Busslinger M, Hurst J, Flavell R A
Cell. 1983 Aug;34(1):197-206. doi: 10.1016/0092-8674(83)90150-2.
We have studied the effect of DNA methylation on human gamma-globin gene expression, using a novel in vitro DNA methylation technique. With this method we have methylated specific segments of the gamma-globin gene and its 5'-flanking region, which were cloned for this purpose in the bacteriophage vector M13mp8. The vitro methylated DNA was introduced into mouse L-cells by cotransfer using the herpes simplex virus thymidine kinase gene as a selective marker. Transformed cell lines were isolated to examine the inheritance of the segmental methylation pattern and its effect on the expression of the gamma-globin gene. Cells transformed with DNA methylated throughout the M13 vector and gamma-globin DNA sequences do not express the gamma-globin gene. Methyl-C residues in either the M13 DNA sequences or the gamma-globin structural gene have, however, no inhibitory effect on globin transcription. In contrast, methylation in the 5' region of the gamma-globin gene (from nucleotides -760 to +100) prevents transcription. These data indicate that DNA methylation in the 5' region of a gene might play a direct role in the regulation of gene expression.
我们使用一种新型的体外DNA甲基化技术，研究了DNA甲基化对人γ-珠蛋白基因表达的影响。通过这种方法，我们对γ-珠蛋白基因及其5'侧翼区域的特定片段进行了甲基化，这些片段为此被克隆到噬菌体载体M13mp8中。体外甲基化的DNA通过共转染被导入小鼠L细胞，使用单纯疱疹病毒胸苷激酶基因作为选择标记。分离出转化细胞系以检查片段甲基化模式的遗传及其对γ-珠蛋白基因表达的影响。用在整个M13载体和γ-珠蛋白DNA序列中都被甲基化的DNA转化的细胞不表达γ-珠蛋白基因。然而，M13 DNA序列或γ-珠蛋白结构基因中的甲基-C残基对珠蛋白转录没有抑制作用。相比之下，γ-珠蛋白基因5'区域（从核苷酸-760到+100）的甲基化会阻止转录。这些数据表明，基因5'区域的DNA甲基化可能在基因表达调控中起直接作用。
