Fig. 3

Transcriptome analysis reveals altered differentiation of SMYD5-depleted ES cells. a K-means clustering analysis of RNA-Seq data from shLuc and shSmyd5 ES cells differentiated without LIF for 14 days. The experimental design is shown on top. Differentially expressed genes (>twofold; RPKM > 1) clustered according to k-means. b Custom tracks of RNA-Seq data in the UCSC genome browser for undifferentiated and differentiated shLuc control and shSmyd5 ES cells. c Principal component analysis (PCA) of differentially expressed genes during EB differentiation of shSmyd5 and shLuc ES cells. d Prediction of differentially expressed genes due to chance or altered differentiation. The percentage of genes that lag behind during differentiation of shSmyd5 ES cells is less than expected. Top each bar represents a group of genes upregulated by at least alpha-fold (X axis) from ESC (0 h) to EB day 6 in the control cells. The percentage of genes with expression values that follow the order: EB day 6 (shLuc) > EB day 6 (shSmyd5) > ES cell is calculated (observed; red bars); error bars are generated by bootstrapping. The expression values of all genes are randomly shuffled independently for EB day 6 (shLuc), EB day 6 (shSmyd5), and ES cells and are repeated many times to give the means and standard deviations for the expectations (expected; blue bars). The red bars represent observed data. Bottom each bar represents a group of genes upregulated by at least alpha-fold (X axis) from ESC (0 h) to EB day 10 in the control cells. The percentage of genes with expression values that follow the order: EB day 10 (shLuc) > EB day 10 (shSmyd5) > ES cell is calculated (observed; red bars); error bars are generated by bootstrapping. The expression values of all genes are randomly shuffled independently for EB day 10 (shLuc), EB day 10 (shSmyd5), and ES cells and are repeated many times to give the means and standard deviations for the expectations (expected; blue bars). The red bars represent observed data. e Gene set enrichment analysis (GSEA) of differentially expressed genes during differentiation of shSmyd5 ES cells relative to ES cells and day 14 EBs. f DAVID gene ontology analysis of differentially expressed genes between shLuc and shSmyd5 ES cells and during EB differentiation. The hierarchical clustering heat map on the right shows enrichment of developmental GO terms. g Correlation matrix of differentially expressed (DE) genes during shSmyd5 ES cell differentiation with promoter binding of transcription factors and epigenetic modifiers. Heat map generated by evaluating pair-wise affinities between differentially expressed (DE) genes during shLuc and shSmyd5 EB differentiation using RNA-Seq datasets generated from this study (0, 24 h, 6, 10, 14 days) and published ChIP-Seq data [14, 24, 27–29, 67]. AutoSOME [68] was used to generate pair-wise affinity values