体外进行甲基化，并通过瞬时表达分析检测其转录活性。用细菌甲基化酶对构建体进行甲基化可使转录活性降低约25倍。启动子内八个潜在的真核甲基化典型位点的位点特异性甲基化极大地降低了转录活性。通过核酸酶敏感性分析转染的pCOL-KT DNA的染色质构象。尽管与内源基因相比，甲基化和未甲基化的转染DNA对DNase I的敏感性均增加，但与未甲基化的转染DNA相比，甲基化的转染DNA对核酸酶的抗性增加，这表明DNA的甲基化改变了染色质构象。我们还测试了不表达I型胶原的人横纹肌肉瘤细胞系支持外源添加的原α1（I）启动子/增强子转录的能力。该转化细胞系能够支持原α1（I）启动子/增强子的转录。用强效DNA甲基化抑制剂5-氮杂胞苷处理该转化细胞系，导致原α1（I）基因的转录激活。这些发现，连同原α1（I）启动子和增强子对甲基化的极端敏感性，表明DNA甲基化可能是间质胶原基因转录失活的重要机制。
