Gutekunst K A, Kashanchi F, Brady J N, Bednarik D P
Centers for Disease Control, Retrovirus Diseases Branch, Atlanta, Georgia.
J Acquir Immune Defic Syndr (1988). 1993 Jun;6(6):541-9.
Transcription from the HIV-1 long terminal repeat (LTR) was shown to be inhibited by DNA CpG methylation both in vivo and in vitro. Enzymatic methylation of CpG sites localized within the LTR decreased the transcription of the CAT reporter gene, chloramphenicol acetyltransferase, as assayed by the transient expression of this gene in tissue culture. The inhibitory effect could be initially overcome, in trans, by the transactivator tat. As a function of time, the presence of tat had no observable effect on transcription, within the limits of detection sensitivity, suggesting that the level of basal transcription was reduced to very low levels. This effect is suggestive of the involvement of cellular CpG methylation-dependent inhibitory factors which have been characterized by other laboratories. These data imply that transactivation is reduced to low levels after longer periods of time when the DNA template is sparsely methylated. The transcriptional inhibitory process may involve proteins such as MeCP which may interact with methylated DNA more slowly and/or weakly. Conversely, densely methylated DNA was transcriptionally repressed immediately which suggests the rapid/strong association of the cellular inhibitory factor(s). The transcriptional inhibitory effect was also observed in an in vitro transcription run-off system. These data suggest that the methylation-mediated inhibition of transcription is directly affected by CpG methylation density and may involve other factors.
研究表明，在体内和体外，HIV-1长末端重复序列（LTR）的转录均受到DNA CpG甲基化的抑制。通过在组织培养中瞬时表达氯霉素乙酰转移酶（CAT）报告基因来检测，定位于LTR内的CpG位点的酶促甲基化降低了该基因的转录。反式激活因子tat最初可以在反式作用中克服这种抑制作用。随着时间的推移，在检测灵敏度范围内，tat的存在对转录没有明显影响，这表明基础转录水平降低到了非常低的水平。这种效应提示存在细胞内依赖CpG甲基化的抑制因子，其他实验室已对其进行了表征。这些数据表明，当DNA模板甲基化程度较低时，较长时间后反式激活作用会降低到较低水平。转录抑制过程可能涉及诸如MeCP之类的蛋白质，它们与甲基化DNA的相互作用可能更缓慢和/或更微弱。相反，高度甲基化的DNA会立即受到转录抑制，这表明细胞抑制因子的快速/强烈结合。在体外转录延伸系统中也观察到了转录抑制作用。这些数据表明，甲基化介导的转录抑制直接受CpG甲基化密度的影响，并且可能涉及其他因素。
