Rest Promotes the Early Differentiation of Mouse ESCs but Is Not Required for Their Maintenance

Abstract

Rest promotes the early differentiation of mouse ESCs *Manuscript Click here to view linked References 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 ES cell 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; vector carrying the floxed last exon of Rest, 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 (Rest 3lox/+, Figure 1A). The transient expression of Cre recombinase generated a Rest floxed ES cell line which lacks a drug selection cassette (Rest 2lox/+). Analyzing the GFP expression allowed us to confirm that Rest is expressed in ESCs (Figure 1B). Rest-/-ESCs were next generated using the floxed Rest ES cell line together with a plasmid expressing Cre recombinase (Figure 1A). After the excision of the floxed Rest gene by the transient transfection of Cre (Rest +/-(1lox)), the second Rest allele was also replaced with the floxed allele (Rest 3lox/-). The transient transfection of Cre into Rest 3lox/-ESCs resulted in the establishment of Rest-/-ESCs that were isogenic to the parental ESCs without any genetic modification except for the Rest alleles. After the recombination of the Rest alleles, the lack of a Rest transcript in such Rest-/-ESCs was confirmed by a Northern blot analysis (Figures 1B, S1A). Consistent with the recombination, a FACS analysis revealed a lack of any GFP signal (F) Forty-eight hrs of the induction of REST causes the ESC differentiation into epithelium-like colonies with a decreased ALP activity. (G) The forced expression of REST in ESCs leads to decreased expression of Nanog, Oct3/4 and Fgf5, whereas it results in increased expression of Gata6. The data are presented as the mean±SD of six independent samples. (H) In vitro differentiation of REST-inducible ESCs into EBs under the absence or presence of doxycycline. The exogenous REST expression results in an increa...

Zebrafish rest regulates developmental gene expression but not neurogenesis

“…Some reports assert that Rest is required for selfrenewal and pluripotency of ES cells (Gupta et al, 2009;Singh et al, 2008), whereas others strongly contest this observation (Buckley et al, 2009;Jørgensen et al, 2009a;Jørgensen et al, 2009b;Yamada et al, 2010). Our results demonstrate that Rest is not required to maintain pluripotency or self-renewal of developing zebrafish blastomeres as germ layer specification proceeds normally in rest mutants (supplementary material Fig.…”

Genetic ablation of Rest leads to in vitro-specific derepression of neuronal genes during neurogenesis

“…Rest 2lox/2lox mice were generated from the Rest 2lox/+ ESC line as described preciously (Yamada et al, 2010). Rosa26::rtTA; Col1a1::tetO-Cre mice (Yamada et al, 2010) and Sox1-Cre/+ mice (Takashima et al, 2007) were bred with Rest 2lox/2lox mice to generate compound transgenic mice.…”

Repressor element 1 silencing transcription factor (REST) controls radial migration and temporal neuronal specification during neocortical development

“…Although the exit of the REST-expressing cells from the NS/P stage could be the result of progression of neuronal differentiation, it is also possible that expression of REST above normal levels itself causes the loss of NS/P identity, as shown for ES cells expressing a higher than normal level of REST (19). To distinguish between these two possibilities, we expressed REST/GFP or GFP alone, using the lentiviral vector pEF1α-IRES-GFP, in a primary culture of NS/P cells in the continuous presence of FGF-2, which maintains cells in a NS/P state.…”