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Figures
- Fig. 1.
KDM5B regulates expression of self-renewal genes in TS cells. (A) TS cells transduced with shLuc (control) or shKdm5b lentiviral particles and stably selected with puromycin. Dotted lines outline boundary of TS cell colony. Representative micrographs from at least three independent experiments are shown. (B) Relative RNA-Seq expression level of Kdm5b in shLuc and shKdm5b TS cells. Kdm5b RNA-Seq mRNA levels (RPKM) were normalized to shLuc TS cells. (C) Scatter plot of RNA-Seq gene expression analysis between shLuc and shKdm5b TS cells. Log2 adjusted differentially expressed genes are plotted (>twofold, RPKM>3). At least two biological replicates were performed for RNA-Seq analyses. (D) Boxplot of RNA-Seq data: upregulated and downregulated genes in shLuc and shKdm5b TS cells (log2 RPKM). (E) Gene set enrichment analysis (GSEA) plot of downregulated (top) and upregulated (bottom) differentially expressed genes in KDM5B-depleted TS cells relative to shLuc TS cells. Note that the expression of the majority of genes downregulated in shKdm5b TS cells is enriched in undifferentiated TS cells (top plot), while expression of genes that are upregulated in shKdm5b TS cells is enriched in differentiated TS cells (bottom plot). A positive enrichment score indicates that expression of genes is enriched in undifferentiated TS cells, while a negative enrichment score indicates that expression of genes is enriched in differentiated TS cells. (F) DAVID gene ontology (GO) functional annotation of differentially expressed genes between shLuc and shKdm5b TS cells. The bottom graph shows significantly enriched placental and trophoblast GO terms. (G) K-means clustering analysis of RNA-Seq data. Differentially expressed genes (>twofold) clustered according to k-means. (H) Principal component analysis (PCA) of RNA-Seq expression between shLuc and shKdm5b TS cells, day 14 differentiated TS cells, ES cells, day 6, 10, and 14 embryoid body (EB) differentiated ES cells, EpiSCs, and MEFs. (I) Network2Canvas (N2C) (Tan et al., 2013) analyses of differentially expressed genes between shLuc and shKdm5b TS cells. In each canvas, each node (square) represents a gene list (shLuc versus Kdm5b DE genes in TS cells) associated with a functional term in a gene-set library (ChIP-X, mouse gene atlas, and MGI phenotype). The terms are organized on the canvas based on the similarity of their gene-set content. The brightness (white) of each node is determined by its P value. (J) Custom tracks of RNA-Seq data in the UCSC genome browser.
- Fig. 2.
KDM5B regulates the H3K4 methylation landscape of TS cells. Depletion of KDM5B leads to altered (A) H3K4me3 and (B) H3K4me2 levels in TS cells (See the Materials and Methods section for SICER-analysis; fold-change >1.5, FDR <0.001). Scatter plots of (C) H3K4me3 and (D) H3K4me2 densities at TSC ChIP-enriched peaks (log2 normalized tag density). (E) Venn diagrams showing overlap of H3K4me3 (left) and H3K4me2 (right) in shLuc and shKdm5b TS cells at SICER-defined islands. (F,G) Boxplots of (F) H3K4me3 and (G) H3K4me2 densities at regions of increased or decreased H3K4me3 or H3K4me2 in shKdm5b TS cells relative to shLuc TS cells. (H,I) Annotation of regions with increased or decreased (H) H3K4me3 or (I) H3K4me2 in KDM5B-depleted TS cells using HOMER software. (J) DAVID gene ontology (GO) functional annotation of genes with altered H3K4me3 in KDM5B-depleted TS cells relative to shLuc TS cells. (K,L) GSEA of genes with (K) increased (left) or decreased (right) H3K4me3 or (L) H3K4me2 in shKdm5b TS cells relative to shLuc TS cells. (M) Custom views of ChIP-Seq data in the UCSC genome browser.
- Fig. 3.
