Volume 20, Issue 12, Pages 2992-3003 (September 2017) DNA Methylome Analysis Identifies Transcription Factor-Based Epigenomic Signatures of Multilineage Competence in Neural Stem/Progenitor Cells  Tsukasa Sanosaka, Takuya Imamura, Nobuhiko Hamazaki, MuhChyi Chai, Katsuhide Igarashi, Maky Ideta-Otsuka, Fumihito Miura, Takashi Ito, Nobuyuki Fujii, Kazuho Ikeo, Kinichi Nakashima  Cell Reports  Volume 20, Issue 12, Pages 2992-3003 (September 2017) DOI: 10.1016/j.celrep.2017.08.086 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 20, 2992-3003DOI: (10.1016/j.celrep.2017.08.086) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Global DNA Methylation Profiles (A) Boxplot of methylation level of all CpG sites in each cell type. The y axis indicates percentage methylation. (B) Distribution of methylation level around gene body. Levels 5 kb upstream of the TSS to 5 kb downstream of the TES are shown. (C) Hierarchical clustering using Pearson correlation across the entire sample set. (D) DNA methylation profiles for several known astrocyte-specific genes’ (Gfap, Kcnj10, and Sox8) loci. The y axis shows DNA methylation level of each CpG site. Black bars indicate genomic scale. Boxplots show the methylation levels of CpG sites within the regions indicated by red frames. Cell Reports 2017 20, 2992-3003DOI: (10.1016/j.celrep.2017.08.086) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 DNA Methylome, but Not Gene Expression, Reflects Cell Lineage Specification (A) Hierarchical clustering and heatmap of gene expression microarray data. Shown are neuron-, astrocyte-, and oligodendrocyte-enriched genes that are expressed at more than a 3-fold higher level than in all other samples. (B) Line plots of methylation level of neuron-, astrocyte-, and oligodendrocyte-enriched genes. The y axis shows mean methylation levels in 100-bp windows. (C) Line plots of mean methylation level of CGIs, shores, and shelves in 100-bp windows. CGIs that annotate genes whose expression is enriched in neurons, astrocytes, and oligodendrocytes were extracted. Cell Reports 2017 20, 2992-3003DOI: (10.1016/j.celrep.2017.08.086) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Identification of Cell-Type-Specific Low-Methylated Regions (A and B) Boxplot showing DNA methylation levels of NS-LMRs (A) and GS-LMRs (B) in each sample. (C and D) DNA methylation profiles of representative regions of NS-LMRs (C; Dlx1,2 and Tbr1) and GS-LMRs (D; Gfap, Nfia, and Olig1), plotting the DNA methylation levels at single-base resolution. The y axis shows DNA methylation level of each CpG site. Red bars show detected LMRs and black bars indicate genomic scale. Cell Reports 2017 20, 2992-3003DOI: (10.1016/j.celrep.2017.08.086) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 NFI Preferentially Binds Glia-Enriched Gene Promoters and Induces Demethylation (A) Enriched motifs found using MEME software in the top 500 of each type of DMR sequence. (B) Line plot shows the frequency of detected motifs in each DMR. (C) Boxplot of methylation levels of NFI-binding regions in all samples. (D) Expression levels of Nfia in NS/PCs and differentiated cells. Data are shown as mean ± SD (n = 3). (E) Percentage of genes that have an NFI-binding site in their promoter region. Cell Reports 2017 20, 2992-3003DOI: (10.1016/j.celrep.2017.08.086) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 DNA Methylome of Cell-Type-Specific Gene Promoters in NS/PCs (A) Heatmap showing average methylation rate of transcription factor-binding sites in different types of DMRs. (B and C) Boxplot of methylation level at the binding sites of each transcription factor (Sox2, Sox21, and Ascl1 in B; Nfia and Tcf3 in C). (D) DNA methylome in NS/PCs dynamically changes during development to regulate cell fate and identity. The first step is demethylation of neuron-specific gene promoters during the transition from ESCs to NS/PCs. The second step is demethylation of glial gene promoters, which occurs during NS/PC development. NS/PCs acquire the potential to differentiate into astrocytes and oligodendrocytes. The last step is de novo methylation of neuronal genes in glial cells to inhibit the expression of neuronal genes. Cell Reports 2017 20, 2992-3003DOI: (10.1016/j.celrep.2017.08.086) Copyright © 2017 The Author(s) Terms and Conditions