Rest promotes the early differentiation of mouse ESCs but is not required for their maintenance.
Rest promotes the early differentiation of mouse ESCs but is not required for their maintenance.
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DOI：
10.1016/j.stem.2009.12.003
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发表时间：
2010-01
期刊：
影响因子：
23.9
通讯作者：
Yasuhiro Yamada;H. Aoki;T. Kunisada;A. Hara
中科院分区：
文献类型：
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作者：
Yasuhiro Yamada;H. Aoki;T. Kunisada;A. Hara
Rest在维持ESC多能性中的功能意义仍然存在争议。我们在此表明Rest对于小鼠ESCs的维持并不是必需的，相反，Rest转录抑制因子在早期ESC分化过程中连接了Oct3/4-Sox2-Nanog核心调控回路。ESCs的多能性是通过包括Oct3/4、Sox2和Nanog在内的核心基因调控回路的协调表达来维持的。Rest（也称为Nrsf）在ESCs中大量表达，是Oct3/4-Sox2-Nanog调控网络的靶点。然而，Rest在维持多能性中的功能意义仍然存在争议。我们已经生成了Rest条件敲除和Rest诱导ESC行。条件消融Rest表明它不是维持多能性所必需的，但它参与了ESCs早期分化过程中自我更新基因的抑制。此外，在ESCs中强制表达REST导致快速分化。这些结果表明，Rest对于小鼠ESCs的维持并不是必需的，相反，Rest转录抑制因子在早期ESC分化过程中与Oct3/4-Sox2-Nanog核心调控回路相连。转录抑制因子Rest是一种锌指蛋白，它与许多编码基本神经元特征的基因中的保守的23 bp基序RE1（抑制因子1，也称为NRSE）结合（Chong et al., 1995; Schoenherr and Anderson, 1995）。Rest在早期发育过程中表达，抑制神经基因的表达，如Syp和Syt4 （Schoenherr et al., 1996）。Rest也在ESCs中表达，并且已被证明是ESCs多能状态调控回路的靶基因之一（Johnson et al., 2008; Sun et al., 2005）。然而，Rest在维持ESCs多能性中的功能意义仍然存在争议（Buckley et al., 2009; Jørgensen et al., 2009a; Singh et al., 2008）。先前的一项研究表明，Rest通过诱导Oct3/4、Nanog和Sox2等自我更新基因来维持多能性（Singh et al., 2008）。相比之下，Jørgensen等人生成了一个Rest null ESC系，并报道与匹配的野生型对照相比，这种Rest null ESC在Oct3/4蛋白水平或碱性磷酸酶活性方面没有实质性变化（Jørgensen等人，2009a, 2009b）。为了阐明Rest在维持多能性中的作用，我们首先生成了含有Rest的条件敲除等位基因的ESC系和小鼠。ESCs中的第一个Rest等位基因(V6。5)被替换为携带rest的最后一个外显子的KO载体，该载体编码coRest结合位点，这对于沉默复合体的产生至关重要（Andres等人，1999；Grimes等人，2000），然后是ires-Gfp来监测修饰等位基因的转录（Rest3lox/+；图1a）。Cre重组酶的瞬时表达产生了缺乏药物选择盒的Rest floxed ESC系（Rest2lox/+）。通过分析GFP表达，我们确认Rest在ESCs中表达（图1 B）。
The functional significance of Rest in the maintenance of ESC pluripotency remains controversial. We herein showed that Rest is not necessary for the maintenance of mouse ESCs, and instead suggested that the Rest transcriptional repressor connects to the Oct3/4-Sox2-Nanog core regulatory circuitry during early ESC differentiation. The pluripotency of ESCs is maintained by coordinated expression of a core regulatory circuit of genes that includes Oct3/4, Sox2, and Nanog. Rest (also called Nrsf) is abundantly expressed in ESCs and is a target of the Oct3/4-Sox2-Nanog regulatory network. However, the functional significance of Rest in the maintenance of pluripotency remains controversial. We have generated Rest conditional knockout and Rest-inducible ESC lines. Conditional ablation of Rest showed that it is not required for maintenance of pluripotency, but it is involved in the suppression of self-renewal genes during early differentiation of ESCs. In addition, forced expression of REST in ESCs results in rapid differentiation. These results indicate that Rest is not necessary for the maintenance of mouse ESCs, and instead suggest that the Rest transcriptional repressor connects to the Oct3/4-Sox2-Nanog core regulatory circuitry during early ESC differentiation. The transcriptional repressor Rest is a zinc finger protein that binds to a conserved 23 bp motif known as RE1 (repressor element 1, also called NRSE) in a number of genes encoding the fundamental neuronal traits (Chong et al., 1995; Schoenherr and Anderson, 1995). Rest is expressed throughout early development where it represses the expression of neural genes, such as Syp and Syt4 (Schoenherr et al., 1996). Rest is also expressed in ESCs and it has also been shown to be one of target genes of the regulatory circuitry of the pluripotent state in ESCs (Johnson et al., 2008; Sun et al., 2005). However, the functional significance of Rest in the maintenance of pluripotency in ESCs still remains controversial (Buckley et al., 2009; Jørgensen et al., 2009a; Singh et al., 2008). A previous study with a heterozygous Rest ESC line combined with an siRNA knockdown indicated that Rest maintains pluripotency through the induction of selfrenewal genes, such as Oct3/4, Nanog, and Sox2 (Singh et al., 2008). In contrast, Jørgensen et al. generated a Rest null ESC line and reported that such Rest null ESCs revealed no substantial change in either the Oct3/4 protein levels or alkaline phosphatase activity in comparison to matched wild-type controls (Jørgensen et al., 2009a, 2009b). In order to elucidate the role of Rest in the maintenance of pluripotency, we first generated an ESC line and mice that contained the conditional knockout alleles of Rest. The first Rest allele in the ESCs (V6. 5) was replaced with the KO vector carrying the floxed last exon ofRest, which encodes the coRest binding site that is essential for the generation of the silencing complex (Andres et al., 1999; Grimes et al., 2000), followed by ires-Gfp to monitor the transcription of the modified allele (Rest3lox/+; Figure 1 A). The transient expression of Cre recombinase generated a Rest floxed ESC line that lacks a drug selection cassette (Rest2lox/+). Analyzing the GFP expression allowed us to confirm that Rest is expressed in ESCs (Figure 1 B).