KDM5B demethylates H3K4me3 at TSC-enriched genes during early differentiation. (A) Bright-field microscopy of day 2–3 differentiated shLuc and shKdm5b TS cells. (B) Percent viability of day 2–3 differentiated shLuc and shKdm5b TS cells. (C) Differentiated KDM5B-depleted TS cells exhibit elevated H3K4me3 ChIP-Seq levels relative to differentiated shLuc TS cells (See the Materials and Methods section for SICER-analysis; fold-change >1.5, FDR <0.001). (D) Venn diagrams showing overlap of H3K4me3 in day 2–4 differentiated shLuc and shKdm5b TS cells at SICER-defined islands. (E) Boxplots of H3K4me3 densities at regions with increased H3K4me3 in day 2, day 3, and day 4 differentiated shKdm5b TS cells. (F) Annotation of regions with increased H3K4me3 in shKdm5b versus shLuc day 2, day 3, or day 4 differentiated TS cells using HOMER software. (G) GSEA of genes with increased H3K4me3 in shKdm5b versus shLuc day 2, day 3, or day 4 differentiated TS cells. (H) Schematic describing the calculation used to determine the differentiation index (DI) at H3K4me3 marked genes in ES cells. The DI is calculated from the ratio of the density of H3K4me3 in differentiated TS cells to the density of H3K4me3 in undifferentiated TS cells. (I) Empirical cumulative distribution for the DI of H3K4me3 across genes marked by H3K4me3 in undifferentiated TS cells for shLuc (black) and shKdm5b (red) TS cells. Y-axis shows the percentage of regions that exhibit a DI less than the value specified by the x-axis. A line shifted to the right means a systematic increase in the differentiation index. P-value for all <2.2E-16 (Kolmogorov–Smirnov test). Note the decreased DI for genes marked by H3K4me3 in shKdm5b TS cells. (J) Custom views of ChIP-Seq data in the UCSC genome browser.
- Fig. 4.
KDM5B demethylates H3K4me3 at TSC-enriched genes during differentiation. (A) Differentiated KDM5B-depleted TS cells exhibit elevated H3K4me3 ChIP-Seq levels relative to differentiated shLuc TS cells (See the Materials and Methods section for SICER-analysis; fold-change >1.5, FDR <0.001). (B) Venn diagrams showing overlap of H3K4me3 in day 14 differentiated shLuc and shKdm5b TS cells at SICER-defined islands. (C) Scatter plot of H3K4me3 densities at TSC ChIP-enriched peaks (log2 normalized tag density). (D) Boxplot of H3K4me3 density at TSC-defined ChIP-Seq peaks in shLuc and shKdm5b day 14 differentiated TS cells. (E) Heat maps of H3K4me3 densities at transcriptional start sites (TSS) in shLuc and shKdm5b day 14 differentiated TS cells (genes were sorted by their expression in undifferentiated TS cells). (F) Average profile of H3K4me3 in shLuc and shKdm5b day 14 differentiated TS cells. (G) Annotation of regions with increased H3K4me3 in shKdm5b versus shLuc day 14 differentiated TS cells using HOMER software. (H) GSEA of genes with increased H3K4me3 in shKdm5b versus shLuc day 14 differentiated TS cells. (I) Empirical cumulative distribution for the differentiation index (DI) of H3K4me3 across genes marked by H3K4me3 in undifferentiated TS cells for shLuc (orange) and shKdm5b (blue) TS cells. Y-axis shows the percentage of regions that exhibit a DI less than the value specified by the x-axis. A line shifted to the right means a systematic increase in the differentiation index. P-value for all <2.2E-16 (Kolmogorov–Smirnov test). Note the decreased DI for genes marked by H3K4me3 in shKdm5b TS cells. (J) Custom views of ChIP-Seq data in the UCSC genome browser.
- Fig. 5.
KDM5B demethylates H3K4me2 at TSC-enriched genes during differentiation. (A) Differentiated KDM5B-depleted TS cells exhibit elevated H3K4me2 ChIP-Seq levels relative to differentiated shLuc TS cells (See the Materials and Methods section for SICER-analysis; fold-change >1.5, FDR <0.001). (B) Venn diagrams showing overlap of H3K4me2 in day 14 differentiated shLuc and shKdm5b TS cells at SICER-defined islands. (C) Scatter plot of H3K4me2 densities at TSC ChIP-enriched peaks (log2 normalized tag density). (D) Boxplot of H3K4me2 density at TSC-defined ChIP-Seq peaks in shLuc and shKdm5b day 14 differentiated TS cells. (E) Heat maps of H3K4me2 densities at transcriptional start sites (TSS) in shLuc and shKdm5b day 14 differentiated TS cells (genes were sorted by their expression in undifferentiated TS cells). (F) Average profile of H3K4me2 in shLuc and shKdm5b day 14 differentiated TS cells. (G) Annotation of regions with increased H3K4me2 in shKdm5b versus shLuc day 14 differentiated TS cells using HOMER software. (H) GSEA of genes with increased H3K4me2 in shKdm5b versus shLuc day 14 differentiated TS cells. (I) Empirical cumulative distribution for the differentiation index (DI) of H3K4me2 across regions marked by H3K4me2 in undifferentiated TS cells for shLuc (orange) and shKdm5b (blue) TS cells. Y-axis shows the percentage of genes that exhibit a DI less than the value specified by the x-axis. A line shifted to the right means a systematic increase in the differentiation index. P-value for all <2.2E-16 (Kolmogorov–Smirnov test). Note the decreased DI for genes marked by H3K4me2 in shKdm5b TS cells. (J) Bright-field microscopy of day 14 differentiated shLuc and shKdm5b TS cells. (K) Percent viability of day 14 differentiated shLuc and shKdm5b TS cells.
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