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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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Cell-Type–Specific Recruitment Patterns of REST as Revealed by ChIP-chip(A) The Venn diagram shows the numbers of bound RE1s in ESC (yellow) and NSC (blue) as determined by ChIP-chip. An additional 253 sites were not occupied in either cell type.(B) Representative qPCR validation of ESC-specific, NSC-specific, and shared sites.(C) Comparison of REST-occupied sites in NIH3T3, ESC, and NSC.(D) Hierarchical clustering of 564 commonly occupied RE1 from (C). Red scale reflects relative enrichment at each RE1.
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pbio-0060256-g002: Cell-Type–Specific Recruitment Patterns of REST as Revealed by ChIP-chip(A) The Venn diagram shows the numbers of bound RE1s in ESC (yellow) and NSC (blue) as determined by ChIP-chip. An additional 253 sites were not occupied in either cell type.(B) Representative qPCR validation of ESC-specific, NSC-specific, and shared sites.(C) Comparison of REST-occupied sites in NIH3T3, ESC, and NSC.(D) Hierarchical clustering of 564 commonly occupied RE1 from (C). Red scale reflects relative enrichment at each RE1.

Mentions: We used RE1 ChIP-chip to compare genome-wide REST occupancy across different cell types. It has been established that REST acts by directly binding and recruiting corepressors to RE1s associated with target genes [27]. It is intriguing that REST is expressed in both lineage-restricted neuronal progenitors [12,14,28] and pluripotent ESC [11,13], given their distinct differentiation potentials. We sought to understand better the role that REST plays in these distinct contexts by applying our ChIP-chip method to compare REST occupancies in these two cell types. The E14 ESC line and the NS5 NSC line [29], which was derived from E14 by in vitro differentiation, provided a genetically matched and developmentally linked pair of stem cell types to compare. One major advantage of the ChIP-chip platform is the ability to perform numerous replicate experiments, quickly and affordably, which thereby provides statistically rigorous results. ChIP material was prepared from five independent biological replicates of both ESC and NSC. These experiments identified 810 and 679 RE1 sites that showed statistically significant binding in ESC and NSC, respectively (Figure 2A). There were 647 sites commonly occupied in both cell types, 163 sites occupied only in ESC, and 32 sites occupied only in NSC. We performed qPCR validation on randomly selected RE1s (Figure 2B). The rate of validation was 8/10 (80%) for commonly bound sites (Figure S4). Seven out of 13 (54%) of ESC-specific RE1s were correctly called by the ChIP-chip (Figure S5). In contrast, we tested all 32 NSC-only sites and found just one that was NSC-specific: the remainder had either detectable recruitment in ESC or no recruitment in NSC (Figure 2B and Figure S6). Statistically, this is not unexpected given the small number of NSC-only sites detected (32 out of 879 total sites or 4.7%), and is consistent with an overall FDR of 10% for the ChIP-chip data. In summary, 60% of all RE1 sites on the ChIP-chip were occupied in both ESC and NSC, approximately 7% were occupied in ESC but not NSC, and there were very few, if any, sites occupied only in NSC. We conclude that the RE1 sites occupied in the NSC are a subset of those occupied 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)

Cell-Type–Specific Recruitment Patterns of REST as Revealed by ChIP-chip(A) The Venn diagram shows the numbers of bound RE1s in ESC (yellow) and NSC (blue) as determined by ChIP-chip. An additional 253 sites were not occupied in either cell type.(B) Representative qPCR validation of ESC-specific, NSC-specific, and shared sites.(C) Comparison of REST-occupied sites in NIH3T3, ESC, and NSC.(D) Hierarchical clustering of 564 commonly occupied RE1 from (C). Red scale reflects relative enrichment at each RE1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2573930&req=5

pbio-0060256-g002: Cell-Type–Specific Recruitment Patterns of REST as Revealed by ChIP-chip(A) The Venn diagram shows the numbers of bound RE1s in ESC (yellow) and NSC (blue) as determined by ChIP-chip. An additional 253 sites were not occupied in either cell type.(B) Representative qPCR validation of ESC-specific, NSC-specific, and shared sites.(C) Comparison of REST-occupied sites in NIH3T3, ESC, and NSC.(D) Hierarchical clustering of 564 commonly occupied RE1 from (C). Red scale reflects relative enrichment at each RE1.
Mentions: We used RE1 ChIP-chip to compare genome-wide REST occupancy across different cell types. It has been established that REST acts by directly binding and recruiting corepressors to RE1s associated with target genes [27]. It is intriguing that REST is expressed in both lineage-restricted neuronal progenitors [12,14,28] and pluripotent ESC [11,13], given their distinct differentiation potentials. We sought to understand better the role that REST plays in these distinct contexts by applying our ChIP-chip method to compare REST occupancies in these two cell types. The E14 ESC line and the NS5 NSC line [29], which was derived from E14 by in vitro differentiation, provided a genetically matched and developmentally linked pair of stem cell types to compare. One major advantage of the ChIP-chip platform is the ability to perform numerous replicate experiments, quickly and affordably, which thereby provides statistically rigorous results. ChIP material was prepared from five independent biological replicates of both ESC and NSC. These experiments identified 810 and 679 RE1 sites that showed statistically significant binding in ESC and NSC, respectively (Figure 2A). There were 647 sites commonly occupied in both cell types, 163 sites occupied only in ESC, and 32 sites occupied only in NSC. We performed qPCR validation on randomly selected RE1s (Figure 2B). The rate of validation was 8/10 (80%) for commonly bound sites (Figure S4). Seven out of 13 (54%) of ESC-specific RE1s were correctly called by the ChIP-chip (Figure S5). In contrast, we tested all 32 NSC-only sites and found just one that was NSC-specific: the remainder had either detectable recruitment in ESC or no recruitment in NSC (Figure 2B and Figure S6). Statistically, this is not unexpected given the small number of NSC-only sites detected (32 out of 879 total sites or 4.7%), and is consistent with an overall FDR of 10% for the ChIP-chip data. In summary, 60% of all RE1 sites on the ChIP-chip were occupied in both ESC and NSC, approximately 7% were occupied in ESC but not NSC, and there were very few, if any, sites occupied only in NSC. We conclude that the RE1 sites occupied in the NSC are a subset of those occupied 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