Normal DNA Methylation Dynamics in DICER1-Deficient Mouse Embryonic Stem Cells
Normal DNA Methylation Dynamics in DICER1-Deficient Mouse Embryonic Stem Cells
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DOI：
10.1371/journal.pgen.1002919
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发表时间：
2012-09-01
期刊：
影响因子：
4.5
通讯作者：
Mann, Jeffrey R.
中科院分区：
文献类型：
--
作者：
Ip, Jonathan;Canham, Paul;Mann, Jeffrey R.
在DICER 1缺陷型小鼠ES细胞中已报道DNA甲基化降低。在着丝粒旁卫星重复序列中观察到的减少表明，siRNA是异染色质正确组装所必需的。最近的研究已经假设甲基化的减少是一种间接效应：Mir 290簇miRNA的丢失导致转录抑制因子RBL 2的上调，其靶向DNA甲基转移酶（Dnmt）基因的下调。然而，观察结果并不一致。我们推测，这种不一致可能与细胞系“年龄”有关，因为DNA甲基化在DNMT缺陷的ES细胞中随着传代而逐渐丧失。因此，我们对Dicer 1（-/-）ES细胞进行了两种实验方案，以严格测试功能性DNMT活性的水平。首先，我们培养他们很长一段时间。如果DNMT活性降低，则会发生甲基化的进一步损失。其次，我们在回弹DNA甲基化测定中测量了它们的DNMT活性：使用靶向Dnmt 1 mRNA的shRNA从Cre/loxP条件突变Dicer 1 ES细胞中剥离DNA甲基化。然后Cre表达将这些细胞转化为Dicer 1（-/-），允许DNMT 1恢复并迫使细胞在不存在RNAi的情况下重新甲基化。在这两种情况下，我们发现功能性DNMT活性是正常的。最后，我们还表明，RBL 2蛋白的水平是不是在过量的Dicer 1（-/-）ES细胞的水平，因为已经假设。这些研究表明，功能性DNMT活性降低不是DICER 1缺陷ES细胞的显著特征。我们认为，有时在这些细胞中观察到的DNA甲基化减少可能是由于DNA甲基化模式的随机改变，这可能在培养中提供生长或存活优势，或者是由于与DNMT通路相反的通路调节异常。
Reduced DNA methylation has been reported in DICER1-deficient mouse ES cells. Reductions seen at pericentric satellite repeats have suggested that siRNAs are required for the proper assembly of heterochromatin. More recent studies have postulated that the reduced methylation is an indirect effect: the loss of Mir290 cluster miRNAs leads to upregulation of the transcriptional repressor RBL2 that targets the downregulation of DNA methyltransferase (Dnmt) genes. However, the observations have been inconsistent. We surmised that the inconsistency could be related to cell line "age,'' given that DNA methylation is lost progressively with passage in DNMT-deficient ES cells. We therefore subjected Dicer1(-/-) ES cells to two experimental regimes to rigorously test the level of functional DNMT activity. First, we cultured them for a prolonged period. If DNMT activity was reduced, further losses of methylation would occur. Second, we measured their DNMT activity in a rebound DNA methylation assay: DNA methylation was stripped from Cre/loxP conditionally mutant Dicer1 ES cells using a shRNA targeting Dnmt1 mRNA. Cre expression then converted these cells to Dicer1(-/-), allowing for DNMT1 recovery and forcing the cells to remethylate in the absence of RNAi. In both cases, we found functional DNMT activity to be normal. Finally, we also show that the level of RBL2 protein is not at excess levels in Dicer1(-/-) ES cells as has been assumed. These studies reveal that reduced functional DNMT activity is not a salient feature of DICER1-deficient ES cells. We suggest that the reduced DNA methylation sometimes observed in these cells could be due to stochastic alterations in DNA methylation patterns that could offer growth or survival advantages in culture, or to the dysregulation of pathways acting in opposition to the DNMT pathway.