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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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Validation of Stem Cell Potency and Microarray Sensitivity(A) Mouse ESC stain for markers of self-renewal. Immunohistochemistry was carried out using antibodies to Oct4, Nanog, and Sox2. For negative control (-ve), the primary antibody was omitted.(B and C) ESC are capable of differentiation. Following treatment with retinoic acid (RA), qRT-PCR was used to assay expression of self-renewal markers (Nanog, Oct4) (B) and differentiation markers (Alpha fetoprotein, Afp; Coup TFII, Nr2f2).(D) NSC are multipotent. Following a standard differentiation protocol (see Text S1), cells were observed to differentiate into neurons, astrocytes and oligodendrocytes, as revealed by staining with Tuj1 (βIII tubulin), GFAP (glial fibrillary acidic protein) and O4 antibodies, respectively. Scale bar: 20 μm.(E) Enrichment for REST occupancy in ESC at the Syt4 RE1 site as determined by ChIP-chip. Locations of seven tiled probes and RE1 site are indicated.(F) Comparative performance of ChIP-chip and qPCR for 150 randomly selected RE1s. Dark blue (occupied) and gray bars (unoccupied) indicate a consistent call by both techniques. Light blue bars denote RE1s that were not consistently called.
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pbio-0060256-g001: Validation of Stem Cell Potency and Microarray Sensitivity(A) Mouse ESC stain for markers of self-renewal. Immunohistochemistry was carried out using antibodies to Oct4, Nanog, and Sox2. For negative control (-ve), the primary antibody was omitted.(B and C) ESC are capable of differentiation. Following treatment with retinoic acid (RA), qRT-PCR was used to assay expression of self-renewal markers (Nanog, Oct4) (B) and differentiation markers (Alpha fetoprotein, Afp; Coup TFII, Nr2f2).(D) NSC are multipotent. Following a standard differentiation protocol (see Text S1), cells were observed to differentiate into neurons, astrocytes and oligodendrocytes, as revealed by staining with Tuj1 (βIII tubulin), GFAP (glial fibrillary acidic protein) and O4 antibodies, respectively. Scale bar: 20 μm.(E) Enrichment for REST occupancy in ESC at the Syt4 RE1 site as determined by ChIP-chip. Locations of seven tiled probes and RE1 site are indicated.(F) Comparative performance of ChIP-chip and qPCR for 150 randomly selected RE1s. Dark blue (occupied) and gray bars (unoccupied) indicate a consistent call by both techniques. Light blue bars denote RE1s that were not consistently called.

Mentions: ChIP DNA was isolated from undifferentiated pluripotent ESC (Figure 1A–1C and Figure S1) and multipotent NSC (Figure 1D and Figure S2) and hybridized to the RE1 ChIP-chip. Statistically significant enrichments were determined by a combination of Gaussian Mixture Model and Single Array Error Model [26] analyses. The false discovery rate (FDR) was selected to optimize sensitivity and selectivity, by comparing both statistical and experimental evaluations of the microarray. The enrichments detected over the tiled regions of Nppa and Syt4 were specific and restricted to small regions around the predicted RE1 sites (Figure 1E). To validate the ChIP-chip performance, quantitative PCR (qPCR) was carried out on a randomly selected set of RE1 sites (Figure 1F and Figure S3). Taking qPCR as the standard, the ChIP-chip correctly called REST occupancy, or lack thereof, at 108 out of 126 RE1 sites (86%). Importantly, 87% of negative control probes were correctly called by the ChIP-chip, indicating a false positive rate of around 13% as expected from the statistical model. With the statistical parameters used in our studies, we found that the ChIP-chip method accurately assessed REST occupancy at the 1,095 predicted RE1 sites. The focused REST ChIP-chip thus provides a robust and rigorous means to compare occupancy of REST across many cell types and under various stages of differentiation, and thereby reveals a more complete and dynamic view of the REST transcriptional regulatory network.


