Dal-Pra Sophie, Hodgkinson Conrad P, Mirotsou Maria, Kirste Imke, Dzau Victor J
From the Mandel Center for Hypertension Research and Division of Cardiovascular Medicine, Department of Medicine, Duke University Medical Center, Durham, NC.
Circ Res. 2017 Apr 28;120(9):1403-1413. doi: 10.1161/CIRCRESAHA.116.308741. Epub 2017 Feb 16.
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 4 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 quantitative polymerase chain reaction array, we found that histone methyltransferases and demethylases that regulate the trimethylation 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 chromatin immunoprecipitation coupled with quantitative polymerase chain reaction. Inhibition of H3K27 methyltransferases or of the PRC2 (Polycomb Repressive Complex 2) 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.
将心脏成纤维细胞直接重编程为心肌细胞最近已成为一种用于再生受损心肌的新颖且有前景的方法。我们之前已经证明了使用我们命名为miR组合的4种微小RNA（miR-1、miR-133、miR-208和miR-499）的组合在体外和体内实现这种方法的可行性。然而，miR组合介导的直接心脏重编程的机制目前尚不清楚。
在此，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。
使用定量聚合酶链反应阵列，我们发现调节H3K27（组蛋白H3赖氨酸27位点三甲基化）三甲基化的组蛋白甲基转移酶和去甲基酶在miR组合处理的成纤维细胞中发生了变化，H3K27me3是一种标志转录抑制的表观遗传修饰。相应地，miR组合处理下调了整体H3K27me3水平。特别是，通过染色质免疫沉淀结合定量聚合酶链反应发现，心脏转录因子的启动子区域显示H3K27me3减少。通过药物抑制或小干扰RNA抑制H3K27甲基转移酶或PRC2（多梳抑制复合物2）可降低H3K27me3水平，并在RNA和蛋白质水平诱导心脏发生标志物，类似于miR组合处理。相反，敲低H3K27去甲基酶Kdm6A和Kdm6B可恢复H3K27me3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，我们证明了去除抑制性标记H3K27me3对于miR组合诱导心脏重编程至关重要。我们的数据不仅突出了在细胞命运转换过程中调节表观遗传格局的重要性，还提供了改进该技术的框架。
