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REST regulates distinct transcriptional networks in embryonic and neural stem cells.

Johnson R, Teh CH, Kunarso G, Wong KY, Srinivasan G, Cooper ML, Volta M, Chan SS, Lipovich L, Pollard SM, Karuturi RK, Wei CL, Buckley NJ, Stanton LW - PLoS Biol. (2008)

Bottom Line: We investigated how these distinct biological roles are effected at a genomic level.In ESC, the REST regulatory network is highly integrated into that of pluripotency factors Oct4-Sox2-Nanog.We propose that an extensive, pluripotency-specific recruitment profile lends REST a key role in the maintenance of the ESC phenotype.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Developmental Biology, Genome Institute of Singapore, Singapore.

ABSTRACT
The maintenance of pluripotency and specification of cellular lineages during embryonic development are controlled by transcriptional regulatory networks, which coordinate specific sets of genes through both activation and repression. The transcriptional repressor RE1-silencing transcription factor (REST) plays important but distinct regulatory roles in embryonic (ESC) and neural (NSC) stem cells. We investigated how these distinct biological roles are effected at a genomic level. We present integrated, comparative genome- and transcriptome-wide analyses of transcriptional networks governed by REST in mouse ESC and NSC. The REST recruitment profile has dual components: a developmentally independent core that is common to ESC, NSC, and differentiated cells; and a large, ESC-specific set of target genes. In ESC, the REST regulatory network is highly integrated into that of pluripotency factors Oct4-Sox2-Nanog. We propose that an extensive, pluripotency-specific recruitment profile lends REST a key role in the maintenance of the ESC phenotype.

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Integration of REST with the ESC Pluripotency Network(A) Venn diagrams show the overlap of target gene sets for REST and the indicated pluripotency transcription factors [35]. Statistical significance was calculated using the hypergeometric function.(B) REST is recruited to a number of pluripotency genes in ESC and coregulates them with the pluripotency transcription factors Nanog, Oct4 and Sox2. Thick lines represent gene exons, thinner lines introns. Colored blocks represent transcription factor recruitment in ESC validated by ChIP-PET (REST) or ChIP-sequencing (Nanog, Oct4, Sox2) [35].(C) Conventional ChIP-qPCR was carried out on independent REST ChIP samples (n = 5) (black bars) to validate recruitment to pluripotency genes. In the case of mir-21, primer sequences were those described in Singh et al. [39]. Error bars represent standard error of the mean. Statistical significance was assessed by comparing data to a non-PET containing region (‘No PET'), using Student's t-test (* p < 0.05). No enrichment was detected using a control non-specific IgG (white bars).
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pbio-0060256-g007: Integration of REST with the ESC Pluripotency Network(A) Venn diagrams show the overlap of target gene sets for REST and the indicated pluripotency transcription factors [35]. Statistical significance was calculated using the hypergeometric function.(B) REST is recruited to a number of pluripotency genes in ESC and coregulates them with the pluripotency transcription factors Nanog, Oct4 and Sox2. Thick lines represent gene exons, thinner lines introns. Colored blocks represent transcription factor recruitment in ESC validated by ChIP-PET (REST) or ChIP-sequencing (Nanog, Oct4, Sox2) [35].(C) Conventional ChIP-qPCR was carried out on independent REST ChIP samples (n = 5) (black bars) to validate recruitment to pluripotency genes. In the case of mir-21, primer sequences were those described in Singh et al. [39]. Error bars represent standard error of the mean. Statistical significance was assessed by comparing data to a non-PET containing region (‘No PET'), using Student's t-test (* p < 0.05). No enrichment was detected using a control non-specific IgG (white bars).

