Lorincz M C, Schübeler D, Groudine M
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Mol Cell Biol. 2001 Dec;21(23):7913-22. doi: 10.1128/MCB.21.23.7913-7922.2001.
The majority of 5-methylcytosine in mammalian DNA resides in endogenous transposable elements and is associated with the transcriptional silencing of these parasitic elements. Methylation also plays an important role in the silencing of exogenous retroviruses. One of the difficulties inherent in the study of proviral silencing is that the sites in which proviruses randomly integrate influence the probability of de novo methylation and expression. In order to compare methylated and unmethylated proviruses at the same genomic site, we used a recombinase-based targeting approach to introduce an in vitro methylated or unmethylated Moloney murine leukemia-based provirus in MEL cells. The methylated and unmethylated states are maintained in vivo, with the exception of the initially methylated proviral enhancer, which becomes demethylated in vivo. Although the enhancer is unmethylated and remodeled, the methylated provirus is transcriptionally silent. To further analyze the repressed state, histone acetylation status was determined by chromatin immunoprecipitation (ChIP) analyses, which revealed that localized histone H3 but not histone H4 hyperacetylation is inversely correlated with proviral methylation density. Since members of the methyl-CpG binding domain (MBD) family of proteins recruit histone deacetylase activity, these proteins may play a role in proviral repression. Interestingly, only MBD3 and MeCP2 are expressed in MEL cells. ChIPs with antibodies specific for these proteins revealed that only MeCP2 associates with the provirus in a methylation-dependent manner. Taken together, our results suggest that MeCP2 recruitment to a methylated provirus is sufficient for transcriptional silencing, despite the presence of a remodeled enhancer.
哺乳动物DNA中大部分5-甲基胞嘧啶存在于内源性转座元件中，并与这些寄生元件的转录沉默相关。甲基化在外源逆转录病毒的沉默中也起着重要作用。原病毒沉默研究中固有的一个困难是，原病毒随机整合的位点会影响从头甲基化和表达的概率。为了在同一基因组位点比较甲基化和未甲基化的原病毒，我们使用了一种基于重组酶的靶向方法，在MEL细胞中引入体外甲基化或未甲基化的莫洛尼氏鼠白血病病毒原病毒。甲基化和未甲基化状态在体内得以维持，但最初甲基化的原病毒增强子除外，它在体内会发生去甲基化。尽管增强子去甲基化并发生重塑，但甲基化的原病毒仍处于转录沉默状态。为了进一步分析这种抑制状态，通过染色质免疫沉淀（ChIP）分析确定组蛋白乙酰化状态，结果显示局部组蛋白H3而非组蛋白H4的高乙酰化与原病毒甲基化密度呈负相关。由于甲基-CpG结合域（MBD）蛋白家族成员募集组蛋白去乙酰化酶活性，这些蛋白可能在原病毒抑制中发挥作用。有趣的是，只有MBD3和MeCP2在MEL细胞中表达。用针对这些蛋白的特异性抗体进行的ChIP分析表明，只有MeCP2以甲基化依赖的方式与原病毒结合。综上所述，我们的结果表明，尽管存在重塑的增强子，但将MeCP2募集到甲基化的原病毒上足以实现转录沉默。
