Stabilization of Foxp3 expression by CRISPR-dCas9-based epigenome editing in mouse primary T cells.
Stabilization of Foxp3 expression by CRISPR-dCas9-based epigenome editing in mouse primary T cells.
复制标题
基于CRISPR - dCas9的表观基因组编辑对小鼠原代T细胞中Foxp3表达的稳定作用
DOI：
10.1186/s13072-017-0129-1
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发表时间：
2017
影响因子：
3.9
通讯作者：
Yoshimura A
中科院分区：
文献类型：
--
作者：
Okada M;Kanamori M;Someya K;Nakatsukasa H;Yoshimura A
表观基因组编辑有望操纵转录和细胞命运，并阐明不同类型细胞中的基因表达机制。对于功能性表观基因组编辑，需要评估人工表观遗传修饰物的染色质上下文相关活性。在这项研究中，我们应用聚簇规则间隔短回文重复序列(CRISPR)-dCas9为基础的表观基因组编辑小鼠原代T细胞，重点关注调节T细胞(Tregs)的主要转录因子Forkhead box P3(Foxp3)基因座。Foxp3基因受组合表观遗传修饰的调控，决定了Foxp3的表达。Foxp3在转化生长因子β(β)诱导的树突状细胞(ITregs)中不稳定表达，而在胸腺来源树突状细胞(TTregs)中稳定表达。为了稳定Foxp3在iTregs中的表达，我们引入了dCas9-TET1CD(dCas9融合到10-11易位双加氧酶1(TET1)的催化区域(CD))和dCas9-p300CD(dCas9融合到p300的CD，组蛋白乙酰转移酶)，并引入了针对Foxp3基因的引导RNA(GRNAs)。虽然dCas9-TET1CD诱导了被称为保守的非编码DNA序列2(CNS2)的增强子区域的部分去甲基化，但没有观察到Foxp3的稳定。相反，针对启动子的dCas9-p300CD即使在体外炎症条件下也能部分维持培养和原代T细胞中Foxp3的转录。此外，dCas9-p300CD可促进Treg信号基因的表达，并增强体外抑制活性。我们的结果表明，人工表观基因组编辑改变了目标基因座的表观遗传状态和基因表达，并结合内源性表观遗传修饰改变了细胞功能，表明这些技术的有效使用，有助于阐明染色质状态和基因表达之间的关系。本文的在线版本(doi：10.1186/s13072-0170129-1)包含补充材料，授权用户可以使用。
Epigenome editing is expected to manipulate transcription and cell fates and to elucidate the gene expression mechanisms in various cell types. For functional epigenome editing, assessing the chromatin context-dependent activity of artificial epigenetic modifier is required. In this study, we applied clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9-based epigenome editing to mouse primary T cells, focusing on the Forkhead box P3 (Foxp3) gene locus, a master transcription factor of regulatory T cells (Tregs). The Foxp3 gene locus is regulated by combinatorial epigenetic modifications, which determine the Foxp3 expression. Foxp3 expression is unstable in transforming growth factor beta (TGF-β)-induced Tregs (iTregs), while stable in thymus-derived Tregs (tTregs). To stabilize Foxp3 expression in iTregs, we introduced dCas9-TET1CD (dCas9 fused to the catalytic domain (CD) of ten-eleven translocation dioxygenase 1 (TET1), methylcytosine dioxygenase) and dCas9-p300CD (dCas9 fused to the CD of p300, histone acetyltransferase) with guide RNAs (gRNAs) targeted to the Foxp3 gene locus. Although dCas9-TET1CD induced partial demethylation in enhancer region called conserved non-coding DNA sequences 2 (CNS2), robust Foxp3 stabilization was not observed. In contrast, dCas9-p300CD targeted to the promoter locus partly maintained Foxp3 transcription in cultured and primary T cells even under inflammatory conditions in vitro. Furthermore, dCas9-p300CD promoted expression of Treg signature genes and enhanced suppression activity in vitro. Our results showed that artificial epigenome editing modified the epigenetic status and gene expression of the targeted loci, and engineered cellular functions in conjunction with endogenous epigenetic modification, suggesting effective usage of these technologies, which help elucidate the relationship between chromatin states and gene expression. The online version of this article (doi:10.1186/s13072-017-0129-1) contains supplementary material, which is available to authorized users.