Levine A, Cantoni G L, Razin A
Department of Cellular Biochemistry, Hebrew University, Hadassah Medical School, Jerusalem, Israel.
Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10119-23. doi: 10.1073/pnas.89.21.10119.
Although the first observations of the inhibitory effect of methylation on gene activity were made almost a decade ago, the mechanism by which methyl groups affect transcription is still obscure. Here we use engineered promoters methylated in vitro in transient transfections to study the mechanism by which methylation mediates promoter repression. The results clearly show that the location of the methyl groups within the promoter region determines the extent of promoter repression. The most effective suppression was observed when methylation was in the preinitiation domain. The results also support a previous suggestion that a mediator protein is involved in the mechanism of promoter inhibition. The suppressor effect of methylation at sequences flanking the TATA box can be partially overcome in the presence of the simian virus 40 enhancer. In addition, results obtained by transient thymidine labeling of Ltk- cells that were transfected with a methylated thymidine kinase gene from herpes simplex virus, at the level of approximately one template per cell, further support the conclusion that methylation affects primarily transcription preinitiation.
尽管甲基化对基因活性的抑制作用早在近十年前就已被首次观察到，但甲基基团影响转录的机制仍不清楚。在此，我们利用在瞬时转染中体外甲基化的工程化启动子来研究甲基化介导启动子抑制的机制。结果清楚地表明，启动子区域内甲基基团的位置决定了启动子抑制的程度。当甲基化位于起始前结构域时，观察到最有效的抑制作用。这些结果还支持了先前的一种观点，即一种中介蛋白参与了启动子抑制机制。在猿猴病毒40增强子存在的情况下，TATA盒侧翼序列处甲基化的抑制作用可被部分克服。此外，用来自单纯疱疹病毒的甲基化胸苷激酶基因转染Ltk-细胞，以每个细胞约一个模板的水平进行瞬时胸苷标记所获得的结果，进一步支持了甲基化主要影响转录起始前阶段的结论。
