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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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ESC-Specific Gene Targeting by REST(A) ESC-specific REST binding sites (green) are defined as those loci having PET10+ in ESC and no evidence (PET1 or no PET) in NSC.(B) For ESC (yellow) or NSC (blue), the fraction of PET clusters of a given size range that also overlap a PET5+ cluster from the other cell type is plotted.(C) The numbers of ESC-specific and common REST PETs shared with kidney cells (from ChIP-SACO [22]), and the numbers overlapping full-length RE1 motifs are shown. Statistical significance was calculated using the Chi-squared test.(D) Genes involved in Wnt and integrin signaling, and genes encoding kinases and chromatin-binding proteins are enriched amongst targets of ESC-specific REST PET clusters. Shown are the numbers of genes and p-value (Chi-square test) for each ontology term among annotated targets of the PET clusters from (A).(E) ESC-specific and ESC-NSC common binding sites have distinct sequence conservation. All REST PET clusters with an identifiable, consensus RE1 were aligned in the same strand orientation. The mean PhastCons sequence conservation score [54] for every position in a 300-bp window around the RE1 is plotted. Statistical significance is based on comparing the mean Phastcons score across every 21mer RE1 (Student's t-test).(F and G) Boxplots show the distribution of (F) RE1 PSSM scores [18] and (G) PET overlap count for ESC-only and common ESC-NSC PET clusters. Statistical significance was calculated using Student's t-test.
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pbio-0060256-g004: ESC-Specific Gene Targeting by REST(A) ESC-specific REST binding sites (green) are defined as those loci having PET10+ in ESC and no evidence (PET1 or no PET) in NSC.(B) For ESC (yellow) or NSC (blue), the fraction of PET clusters of a given size range that also overlap a PET5+ cluster from the other cell type is plotted.(C) The numbers of ESC-specific and common REST PETs shared with kidney cells (from ChIP-SACO [22]), and the numbers overlapping full-length RE1 motifs are shown. Statistical significance was calculated using the Chi-squared test.(D) Genes involved in Wnt and integrin signaling, and genes encoding kinases and chromatin-binding proteins are enriched amongst targets of ESC-specific REST PET clusters. Shown are the numbers of genes and p-value (Chi-square test) for each ontology term among annotated targets of the PET clusters from (A).(E) ESC-specific and ESC-NSC common binding sites have distinct sequence conservation. All REST PET clusters with an identifiable, consensus RE1 were aligned in the same strand orientation. The mean PhastCons sequence conservation score [54] for every position in a 300-bp window around the RE1 is plotted. Statistical significance is based on comparing the mean Phastcons score across every 21mer RE1 (Student's t-test).(F and G) Boxplots show the distribution of (F) RE1 PSSM scores [18] and (G) PET overlap count for ESC-only and common ESC-NSC PET clusters. Statistical significance was calculated using Student's t-test.

Mentions: REST binding profiles in ESC and NSC, as determined by ChIP-PET, were compared to discover cell-type specific binding sites. Of the 2,460 high-confidence sites (PET5+) found in ESC, 1,365 were also identified in NSC. Thus, by this comparative PET analysis there were 1,095 sites (45%) occupied uniquely in ESC (Figure 4A). Conversely, among the 857 high-confidence sites found in NSC, only ten were not also found in ESC. If the stringency of our cutoffs was raised to PET10+, then for ESC we found 153 sites (19%) that were not in enriched in NSC, and there were no sites enriched only in NSC. To assess the most appropriate threshold for a comparative analysis, the degrees of PET overlaps from the two libraries were cumulatively assessed at each cutoff from PET2+ to PET10+ (Figure 4B). For a threshold above PET5+ in the NSC, there was complete overlap with ESC PETs. The converse was not true: even at the most stringent cutoff, PET10+, only 80% of ESC PET10+ sites have an equivalent in NSC. qPCR carried out on randomly selected sites confirmed the common (19/19, 100%; Figure S11) and ESC-specific binding sites (13/20, 65%; Figure S12). Thus, the ChIP-PET results were consistent with the results obtained in our ChIP-chip experiments, which indicated that up to 30% of REST binding sites are selectively bound in ESC relative to NSC and that there were few, if any, sites exclusively bound in NSC.