Mentions: REST had been implicated previously in the transcriptional regulatory networks that regulate ESC pluripotency, as the Rest gene is a target of Oct4, Sox2, and Nanog binding [2,5]. We explored more fully the connection between REST and the pluripotency transcription factors Oct4, Sox2, and Nanog. Recently our colleagues completed a detailed mapping of binding sites for Oct4, Sox2, and Nanog in ESC [35]. Very deep sequencing of ChIP DNA from ESC identified 1,834, 1,765, and 3,317 genes with binding sites for Oct4, Sox2, and Nanog, respectively, either within the gene itself or not more than 10 kb upstream of the target gene. These data confirm and extend earlier studies [2] in which these three transcription factors represent a core regulatory complex in ESC. By comparing the list of REST target genes with those of Nanog, Oct4, and Sox2, we found a statistically significant integration of target gene repertoires (Figure 7A). Of the 1,287 REST target genes, 270 (21%), 238 (18%), and 399 (31%) of these were also targets of Oct4, Sox2, and Nanog, respectively. In addition, 107 genes were targets of all four factors in ESC, including Rest itself (having three REST PET clusters), and several transcription factors implicated in ESC self renewal, such as Nanog [36,37] and Zfp206 [5,23] (Figure 7B). We also noted that the gene of the reprogramming factor Lin28 [38] contains a REST PET cluster in the proximal upstream region (Figure 7B). Using conventional ChIP-qPCR, we validated REST recruitment to Nanog, Zfp206, Zfp281, Esrrb, and Lin28, in addition to the Rest gene itself (Figure 7C). Furthermore, expression of Zfp206, Zfp281, and Lin28 was induced by DN:REST (Figure S14). Surprisingly, we could find no evidence for recruitment of REST to the gene for the microRNA mir-21, as was reported recently [39] (Figure 7C). Oct4, Sox2, and Nanog have been shown to form an autoregulatory circuit where every factor regulates its own gene and that of the other two [5,40]. Our mapping data indicate that REST also forms such an autoregulatory circuit. These results support the hypothesis that REST is a component of the pluripotency network that includes Oct4, Sox2, and Nanog, which together control differentiation and pluripotency in ESC.


REST regulates distinct transcriptional networks in embryonic and neural stem cells.

Johnson R, Teh CH, Kunarso G, Wong KY, Srinivasan G, Cooper ML, Volta M, Chan SS, Lipovich L, Pollard SM, Karuturi RK, Wei CL, Buckley NJ, Stanton LW - PLoS Biol. (2008)

