Fig. 3

Expression analyses for identification of the potential driver gene in the 13q12.2 amplicon. a Box plots showing significant positive correlation between gene copy number and mRNA expression (log10 (normalized RSEM value + 1)) in 5 genes (i.e., LNX2, POLR1D, CDX2, PDX1, and PAN3) in the TCGA cohort. Control/matched normal tissue, n = 51; Balanced, n = 196; Gain, n = 129; Amplification, n = 46. b Scatter plots illustrating positive correlation in gene copy number and mRNA expression (log2 (TPM + 1)) in 5 genes (i.e., LNX2, POLR1D, CDX2, PDX1, and PAN3) in 58 CRC cell lines. R values and p values were calculated using Pearson’s correlation test. The red line represents the noise threshold (TPM = 1). c The bar chart illustrates cell viability changes after knockdown of 5 genes (i.e., CDX2, LNX2, PAN3, PDX1, and POLR1D) in 2 CRC cell lines (i.e., HT29 and SW480). Silencing of POLR1D in both cell lines showed reduction in cell viability over 15%. p values calculated by t test are shown above the bar. d Silencing of POLR1D with 3 different siRNA constructs. RT-PCR showing that silencing provided sufficient knockdown of POLR1D expression in both cell lines. e Cell viability time curve illustrating significant reduction of cell viability after knockdown of POLR1D expression in HT29 and SW480 cells (*p < 0.1; **p < 0.05; ***p < 0.01; t test). f Box plot illustrating the different expression (normalized DESeq2 read count) of VEGFA and EREG between negative control (SCR, a scrambled siRNA) and POLR1D knockdown in SW480 (SCR, n = 6; siPOLR1D2, n = 3; siPOLR1D3, n = 3) and HT29 (SCR, n = 4; siPOLR1D2, n = 2; siPOLR1D3, n = 2) cell lines. VEGFA and EREG expression was suppressed after POLR1D silencing. Adjusted p values were calculated by DESeq2, an R package. g Violin plots of VEGFA and EREG expression (normalized RSEM value) of TCGA cases. Samples with chr13q12.2 gain (n = 129) or amplification (n = 46) showed a significant upregulation compared to balanced cases (n = 196). h Schematic presentation how nucleosome organization around promoters of repressed and active genes differ in their promoter regions. Promoters of active genes have a nucleosome-depleted region (NDR, dark blue line), whereas the nucleosome organization of promoters of repressed genes is not well-defined, resulting in different nucleosome footprints at transcription start sites. We leveraged these differences by employing our previously published nucleosome positioning [38] to determine the expression status of genes within the 13q12.2 amplicon. In addition to the genes discussed in the text, we added the gene GSX1 (light blue) as an example for a repressed gene (part of the figure adapted from [49])