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)

ESC-Specific Gene Targeting by REST(A) ESC-specific REST binding sites (green) are defined as those loci having PET10+ in ESC and no evidence (PET1 or no PET) in NSC.(B) For ESC (yellow) or NSC (blue), the fraction of PET clusters of a given size range that also overlap a PET5+ cluster from the other cell type is plotted.(C) The numbers of ESC-specific and common REST PETs shared with kidney cells (from ChIP-SACO [22]), and the numbers overlapping full-length RE1 motifs are shown. Statistical significance was calculated using the Chi-squared test.(D) Genes involved in Wnt and integrin signaling, and genes encoding kinases and chromatin-binding proteins are enriched amongst targets of ESC-specific REST PET clusters. Shown are the numbers of genes and p-value (Chi-square test) for each ontology term among annotated targets of the PET clusters from (A).(E) ESC-specific and ESC-NSC common binding sites have distinct sequence conservation. All REST PET clusters with an identifiable, consensus RE1 were aligned in the same strand orientation. The mean PhastCons sequence conservation score [54] for every position in a 300-bp window around the RE1 is plotted. Statistical significance is based on comparing the mean Phastcons score across every 21mer RE1 (Student's t-test).(F and G) Boxplots show the distribution of (F) RE1 PSSM scores [18] and (G) PET overlap count for ESC-only and common ESC-NSC PET clusters. Statistical significance was calculated using Student's t-test.
© Copyright Policy
Related In: Results  -  Collection

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pbio-0060256-g004: ESC-Specific Gene Targeting by REST(A) ESC-specific REST binding sites (green) are defined as those loci having PET10+ in ESC and no evidence (PET1 or no PET) in NSC.(B) For ESC (yellow) or NSC (blue), the fraction of PET clusters of a given size range that also overlap a PET5+ cluster from the other cell type is plotted.(C) The numbers of ESC-specific and common REST PETs shared with kidney cells (from ChIP-SACO [22]), and the numbers overlapping full-length RE1 motifs are shown. Statistical significance was calculated using the Chi-squared test.(D) Genes involved in Wnt and integrin signaling, and genes encoding kinases and chromatin-binding proteins are enriched amongst targets of ESC-specific REST PET clusters. Shown are the numbers of genes and p-value (Chi-square test) for each ontology term among annotated targets of the PET clusters from (A).(E) ESC-specific and ESC-NSC common binding sites have distinct sequence conservation. All REST PET clusters with an identifiable, consensus RE1 were aligned in the same strand orientation. The mean PhastCons sequence conservation score [54] for every position in a 300-bp window around the RE1 is plotted. Statistical significance is based on comparing the mean Phastcons score across every 21mer RE1 (Student's t-test).(F and G) Boxplots show the distribution of (F) RE1 PSSM scores [18] and (G) PET overlap count for ESC-only and common ESC-NSC PET clusters. Statistical significance was calculated using Student's t-test.
Mentions: REST binding profiles in ESC and NSC, as determined by ChIP-PET, were compared to discover cell-type specific binding sites. Of the 2,460 high-confidence sites (PET5+) found in ESC, 1,365 were also identified in NSC. Thus, by this comparative PET analysis there were 1,095 sites (45%) occupied uniquely in ESC (Figure 4A). Conversely, among the 857 high-confidence sites found in NSC, only ten were not also found in ESC. If the stringency of our cutoffs was raised to PET10+, then for ESC we found 153 sites (19%) that were not in enriched in NSC, and there were no sites enriched only in NSC. To assess the most appropriate threshold for a comparative analysis, the degrees of PET overlaps from the two libraries were cumulatively assessed at each cutoff from PET2+ to PET10+ (Figure 4B). For a threshold above PET5+ in the NSC, there was complete overlap with ESC PETs. The converse was not true: even at the most stringent cutoff, PET10+, only 80% of ESC PET10+ sites have an equivalent in NSC. qPCR carried out on randomly selected sites confirmed the common (19/19, 100%; Figure S11) and ESC-specific binding sites (13/20, 65%; Figure S12). Thus, the ChIP-PET results were consistent with the results obtained in our ChIP-chip experiments, which indicated that up to 30% of REST binding sites are selectively bound in ESC relative to NSC and that there were few, if any, sites exclusively bound in NSC.

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