Keshet I, Lieman-Hurwitz J, Cedar H
Cell. 1986 Feb 28;44(4):535-43. doi: 10.1016/0092-8674(86)90263-1.
To study the mechanism of gene repression by DNA methylation, M13 gene constructs were methylated to completion and inserted into mouse L cells by DNA-mediated gene transfer. All unmethylated sequences, regardless of their source, integrated into the DNA in a potentially active DNAase I-sensitive conformation. Total CpG methylation prevented the formation of this structure and rendered these sequences DNAase I-insensitive over the entire methylated domain. Whereas unmethylated DNA demonstrated additional conformational features of active genes, such as DNAase I hypersensitivity and restriction endonuclease-sensitive segments, these markers were not present when methylated DNA was used for transfection. The use of micrococcal nuclease to probe for active or inactive supranucleosome particles also showed that DNA methylation directs DNA into an inactive type of structure. The results suggest that DNA methylation may exert its effect on gene transcription by altering both specific and nonspecific interactions between DNA and nuclear proteins.
为研究DNA甲基化导致基因抑制的机制，将M13基因构建体完全甲基化，然后通过DNA介导的基因转移插入小鼠L细胞。所有未甲基化的序列，无论其来源如何，均以潜在的活性DNA酶I敏感构象整合到DNA中。完全的CpG甲基化阻止了这种结构的形成，并使这些序列在整个甲基化区域对DNA酶I不敏感。未甲基化的DNA表现出活性基因的其他构象特征，如DNA酶I超敏性和限制性内切酶敏感片段，而当使用甲基化DNA进行转染时，这些标记不存在。使用微球菌核酸酶探测活性或非活性超核小体颗粒也表明，DNA甲基化将DNA导向一种非活性结构类型。结果表明，DNA甲基化可能通过改变DNA与核蛋白之间的特异性和非特异性相互作用来影响基因转录。
