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首页> 外文期刊>Epigenetics & Chromatin >Widespread recovery of methylation at gametic imprints in hypomethylated mouse stem cells following rescue with DNMT3A2
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Widespread recovery of methylation at gametic imprints in hypomethylated mouse stem cells following rescue with DNMT3A2

机译：DNMT3A2拯救后，低甲基化的小鼠干细胞中配子印迹处的甲基化得到广泛恢复

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Background Imprinted loci are paradigms of epigenetic regulation and are associated with a number of genetic disorders in human. A key characteristic of imprints is the presence of a gametic differentially methylated region (gDMR). Previous studies have indicated that DNA methylation lost from gDMRs could not be restored by DNMT1, or the de novo enzymes DNMT3A or 3B in stem cells, indicating that imprinted regions must instead undergo passage through the germline for reprogramming. However, previous studies were non-quantitative, were unclear on the requirement for DNMT3A/B and showed some inconsistencies. In addition, new putative gDMR has recently been described, along with an improved delineation of the existing gDMR locations. We therefore aimed to re-examine the dependence of methylation at gDMRs on the activities of the methyltransferases in mouse embryonic stem cells (ESCs). Results We examined the most complete current set of imprinted gDMRs that could be assessed using quantitative pyrosequencing assays in two types of ESCs: those lacking DNMT1 (1KO) and cells lacking a combination of DNMT3A and DNMT3B (3abKO). We further verified results using clonal analysis and combined bisulfite and restriction analysis. Our results showed that loss of methylation was approximately equivalent in both cell types. 1KO cells rescued with a cDNA-expressing DNMT1 could not restore methylation at the imprinted gDMRs, confirming some previous observations. However, nearly all gDMRs were remethylated in 3abKO cells rescued with a DNMT3A2 expression construct (3abKO?+?3a2). Transcriptional activity at the H19/Igf2 locus also tracked with the methylation pattern, confirming functional reprogramming in the latter. Conclusions These results suggested (1) a vital role for DNMT3A/B in methylation maintenance at imprints, (2) that loss of DNMT1 and DNMT3A/B had equivalent effects, (3) that rescue with DNMT3A2 can restore imprints in these cells. This may provide a useful system in which to explore factors influencing imprint reprogramming.
机译：背景印迹基因座是表观遗传调控的范例，与人类的许多遗传疾病有关。烙印的关键特征是配子差异甲基化区域（gDMR）的存在。先前的研究表明，DNMT1或干细胞中的从头酶DNMT3A或3B无法恢复从gDMR中丢失的DNA甲基化，这表明印迹区域必须通过种系进行重新编程。但是，先前的研究是非定量的，对DNMT3A / B的要求尚不清楚，并且显示出一些不一致之处。另外，最近已经描述了新的推定gDMR，以及对现有gDMR位置的改进描述。因此，我们旨在重新检查gDMR上甲基化对小鼠胚胎干细胞（ESC）中甲基转移酶活性的依赖性。结果我们检查了目前最完整的印迹gDMR，可以使用定量焦磷酸测序法在两种类型的ESC中进行评估：缺少DNMT1（1KO）的那些和缺少DNMT3A和DNMT3B（3abKO）的细胞。我们使用克隆分析以及亚硫酸氢盐和限制分析的组合进一步验证了结果。我们的结果表明，两种细胞类型的甲基化损失均大致相同。用表达cDNA的DNMT1拯救的1KO细胞无法恢复印迹的gDMR处的甲基化，证实了以前的观察结果。但是，几乎所有的gDMR在用DNMT3A2表达构建体（3abKOβ+β3a2）拯救的3abKO细胞中都被甲基化。 H19 / Igf2位点的转录活性也以甲基化模式进行跟踪，从而证实了后者的功能重编程。结论这些结果表明（1）DNMT3A / B在印迹的甲基化维持中起着至关重要的作用;（2）DNMT1和DNMT3A / B的丧失具有同等的作用;（3）DNMT3A2的抢救可以恢复这些细胞中的印迹。这可以提供有用的系统，在其中探索影响印记重编程的因素。

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