Figure 3. Integrative analysis of RNA and ATAC sequencing shows well-known and novel genes deregulated in NK cell resistant B-ALL cells.

The (A-D and G) transcriptome and (E-I) chromatin accessibility analysis of B-ALL cells co-cultured with NK cells for 4 (time point 1 (TP1)) or 14 (TP2) days were analysed and compared to the B-ALL alone cells on TP1 (n=3-4 independent experiments). (A) The volcano plot shows the significantly up- or downregulated genes in B-ALL cell lines A and B after co-culture with NK cells for 14 days (n=3 experiments). Statistics were calculated with the Wald test. (B) The dot plot illustrates the enrichment analysis using the hyperR tool (28) for the Gene Ontology Biological Process (GOBP) terms associated with NK cell co-cultured B-ALL cells shown in panel (A). X-axis and dot colour depicts the false discovery rate (FDR) and y-axis the corresponding biological processes. Dot size illustrates the number of genes associated with the biological processes. (C) The Euler diagrams display up- or downregulated genes in cell lines A/B and/or C/D after 14 days of NK cell co-culture. (D) The heatmap shows the expression of selected overlapping genes (top-20 upregulated, top-10 downregulated; ranked by p-value) identified in (C) (n=3-4 experiments, 4 B-ALL cell lines). The row-scaled normalised counts represent the log₂(fold change). (E) The volcano plot shows DARs in B-ALL cells A/B after co-culture with NK cells for 14 days (n=3 experiments). Statistics were calculated with the Wald test. (F) The Euler diagrams highlight genes whose promoters (TSS ± 3kb) had higher or lower chromatin accessibility in cell lines A/B and/or C/D after 14 days of NK cell co-culture in n=3-4 experiments. Gene promoters with the highest log₂(fold change) are highlighted. (G) Integration of RNA and ATAC sequencing data shows that 37 genes are up- and 16 downregulated in cell lines A/B; the top 10 up- or downregulated genes are highlighted on the right. (H) Analysis of differential transcription factors activity using (diffTF) (38) highlighted activator (green) and repressor (red) TFs that were differentially expressed in B-ALL cell lines A/B after NK cell co-culture. (I) Integrative Genomics Viewer (IGV) genome browser tracks display representative ATAC sequencing signal densities (n=3 experiments) at the Ly6a and Plaat3 loci in B-ALL cells with or without NK cell co-culture. Shown are the gene bodies including a 3 kb upstream promoter region, as well as binding sites of TFs identified in (H). Database HOCOMOCO (v10) (39) was used and depicted are counts per million (CPM) with a range of [0-1.37].