Extended Data Fig. 12: OAT silencing results in transcriptional and epigenetic changes similar to those of ODC1 knockdown.

a, Venn diagrams showing a higher overlapping number of differentially expressed genes (q < 0.05) in AsPC-1 cells with knockdown of OAT or ODC1 (608 down-regulated and 599 up-regulated), as compared to ARG2 and ODC1 or ARG2 and OAT. Numbers reflect differential expression common to 2 hairpins per gene. b, ATAC-Seq data showing significant changes (q < 0.05) in chromatin access at enhancers located < 20 kb from transcription start sites in AsPC-1 cells with knockdown of ODC1, compared to control Scramble (Scr), OAT, or ARG2 (2 hairpins per gene). n = 2 biological replicates per hairpin. Significant changes detected include losses and gains of chromatin accessibility near 175 and 135 enhancers, respectively. c, Correlation of changes in gene expression and nearby (< 25 kb) chromatin accessibility for 403 genes that were consistently up-regulated upon ODC1 and OAT silencing in AsPC-1 cells (cluster V, Fig. 4h). Each dot represents a gene. Gene-linked enhancers (< 25 kb) show gains in accessibility upon knockdown of ODC1 or OAT, but not ARG2. d, List of top 18 pathways in descending significance (FDR 0.003-0.30) that were differentially altered at the transcriptional level (either negative or positive enrichment) upon ODC1 knockdown compared to Scr control in AsPC-1 PDA cells described in a, b. Negatively enriched pathways related to growth factors, cytokines and response to starvation are highlighted in yellow and those involved in cell shape, migration, differentiation and ion transport are highlighted in blue. Asterisks indicate representative pathways illustrated in e and Fig. 4j. e, Heatmaps of genes from representative pathways (blue) in d displaying negative enrichment upon knockdown of ODC1 compared to Scr control (n = 3), with corresponding GSEA plots. NES normalized enrichment scores, FDR false discovery rate, Nom. Nominal. f, List of differentially expressed genes from top 18 negatively enriched pathways in d, that are part of Cluster I in Fig. 4h and are indicated with dark blue dots in the correlation plots of Fig. 4i. Genes in Quadrant (Q) III of Fig. 4i plots associate with decreased chromatin accessibility, and are more numerous in cells with knockdown of ODC1 (37/58) or OAT (44/58), than ARG2 (22/58). g, Relative mRNA levels (by qPCR) of 7 representative genes in AsPC-1 cells with knockdown of ODC1, OAT, or ARG2 (2 hairpins per gene) compared to control Scr, that were maintained for 72 h in the presence or absence of 10 μM Putrescine. Genes were randomly chosen from Q III described in f and Fig. 4i, and display common decreases in expression upon OAT or ODC1 knockdown but not ARG2, along with concordant decreases in chromatin access. Data represent the mean ± s.d. of 3 biological replicates from 3 independent experiments. p-values were obtained by two-way ANOVA followed by Tukey test. Statistical significance is for knockdown of ODC1, OAT or ARG2 vs. Scr under similar putrescine condition, unless otherwise indicated by lines.