NUDT2 Disruption Elevates Diadenosine Tetraphosphate (Ap(4)A) and Down-Regulates Immune Response and Cancer Promotion Genes

Marriott, Andrew S.; Vasieva, Olga; Fang, Yongxiang; Copeland, Nikki A.; McLennan, Alexander G.; Jones, Nigel J.

In order to place the gene expression data into a biological context, Ingenuity 1 Pathway Analysis (IPA 1 ) software was used to assign the DEGs to known canonical pathways and functional networks in order to predict the biological functions of the transcriptional changes. For simplicity, the initial analysis included only genes that were up-or down-regulated by 2-fold (P < 0.05); however, where present in the resulting pathways and networks, genes up-or down-regulated by 1.2 were also considered to be of potential interest as there is no biological justification for a cut-off value of 2. It was found that the DEGs mapped to a large number of pathways with a significant enrichment score (-log(P-value)) (S4 Table) . Top ranked within both up-and down-regulated gene sets were signaling pathways related to immunity and inflammation. Pathways associated primarily with the innate immune response, such as activation of interferon regulatory factors (IRFs) by pattern recognition receptors (PRRs), and inflammation were specifically enriched in the down-regulated set of genes while functions associated with MHC class II antigens were specific for the set of up-regulated genes ( Table 4 ). The predominance of these pathways in the dataset may reflect the myeloid nature of the KBM-7 cell line [37] . These pathways are discussed in detail below. Interferon response and innate immunity. Interferons are important mediators of the innate immune response, which provides an initial vital defence against invading pathogens (viruses, bacteria, protozoa) following interaction of pathogen components with PRRs in various cellular compartments. They can also inhibit cell proliferation, modulate the adaptive immune response, and be pro-or anti-inflammatory, depending on context [48] [49] [50] [51] . Submission of the set of 4,835 DEGs with fold change 1.2 to the Interferome database (v2.01) [52] revealed a subset of at least 1,038 DEGs known to be regulated by Type I IFNs (IFNα and IFNβ) in other systems. Roughly half of these overlapped with the set of 944 showing known regulation by Type II IFNs (IFNγ). Some (56) also showed Type III (IFNλ) regulation with 15 of these potentially unique to Type III (S5 Table) . Table) . The JAK-STAT signaling pathways are central to the interferon response. In Type II interferon signaling, activated STAT1 homodimers bind to the GAS (Interferon Gamma Activated Sequence) promoter and induce gene expression while Type I signaling involves the combination of STAT1-STAT2 heterodimers with IRF9 (Interferon Response Factor 9) forming ISGF3 (Interferon Stimulated Gene Factor), which then binds to the ISRE (Interferon-Stimulated Response Element) promoter. STAT1, STAT2 and IRF9 were all down-regulated (1.4-, 1.8-and 3.7-fold respectively) while the pathway suppressors SOCS1 and PTPN2 were up-regulated (1.2-1.4-fold). Though individually slight, the combined effect of these changes could nevertheless be significant. The increases in SOCS1 and PTPN2 also show that there is not just a general suppression of gene expression but that negative feedback via these genes is preserved. Finally, several of the STATcontrolled genes that are down-regulated are themselves activators of further IFN response genes, e.g. IRF1, IRF7 and IRF9 (1.2-, 3.8-and 3.7-fold respectively).