O'Geen Henriette, Ren Chonghua, Nicolet Charles M, Perez Andrew A, Halmai Julian, Le Victoria M, Mackay Joel P, Farnham Peggy J, Segal David J
Genome Center and Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616, USA.
Guangzhou Key Laboratory of Insect Development Regulation and Application Research, School of Life Sciences, South China Normal University, Guangzhou 510631, China.
Nucleic Acids Res. 2017 Sep 29;45(17):9901-9916. doi: 10.1093/nar/gkx578.
Distinct epigenomic profiles of histone marks have been associated with gene expression, but questions regarding the causal relationship remain. Here we investigated the activity of a broad collection of genomically targeted epigenetic regulators that could write epigenetic marks associated with a repressed chromatin state (G9A, SUV39H1, Krüppel-associated box (KRAB), DNMT3A as well as the first targetable versions of Ezh2 and Friend of GATA-1 (FOG1)). dCas9 fusions produced target gene repression over a range of 0- to 10-fold that varied by locus and cell type. dCpf1 fusions were unable to repress gene expression. The most persistent gene repression required the action of several effector domains; however, KRAB-dCas9 did not contribute to persistence in contrast to previous reports. A 'direct tethering' strategy attaching the Ezh2 methyltransferase enzyme to dCas9, as well as a 'recruitment' strategy attaching the N-terminal 45 residues of FOG1 to dCas9 to recruit the endogenous nucleosome remodeling and deacetylase complex, were both successful in targeted deposition of H3K27me3. Surprisingly, however, repression was not correlated with deposition of either H3K9me3 or H3K27me3. Our results suggest that so-called repressive histone modifications are not sufficient for gene repression. The easily programmable dCas9 toolkit allowed precise control of epigenetic information and dissection of the relationship between the epigenome and gene regulation.
组蛋白标记的不同表观基因组图谱与基因表达相关，但关于因果关系的问题仍然存在。在这里，我们研究了一系列基因组靶向表观遗传调控因子的活性，这些调控因子可以写入与抑制性染色质状态相关的表观遗传标记（G9A、SUV39H1、Krüppel相关盒（KRAB）、DNMT3A以及Ezh2和GATA-1之友（FOG1）的首个可靶向版本）。dCas9融合蛋白在0至10倍的范围内产生了靶基因抑制，其抑制程度因基因座和细胞类型而异。dCpf1融合蛋白无法抑制基因表达。最持久的基因抑制需要几个效应结构域的作用；然而，与之前的报道相反，KRAB-dCas9对持久性没有贡献。一种将Ezh2甲基转移酶与dCas9连接的“直接 tethering”策略，以及一种将FOG1的N端45个残基与dCas9连接以募集内源性核小体重塑和去乙酰化酶复合物的“招募”策略，都成功地实现了H3K27me3的靶向沉积。然而，令人惊讶的是，抑制与H3K9me3或H3K27me3的沉积均不相关。我们的结果表明，所谓的抑制性组蛋白修饰不足以实现基因抑制。易于编程的dCas9工具包允许对表观遗传信息进行精确控制，并剖析表观基因组与基因调控之间的关系。
