Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.
Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.
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使用工程 TALE 和 Cas9 转录因子直接激活人类和小鼠 Oct4 基因。
DOI：
10.1093/nar/gku109
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发表时间：
2014-04
影响因子：
14.9
通讯作者：
Feng B
中科院分区：
文献类型：
--
作者：
Hu J;Lei Y;Wong WK;Liu S;Lee KC;He X;You W;Zhou R;Guo JT;Chen X;Peng X;Sun H;Huang H;Zhao H;Feng B
新开发的转录激活因子样效应蛋白（TALE）和成簇的规则间隔短回文重复序列/Cas9转录因子（TF）为调节基因表达提供了一种强大而精确的方法。在这篇文章中，我们系统地研究了这些新工具在小鼠和人体细胞中激活严格沉默的多能性基因Oct 4（Pou 5 f1）的潜力。首先，利用靶向小鼠和人类Oct 4启动子中不同区域的许多TALE和sgRNA，我们发现最有效的TALE-VP 64结合在-120到-80 bp左右，而高效的sgRNA靶向转录起始位点上游的-147到-89 bp，以诱导荧光素酶报告基因的高活性。此外，我们观察到显着的转录协同作用时，多个TF同时应用。尽管单个TF表现出上调内源性基因表达的边缘活性，但TALE-VP 64的优化组合可在小鼠NIH 3 T3细胞中将内源性Oct 4转录提高高达30倍，在人HEK 293 T细胞中提高20倍。更重要的是，OCT 4转录的增强最终产生了OCT 4蛋白。此外，不同表观遗传修饰剂的检查显示组蛋白乙酰转移酶p300可以增强TALE-VP 64和sgRNA/dCas 9-VP 64诱导的内源OCT 4的转录。总之，我们的研究表明，工程化的TALE-TF和dCas 9-TF是用于调节哺乳动物细胞中基因表达的有用工具。
The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around −120 to −80 bp, while highly effective sgRNAs targeted from −147 to −89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.