Nature：体细胞重编程的调控因子

来自Dana-Farber癌症研究所和波士顿儿童医院以及哈佛医学院的研究人员利用短发夹RNAs (shRNAs)靶向DNA和组蛋白甲基化信号通路中的基因的方法，鉴别出了iPSC生成的正负调控因子。研究论文发表在3月4日的Nature杂志上。

　　

生物通报道  通过体细胞重编程生成诱导多能干细胞(iPSCs)涵盖了广泛的表观遗传学重塑。过去的研究表明一些参与调控染色质修饰的蛋白与重编程前后不同的表观遗传学状态相关，然而直到现在科学家们还不清楚在重编程过程中特异染色质修饰酶的功能。

为了确定这些染色质修饰蛋白是如何影响体细胞重编程的，来自Dana-Farber癌症研究所和波士顿儿童医院以及哈佛医学院的研究人员利用短发夹RNAs (shRNAs)靶向DNA和组蛋白甲基化信号通路中的基因的方法，鉴别出了iPSC生成的正负调控因子。研究人员发现抑制多梳抑制复合物(Polycomb repressive complex）PRC1 和PRC2核心元件，包括H3组蛋白第27位赖氨酸的三甲基化(H3K27me3)甲基转移酶EZH2，会降低重编程效率；抑制SUV39H1、 YY1 和 DOT1L可促进重编程。研究人员证实利用shRNA特异性靶向抑制H3K79组蛋白甲基转移酶DOT1L，可取代KLF4和 c-Myc显著加快重编程速度，增加iPSC克隆数量。在重编程过程早期抑制DOT1L与两种在重编程增强作用中发挥关键性功能作用的因子NANOG 、LIN28表达上调有关。全基因组分析H3K79me2分布揭示与上皮间质转化相关的成纤维细胞特异性基因在重编程的初期失去了H3K79me2。DOT1L抑制推动了在多能状态中受到抑制的基因H3K79me2标记的丢失。这些研究发现表明特异的染色质修饰酶可在体细胞重编程中充当阻碍因子或是促进因子，揭示了如何通过调节这些染色质修饰酶来减少外源转录因子，促进更高效地生成iPSCs。

研究结果：
1.为了检测染色质修饰因子对体细胞重编程的影响，研究人员利用shRNA功能丧失分析系统检测了DNA和组蛋白甲基化信号通路中的22个候选基因的作用。其中OCT4和两种H3K3甲基转移酶EHMT1、SETDB1抑制将导致重编程效率降低。抑制PRC1（BMI1，RING1）和PRC2元件（EZH1，EED,SUZ12）可显著降低重编程效率。而抑制UV39H1、 YY1 和 DOT1L三种基因却能显著促进重编程。
2. DOT1L抑制可替代KLF4和Myc增进重编程效率。DOT1L抑制导致的重编程增强效应与NANOG和LIN28表达上调有关。
3.全基因组分析重编程过程中H3K79me2标记。在胚胎干细胞和成纤维细胞中，H3K79me2与转录活性基因正相关，与H3K27me3标记基因负相关。在ESC中，H3K79me2标记的特异性基因包括多能性因子及下游靶基因，以及与上皮细胞粘附相关的基因。而在成纤维细胞中，H3K79me2标记富集在上皮间质转化过程中诱导的基因上。

讨论：
功能丧失分析证实染色质修饰酶在分化细胞重编程为多能细胞的全面表观遗传学重塑过程中起着重激活沉默位点，及调整异染色质结构的作用，揭示了作为细胞命运转变促进因子或阻碍因子的特异性酶。表明通过调控这些特异的染色质修饰因子可在减少外源介导转录因子的情况下更高效地生成iPSCs。

（生物通：何嫱）

生物通推荐原文摘要：

Chromatin-modifying enzymes as modulators of reprogramming

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling1. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming2, 3, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.