Department of Implantation, School of Stomatology, Fourth Military Medical University, Xi'an, 710032, Shaanxi, China.
Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an, 710032, Shaanxi, China.
Biochem Biophys Res Commun. 2019 Apr 2;511(2):246-252. doi: 10.1016/j.bbrc.2019.02.011. Epub 2019 Feb 20.
Cell phenotypes are closely related to the epigenome, which could be precisely regulated by the targeted manipulation of epigenetic marks. Here, we have successfully produced a targeted histone methylation system, which consists of nuclease-null dCas9 protein, the sgRNA fused with PP7 RNA aptamers and the Enhancer of Zeste Homolog 2 (EZH2) fused to PP7 coat protein (PCP). Guided by the dCas9/sgRNA-PP7, the PCP-EZH2 can specifically target gene loci to catalyze 3 methylation of histone H3 lysine 27, resulting in the inhibition of gene expression. This kind of gene inhibition system is supposed to be highly effective, specific and flexible. As a proof-of-concept study, sgRNA targeting C/ebpα promoter region was designed. In the cells co-infected with the dCas9, sgRNA/C/ebpα-PP7 and PCP-EZH2, the expression of C/ebpα gene was significantly reduced via induction of trimethylation to H3K27 on C/ebpα promoter, with the results epigenetically inherited in the daughter cells. In conclusion, our results successfully established a gene modification system consisting of dCas9/sgRNA-PP7 and PCP-EZH2, providing a robust tool for targeted manipulation of gene epigenetic modification and expression.
细胞表型与表观基因组密切相关，后者可以通过对表观遗传标记的靶向操作进行精确调控。在这里，我们成功构建了一个靶向组蛋白甲基化系统，该系统由无核酸酶的 dCas9 蛋白、融合了 PP7 RNA 适体的 sgRNA 和融合到 PP7 外壳蛋白（PCP）的 Enhancer of Zeste Homolog 2（EZH2）组成。在 dCas9/sgRNA-PP7 的引导下，PCP-EZH2 可以特异性地靶向基因座，催化组蛋白 H3 赖氨酸 27 的 3 位甲基化，从而抑制基因表达。这种基因抑制系统应该具有高效、特异性和灵活性。作为概念验证研究，设计了靶向 C/ebpα 启动子区域的 sgRNA。在共感染 dCas9、sgRNA/C/ebpα-PP7 和 PCP-EZH2 的细胞中，通过诱导 C/ebpα 启动子上 H3K27 的三甲基化，C/ebpα 基因的表达显著降低，并且该结果在子细胞中被表观遗传继承。总之，我们的结果成功建立了一个由 dCas9/sgRNA-PP7 和 PCP-EZH2 组成的基因修饰系统，为靶向操纵基因表观遗传修饰和表达提供了一个强大的工具。