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)

Validation of Stem Cell Potency and Microarray Sensitivity(A) Mouse ESC stain for markers of self-renewal. Immunohistochemistry was carried out using antibodies to Oct4, Nanog, and Sox2. For negative control (-ve), the primary antibody was omitted.(B and C) ESC are capable of differentiation. Following treatment with retinoic acid (RA), qRT-PCR was used to assay expression of self-renewal markers (Nanog, Oct4) (B) and differentiation markers (Alpha fetoprotein, Afp; Coup TFII, Nr2f2).(D) NSC are multipotent. Following a standard differentiation protocol (see Text S1), cells were observed to differentiate into neurons, astrocytes and oligodendrocytes, as revealed by staining with Tuj1 (βIII tubulin), GFAP (glial fibrillary acidic protein) and O4 antibodies, respectively. Scale bar: 20 μm.(E) Enrichment for REST occupancy in ESC at the Syt4 RE1 site as determined by ChIP-chip. Locations of seven tiled probes and RE1 site are indicated.(F) Comparative performance of ChIP-chip and qPCR for 150 randomly selected RE1s. Dark blue (occupied) and gray bars (unoccupied) indicate a consistent call by both techniques. Light blue bars denote RE1s that were not consistently called.
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pbio-0060256-g001: Validation of Stem Cell Potency and Microarray Sensitivity(A) Mouse ESC stain for markers of self-renewal. Immunohistochemistry was carried out using antibodies to Oct4, Nanog, and Sox2. For negative control (-ve), the primary antibody was omitted.(B and C) ESC are capable of differentiation. Following treatment with retinoic acid (RA), qRT-PCR was used to assay expression of self-renewal markers (Nanog, Oct4) (B) and differentiation markers (Alpha fetoprotein, Afp; Coup TFII, Nr2f2).(D) NSC are multipotent. Following a standard differentiation protocol (see Text S1), cells were observed to differentiate into neurons, astrocytes and oligodendrocytes, as revealed by staining with Tuj1 (βIII tubulin), GFAP (glial fibrillary acidic protein) and O4 antibodies, respectively. Scale bar: 20 μm.(E) Enrichment for REST occupancy in ESC at the Syt4 RE1 site as determined by ChIP-chip. Locations of seven tiled probes and RE1 site are indicated.(F) Comparative performance of ChIP-chip and qPCR for 150 randomly selected RE1s. Dark blue (occupied) and gray bars (unoccupied) indicate a consistent call by both techniques. Light blue bars denote RE1s that were not consistently called.
Mentions: ChIP DNA was isolated from undifferentiated pluripotent ESC (Figure 1A–1C and Figure S1) and multipotent NSC (Figure 1D and Figure S2) and hybridized to the RE1 ChIP-chip. Statistically significant enrichments were determined by a combination of Gaussian Mixture Model and Single Array Error Model [26] analyses. The false discovery rate (FDR) was selected to optimize sensitivity and selectivity, by comparing both statistical and experimental evaluations of the microarray. The enrichments detected over the tiled regions of Nppa and Syt4 were specific and restricted to small regions around the predicted RE1 sites (Figure 1E). To validate the ChIP-chip performance, quantitative PCR (qPCR) was carried out on a randomly selected set of RE1 sites (Figure 1F and Figure S3). Taking qPCR as the standard, the ChIP-chip correctly called REST occupancy, or lack thereof, at 108 out of 126 RE1 sites (86%). Importantly, 87% of negative control probes were correctly called by the ChIP-chip, indicating a false positive rate of around 13% as expected from the statistical model. With the statistical parameters used in our studies, we found that the ChIP-chip method accurately assessed REST occupancy at the 1,095 predicted RE1 sites. The focused REST ChIP-chip thus provides a robust and rigorous means to compare occupancy of REST across many cell types and under various stages of differentiation, and thereby reveals a more complete and dynamic view of the REST transcriptional regulatory network.

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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