Fig. 3

Genome-wide transcriptional changes after ectopic ASCL1 expression in NB cells. a PCA of RNA-seq data from NB cells expressing uninduced levels of ASCL1 (endogenous), or overexpressing WT or S-A ASCL1 after 24 h induction with 1 μg/ml doxycycline (n = 10). Data from two separate clones is shown for each condition. b Venn diagram showing the number of WT or S-A ASCL1 response genes which are up- or down-regulated compared to WT ASCL1 uninduced cells after 24 h induction with 1 μg/ml doxycycline (DESeq2 padj < 0.05, n = 10), and the number of target genes which are shared by WT and S-A ASCL1. Proportion and actual numbers of genes shown in each segment. c Heatmap showing relative expression of genes significantly differentially expressed between WT uninduced with endogenous levels of ASCL1, and WT and S-A ASCL1 induced NB clones after 24 h induction with 1 μg/ml doxycycline (DESeq2 padj < 0.05, n = 10). Z-scaled RPKM values for endogenous WT uninduced (grey), WT induced (green), and S-A induced (blue) clones are shown. d Violin plot showing relative expression of WT or S-A ASCL1 target genes associated with significantly increased binding of S-A ASCL1 compared to WT (DESeq2 padj < 0.05, n = 8). Genes are grouped on the x-axis by gene activation or repression. Significance between WT and S-A groups was determined by two-tailed unpaired t-test. e Dotplot showing the correlation between average logFC gene expression (y axis), and average ASCL1 binding strength (x axis) in NB cells overexpressing WT or S-A ASCL1 after 24 h induction with 1 μg/ml doxycycline. Points represents equal sized groups of ASCL1 response genes with an ASCL1 binding site within 3 kb of the promoter, divided into bins based on ASCL1 binding strength. Activated genes (left), repressed genes (right)