Demethylation of H3K27 Is Essential for the Induction of Direct Cardiac Reprogramming by miR Combo.
Demethylation of H3K27 Is Essential for the Induction of Direct Cardiac Reprogramming by miR Combo.
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DOI：
10.1161/circresaha.116.308741
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发表时间：
2017-04-28
影响因子：
20.1
通讯作者：
Dzau VJ
中科院分区：
文献类型：
--
作者：
Dal-Pra S;Hodgkinson CP;Mirotsou M;Kirste I;Dzau VJ
心脏成纤维细胞直接重编程为心肌细胞最近已成为一种新颖且有前途的受损心肌再生方法。我们之前已经使用四种 microRNA（miR-1、miR-133、miR-208 和 miR-499）的组合（我们将其命名为 miR 组合）在体外和体内证明了这种方法的可行性。然而，miR组合介导的直接心脏重编程的机制目前尚不清楚。在这里，我们研究了 miR 组合通过表观遗传机制启动直接心脏重编程的可能性。使用 qPCR 阵列，我们发现调节 H3K27 (H3K27me3) 三甲基化的组蛋白甲基转移酶和去甲基化酶（一种标记转录抑制的表观遗传修饰）在 miR 组合处理的成纤维细胞中发生了变化。因此，miR 组合治疗下调了整体 H3K27me3 水平。特别是，ChIP-qPCR 显示，心脏转录因子的启动子区域显示 H3K27me3 减少。通过药物抑制或 siRNA 抑制 H3K27 甲基转移酶或多梳抑制复合物 2 (PRC2) 可降低 H3K27me3 的水平，并在 RNA 和蛋白质水平上诱导心肌标志物，与 miR 组合治疗类似。相比之下，敲除 H3K27 去甲基酶 Kdm6A 和 Kdm6B 可恢复 H3K27me3 的水平，并阻断 miR 组合处理的成纤维细胞中心脏基因表达的诱导。总之，我们证明去除抑制标记 H3K27me3 对于 miR 组合诱导心脏重编程至关重要。我们的数据不仅强调了在细胞命运转换过程中调节表观遗传景观的重要性，而且还提供了改进该技术的框架。
Direct reprogramming of cardiac fibroblasts to cardiomyocytes has recently emerged as a novel and promising approach to regenerate the injured myocardium. We have previously demonstrated the feasibility of this approach in vitro and in vivo using a combination of four microRNAs (miR-1, miR-133, miR-208 and miR-499) that we named miR combo. However, the mechanism of miR combo mediated direct cardiac reprogramming is currently unknown. Here we investigated the possibility that miR combo initiated direct cardiac reprogramming through an epigenetic mechanism. Using a qPCR array, we found that histone methyltransferases and demethylases that regulate the tri-methylation of H3K27 (H3K27me3), an epigenetic modification that marks transcriptional repression, were changed in miR combo treated fibroblasts. Accordingly, global H3K27me3 levels were downregulated by miR combo treatment. In particular, the promoter region of cardiac transcription factors showed decreased H3K27me3 as revealed by ChIP-qPCR. Inhibition of H3K27 methyltransferases or of the Polycomb Repressive Complex 2 (PRC2) by pharmaceutical inhibition or siRNA reduced the levels of H3K27me3 and induced cardiogenic markers at the RNA and protein level, similarly to miR combo treatment. In contrast, knockdown of the H3K27 demethylases Kdm6A and Kdm6B restored the levels of H3K27me3 and blocked the induction of cardiac gene expression in miR combo treated fibroblasts. In summary, we demonstrated that removal of the repressive mark H3K27me3 is essential for the induction of cardiac reprogramming by miR combo. Our data not only highlight the importance of regulating the epigenetic landscape during cell fate conversion but also provide a framework to improve this technique.