In vitro methylation inhibits the promotor activity of a cloned intracisternal A-particle LTR.
In vitro methylation inhibits the promotor activity of a cloned intracisternal A-particle LTR.
复制标题
体外甲基化抑制克隆的脑池内 A 粒子 LTR 的启动子活性。
作者：
A. Feenstra;J. Fewell;K. Lueders;E. Kuff
我们研究了LTR甲基化与小鼠内源性a -颗粒（IAP）相关的内源性逆转录病毒样元件家族表达之间的关系。比较基因组DNA的HpaII/MspI和HhaI限制性内切分析表明，在IAP基因表达水平较低的细胞和组织中，5' LTR内的HpaII和HhaI位点被重度甲基化，而在IAP基因表达丰富的细胞中，20 - 30%的5' LTR位点在这些位点被去甲基化。利用与氯霉素乙酰转移酶（chloramphenicol acetyl transferase， CAT）基因连接的质粒pMIA5’L-cat，直接研究了甲基化对克隆的IAP 5’LTR启动子活性的影响。体外甲基化位于-137和-205 bp之间的三个HhaI位点，使pMIA5' L-cat的启动子活性完全失活，pMIA5' L-cat转染到COS7细胞。位于RNA起始位点下游94 bp处的HpaII位点的甲基化使启动子活性降低75%。结果表明，RNA起始位点上游和下游的甲基化深刻影响了该LTR的启动子活性，表明5' LTR的甲基化可以调节IAP基因在体内的表达。
We studied the relation between LTR methylation and expression of the family of endogenous retrovirus-like elements related to mouse intracisternal A-particles (IAP). Comparative HpaII/MspI and HhaI restriction analysis of genomic DNA's showed that in cells and tissues with a low level of IAP gene expression, HpaII and HhaI sites within the 5' LTR were heavily methylated, while in cells abundantly expressing IAP's 20 to 30% of the 5' LTRs were demethylated at these sites. The effects of methylation on the promoter activity of a cloned IAP 5' LTR was studied directly, using the plasmid pMIA5' L-cat in which this LTR was linked to the chloramphenicol acetyl transferase (CAT) gene. In vitro methylation of three HhaI sites located between -137 and -205 bp from the RNA start site of this LTR completely inactivated the promoter activity of pMIA5' L-cat transfected into COS7 cells. Methylation of a HpaII site located 94 bp downstream from the RNA start site reduced the promoter activity by 75%. The results show that methylation at sites both upstream and downstream from the RNA start site profoundly effects the promoter activity of this LTR and suggest that methylation within the 5' LTR can serve to regulate IAP gene expression in vivo.