Integration of REST with the ESC Pluripotency Network(A) Venn diagrams show the overlap of target gene sets for REST and the indicated pluripotency transcription factors [35]. Statistical significance was calculated using the hypergeometric function.(B) REST is recruited to a number of pluripotency genes in ESC and coregulates them with the pluripotency transcription factors Nanog, Oct4 and Sox2. Thick lines represent gene exons, thinner lines introns. Colored blocks represent transcription factor recruitment in ESC validated by ChIP-PET (REST) or ChIP-sequencing (Nanog, Oct4, Sox2) [35].(C) Conventional ChIP-qPCR was carried out on independent REST ChIP samples (n = 5) (black bars) to validate recruitment to pluripotency genes. In the case of mir-21, primer sequences were those described in Singh et al. [39]. Error bars represent standard error of the mean. Statistical significance was assessed by comparing data to a non-PET containing region (‘No PET'), using Student's t-test (* p < 0.05). No enrichment was detected using a control non-specific IgG (white bars).
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pbio-0060256-g007: Integration of REST with the ESC Pluripotency Network(A) Venn diagrams show the overlap of target gene sets for REST and the indicated pluripotency transcription factors [35]. Statistical significance was calculated using the hypergeometric function.(B) REST is recruited to a number of pluripotency genes in ESC and coregulates them with the pluripotency transcription factors Nanog, Oct4 and Sox2. Thick lines represent gene exons, thinner lines introns. Colored blocks represent transcription factor recruitment in ESC validated by ChIP-PET (REST) or ChIP-sequencing (Nanog, Oct4, Sox2) [35].(C) Conventional ChIP-qPCR was carried out on independent REST ChIP samples (n = 5) (black bars) to validate recruitment to pluripotency genes. In the case of mir-21, primer sequences were those described in Singh et al. [39]. Error bars represent standard error of the mean. Statistical significance was assessed by comparing data to a non-PET containing region (‘No PET'), using Student's t-test (* p < 0.05). No enrichment was detected using a control non-specific IgG (white bars).
Mentions: REST had been implicated previously in the transcriptional regulatory networks that regulate ESC pluripotency, as the Rest gene is a target of Oct4, Sox2, and Nanog binding [2,5]. We explored more fully the connection between REST and the pluripotency transcription factors Oct4, Sox2, and Nanog. Recently our colleagues completed a detailed mapping of binding sites for Oct4, Sox2, and Nanog in ESC [35]. Very deep sequencing of ChIP DNA from ESC identified 1,834, 1,765, and 3,317 genes with binding sites for Oct4, Sox2, and Nanog, respectively, either within the gene itself or not more than 10 kb upstream of the target gene. These data confirm and extend earlier studies [2] in which these three transcription factors represent a core regulatory complex in ESC. By comparing the list of REST target genes with those of Nanog, Oct4, and Sox2, we found a statistically significant integration of target gene repertoires (Figure 7A). Of the 1,287 REST target genes, 270 (21%), 238 (18%), and 399 (31%) of these were also targets of Oct4, Sox2, and Nanog, respectively. In addition, 107 genes were targets of all four factors in ESC, including Rest itself (having three REST PET clusters), and several transcription factors implicated in ESC self renewal, such as Nanog [36,37] and Zfp206 [5,23] (Figure 7B). We also noted that the gene of the reprogramming factor Lin28 [38] contains a REST PET cluster in the proximal upstream region (Figure 7B). Using conventional ChIP-qPCR, we validated REST recruitment to Nanog, Zfp206, Zfp281, Esrrb, and Lin28, in addition to the Rest gene itself (Figure 7C). Furthermore, expression of Zfp206, Zfp281, and Lin28 was induced by DN:REST (Figure S14). Surprisingly, we could find no evidence for recruitment of REST to the gene for the microRNA mir-21, as was reported recently [39] (Figure 7C). Oct4, Sox2, and Nanog have been shown to form an autoregulatory circuit where every factor regulates its own gene and that of the other two [5,40]. Our mapping data indicate that REST also forms such an autoregulatory circuit. These results support the hypothesis that REST is a component of the pluripotency network that includes Oct4, Sox2, and Nanog, which together control differentiation and pluripotency in ESC.

Bottom Line: We investigated how these distinct biological roles are effected at a genomic level.In ESC, the REST regulatory network is highly integrated into that of pluripotency factors Oct4-Sox2-Nanog.We propose that an extensive, pluripotency-specific recruitment profile lends REST a key role in the maintenance of the ESC phenotype.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Developmental Biology, Genome Institute of Singapore, Singapore.

ABSTRACT
The maintenance of pluripotency and specification of cellular lineages during embryonic development are controlled by transcriptional regulatory networks, which coordinate specific sets of genes through both activation and repression. The transcriptional repressor RE1-silencing transcription factor (REST) plays important but distinct regulatory roles in embryonic (ESC) and neural (NSC) stem cells. We investigated how these distinct biological roles are effected at a genomic level. We present integrated, comparative genome- and transcriptome-wide analyses of transcriptional networks governed by REST in mouse ESC and NSC. The REST recruitment profile has dual components: a developmentally independent core that is common to ESC, NSC, and differentiated cells; and a large, ESC-specific set of target genes. In ESC, the REST regulatory network is highly integrated into that of pluripotency factors Oct4-Sox2-Nanog. We propose that an extensive, pluripotency-specific recruitment profile lends REST a key role in the maintenance of the ESC phenotype.

Show MeSH