Figure 1.
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.
Figure 2.
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.
Figure 3.
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).
Figure 4.
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.
Figure 5.
Diverse Outcomes of REST Function on ESC and NSC Gene Expression Profiles
(A and B) The numbers of genes decreasing (“down”) and increasing (“up”) in response to DN:REST treatment are shown (p < 0.01 threshold for statistical significance) for (A) NSC and (B) ESC.
(C and D) Genes with significant (p < 0.01) changes (≥3-fold) in expression were tabulated for (C) NSC and (D) ESC in response to DN:REST. Gray shaded genes are those with associated REST binding sites at the indicated distance (“Dist.”) from the TSS. Unshaded genes have no REST binding sites within 100 kbp. Common genes are printed in blue.
Figure 6.
Recruitment of REST to Repressed Genes
Significantly changing genes (p < 0.01) were ranked by their fold expression change in response to DN:REST (green, increased by DN:REST; red, decreased). For a sliding window, the fraction of genes within 100 kb of a REST PET cluster is plotted. The dashed line represents the mean value for all genes in the genome.
Figure 7.
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).
Figure 8.
A Model of REST Regulation of Pluripotency and Differentiation in ESC
Yellow and orange panels contain a selection of genes that are commonly targeted by REST, Nanog, Oct4 and Sox2. Red panel contains Wnt pathway-related genes (as identified by the Panther database) that are bound by REST. Activating (arrows) and repressive (bars) regulation is inferred from ChIP binding data.