Change in gene expression profile during ESC neural induction.

<p>A) Global unsupervised clustering performed on the entire pool of 19,204 genes indicates that, at the transcriptional level, ESCs and NESCs are two distinct cell populations. Red boxes highlight reproducible clusters that are strongly supported by data (Bootstrap Probability value ≥95%). B) Heat map of the subset of 2,413 genes that change their expression levels in ESCs as compared to NESCs (at False Discovery Rate <0.01 and absolute FC level ≥2). Expression levels are presented as row-wise standardized values (log₂ fold change). C) Functional enrichment of the 2,413 differentially expressed genes obtained using DAVID GO annotation. Upper plot reports the GO categories associated to genes up-regulated in ESCs, while in the lower plots are the functional enrichment of genes over-expressed during neural induction, i.e. in NESCs.</p

Transcribed enhancers during ESC neural commitment.

<p>The table lists the non-annotated CAGE promoters or promoters annotated to ncRNAs active in ESCs, NESCs or both cell types (maintaining active transcription during ESC neural commitment), located inside ESC-specific, NESC-specific or common enhancer regions.</p><p>Transcribed enhancers during ESC neural commitment.</p

Networks of genes associated to NESC-specific and up-regulated CAGE promoters.

<p>The networks visually represent the connections between the genes associated to NESC-specific (up) and up-regulated CAGE promoters (down). Most of the genes are included in the regulatory pathways of axonal guidance signaling, ESC pluripotency and signal transduction. Purple arrows indicate the connections between genes based on the Ingenuity Knowledge Base dataset (dotted or solid lines for indirect and direct relationships respectively). Then, genes included in IPA canonical pathways (CP) are indicated by grey arrows. The shape of the gene symbol indicates the corresponding protein function, while the color (from white to red) represents the CAGE expression level of the promoter associated to the gene (for NESC-specific promoters) or its ratio between ESCs and NESCs (for upregulated CAGE promoters). For a complete IPA legend see <a href="http://ingenuity.force.com/ipa/articles/Feature_Description/Legend" target="_blank">http://ingenuity.force.com/ipa/articles/Feature_Description/Legend</a></p

Neural induction of human ESCs to NESCs.

<p>A) Schematic representation of the protocol used to differentiate the ESC line H9 to NESCs, entailing the formation of embryoid bodies, followed by neural rosette formation and mechanical isolation. Bar scale 100 μm. B) NESCs cultured in EGF and FGF2 containing medium staining positive for SOX2 in the nucleus (red) and NESTIN in the cytoplasm (green), bar scale 100 μm. NESCs showed a tendency to distribute in rosettes-like structures (white arrow), with the nuclei located at the center and the cytoplasms at the periphery. C) Expression level of pluripotency and neural factors in the three independent cultures of ESCs and NESCs as obtained from the microarray analysis. Genes marked by asterisks were significantly differentially expressed (p ≤0.001***, p ≤0.0001 ****, unpaired t test with Bonferroni correction).</p

Genome-wide mapping of epigenetically-defined promoters and enhancers in ESCs and NESCs.

<p>A) Heat maps showing the distribution of promoter (left) and enhancer (right) regions in a window of ±5-kb from CAGE-mapped TSSs in ESCs and NESCs. Promoter islands are defined as H3K4me3^+/high/me1^-/low, enhancers as H3K4me1^+/high/me3^-/low. Promoter islands are clustered around CAGE-mapped TSSs, whereas enhancers are spread out. B) Average profile of single H3K4me3 (orange) and H3K4me1 (green) peaks around the TSS, in a ±3-kb. Normalized read count means histone modification read count per million mapped reads. C) Epigenetic state of CAGE promoters in ESCs. The histogram shows the fraction of ESC-specific, down-regulated, up-regulated and NESC-specific promoters with the epigenetic signature of active (H3K4me3⁺, red) or poised (H3K4me3⁺/H3K27me3⁺, grey) promoter. The most part of up-regulated and NESC-specific promoters are poised for transcription in ESCs. D) Pie-diagrams showing the fraction of common and cell-specific total enhancers mapped in ESCs and NESCs. E) Analysis of putative TFBS enrichment within cell-specific enhancers as determined by HOMER tool. ESC-specific enhancers showed enrichment of binding motifs for the pluripotency TFs, as observed for cell-specific promoters, and for ETS family factors. NESC-specific enhancers were enriched for ETS-family, RFX-family and Jun/AP1 factors.</p

Differential Pol-II promoter usage during differentiation of ESCs to NESCs.

<p>A) CAGE mapping of transcription start sites (TSSs) to intergenic regions or to promoters (500 bp around the RefSeq TSS), 5’ UTRs, exons, introns and 3’ UTRs of protein-coding and non-coding RefSeq transcripts annotated on the hg19 assembly of the human genome. Bars indicate the percentage of each category of CAGE-defined TSSs in the sense (above the X axis) or anti-sense (below the X axis) transcriptional orientation with respect to the annotated transcript. B) Venn diagram showing the fraction of common and cell-specific CAGE promoters mapped in ESCs and NESCs. For the common promoters, a histogram indicates the number of promoters (left Y axis) distributed in different categories of differential expression during ESC neural induction, with FC from -12 to 0 (down-regulated promoters) and from 0 to +12 (up-regulated promoters). For each category, the fraction of promoters differentially expressed at a statistically significant level (χ² test, p ≤0.01) is indicated (right Y axis). C) Proportion of all, cell-specific, upregulated and down-regulated CAGE promoters annotated to RefSeq protein-coding (yellow) or non-coding (light brown) genes. The fraction of unannotated promoters is shown in dark brown, and significantly increases in the regulated and cell-specific categories. D) Example of a gene (HNRNPF) associated to multiple alternative promoters in both cell types. All 5 known HNRNPF promoters were mapped in ESCs (red bars) and NESCs (blue bars) by CAGE-seq, which identified also two novel promoters in the last exon of the gene. The height of the bars reflects the promoter strength expressed in tpm (scale on the right).</p