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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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ChIP-PET Identification of Noncanonical REST Binding Motifs(A) Sequencing and mapping statistics for REST ChIP-PET in ESC and NSC.(B) REST binding sites identified by ChIP-PET were classified by similarity to the full-length RE1 motif, and orientation of the left (red) and right (blue) RE1 half-sites.(C) Number of sites with altered spacing between the left and right half sites, compared with the canonical 2-bp spacer. Shown is a histogram of the number of instances for each spacer size, for the PET5+ sites from ESC (yellow) and NSC (blue). NA, not applicable (i.e., canonical 2-bp spacer).
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pbio-0060256-g003: ChIP-PET Identification of Noncanonical REST Binding Motifs(A) Sequencing and mapping statistics for REST ChIP-PET in ESC and NSC.(B) REST binding sites identified by ChIP-PET were classified by similarity to the full-length RE1 motif, and orientation of the left (red) and right (blue) RE1 half-sites.(C) Number of sites with altered spacing between the left and right half sites, compared with the canonical 2-bp spacer. Shown is a histogram of the number of instances for each spacer size, for the PET5+ sites from ESC (yellow) and NSC (blue). NA, not applicable (i.e., canonical 2-bp spacer).

Mentions: The RE1 ChIP-chip approach described above interrogated high-quality RE1 motifs that had a strong match to the RE1 PSSM. However, recent reports have identified noncanonical RE1-like motifs that can effectively recruit REST in vivo [22,23,30]. To extend our study to the unbiased identification of REST binding sites, we generated a genome-wide map of REST binding in ESC using ChIP-PET technology, which combines ChIP with deep DNA sequencing [31]. Briefly, a library was prepared from ChIP DNA in a manner that produced paired-end tags (PETs) for each DNA fragment. The PETs were sequenced and mapped to the mouse genome to define the chromosomal locations where REST is bound in vivo. This ChIP-PET technique has been used previously to map binding sites for several transcription factors in ESC, including Oct4 and Nanog, and has been shown to be accurate and sensitive [5,31]. ChIP-PET mapping of REST in ESC generated 713,713 nonredundant PETs that clustered (i.e., overlapped at the same genomic location) into 2,460 high-confidence REST binding sites (Figure 3A). A confidence threshold was set at PET clusters of five or more unique and overlapping members (PET5+) based upon ChIP-PCR validation of sets of 20 genomic loci, each randomly selected from clusters of size three to ten (PET3–PET10) (Figure S8). Seventy-five percent (39/52) of PET5–PET7 clusters we tested had detectable REST binding by qPCR. Further evidence for the efficacy of the method is demonstrated by the fact that PET5+ clusters overlap 91% (649/714) of the RE1s identified by ChIP-chip in ESC (Figure S9).


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)

ChIP-PET Identification of Noncanonical REST Binding Motifs(A) Sequencing and mapping statistics for REST ChIP-PET in ESC and NSC.(B) REST binding sites identified by ChIP-PET were classified by similarity to the full-length RE1 motif, and orientation of the left (red) and right (blue) RE1 half-sites.(C) Number of sites with altered spacing between the left and right half sites, compared with the canonical 2-bp spacer. Shown is a histogram of the number of instances for each spacer size, for the PET5+ sites from ESC (yellow) and NSC (blue). NA, not applicable (i.e., canonical 2-bp spacer).
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0060256-g003: ChIP-PET Identification of Noncanonical REST Binding Motifs(A) Sequencing and mapping statistics for REST ChIP-PET in ESC and NSC.(B) REST binding sites identified by ChIP-PET were classified by similarity to the full-length RE1 motif, and orientation of the left (red) and right (blue) RE1 half-sites.(C) Number of sites with altered spacing between the left and right half sites, compared with the canonical 2-bp spacer. Shown is a histogram of the number of instances for each spacer size, for the PET5+ sites from ESC (yellow) and NSC (blue). NA, not applicable (i.e., canonical 2-bp spacer).
Mentions: The RE1 ChIP-chip approach described above interrogated high-quality RE1 motifs that had a strong match to the RE1 PSSM. However, recent reports have identified noncanonical RE1-like motifs that can effectively recruit REST in vivo [22,23,30]. To extend our study to the unbiased identification of REST binding sites, we generated a genome-wide map of REST binding in ESC using ChIP-PET technology, which combines ChIP with deep DNA sequencing [31]. Briefly, a library was prepared from ChIP DNA in a manner that produced paired-end tags (PETs) for each DNA fragment. The PETs were sequenced and mapped to the mouse genome to define the chromosomal locations where REST is bound in vivo. This ChIP-PET technique has been used previously to map binding sites for several transcription factors in ESC, including Oct4 and Nanog, and has been shown to be accurate and sensitive [5,31]. ChIP-PET mapping of REST in ESC generated 713,713 nonredundant PETs that clustered (i.e., overlapped at the same genomic location) into 2,460 high-confidence REST binding sites (Figure 3A). A confidence threshold was set at PET clusters of five or more unique and overlapping members (PET5+) based upon ChIP-PCR validation of sets of 20 genomic loci, each randomly selected from clusters of size three to ten (PET3–PET10) (Figure S8). Seventy-five percent (39/52) of PET5–PET7 clusters we tested had detectable REST binding by qPCR. Further evidence for the efficacy of the method is demonstrated by the fact that PET5+ clusters overlap 91% (649/714) of the RE1s identified by ChIP-chip in ESC (Figure S9).

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