Theme of Genetic Disorders, Murdoch Childrens Research Institute, The Royal Children's Hospital, Parkville, Victoria, Australia.
PLoS Genet. 2012 Sep;8(9):e1002919. doi: 10.1371/journal.pgen.1002919. Epub 2012 Sep 6.
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.
DICER1 缺陷型小鼠胚胎干细胞中的 DNA 甲基化减少已被报道。在着丝粒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。最近的研究假设，减少的甲基化是一种间接效应：Mir290 簇 miRNA 的缺失导致转录抑制因子 RBL2 的上调，从而靶向 DNA 甲基转移酶（Dnmt）基因的下调。然而，这些观察结果并不一致。我们推测，这种不一致可能与细胞系的“年龄”有关，因为在 DNMT 缺陷型胚胎干细胞中，随着传代的进行，DNA 甲基化逐渐丢失。因此，我们让 Dicer1(-/-) 胚胎干细胞经历两种实验方案，以严格测试功能性 DNMT 活性的水平。首先，我们长时间培养它们。如果 DNMT 活性降低，进一步的甲基化损失将会发生。其次，我们在反弹 DNA 甲基化测定中测量它们的 DNMT 活性：使用靶向 Dnmt1 mRNA 的 shRNA 从 Cre/loxP 条件性突变的 Dicer1 胚胎干细胞中去除 DNA 甲基化。然后 Cre 表达将这些细胞转化为 Dicer1(-/-)，允许 DNMT1 恢复，并在没有 RNAi 的情况下迫使细胞重新甲基化。在这两种情况下，我们都发现功能性 DNMT 活性正常。最后，我们还表明，Dicer1(-/-) 胚胎干细胞中 RBL2 蛋白的水平并没有像假设的那样过高。这些研究揭示了减少的功能性 DNMT 活性不是 DICER1 缺陷型胚胎干细胞的显著特征。我们认为，这些细胞中有时观察到的 DNA 甲基化减少可能是由于 DNA 甲基化模式的随机改变，这些改变可能在培养中提供生长或生存优势，或者是由于与 DNMT 途径相反的途径失调。
