Fig 2: SOD1 regulates expression of numerous genes in transcriptional phases. (A) A hierarchical clustering analysis of the differentially expressed genes (DEGs) caused by DNA binding of SOD1. After overlapping the genes in control group of ChIP-Seq and the DEGs in SOD1 knockdown group of RNA-Seq, the heat map was preparation by normalized log10(FPKM+1) of the DEGs. (B) A volcano plot of log2(fold change) vs. the -log10(padj) for the DEGs in (A). (C, E and F) Relative FPKM values of some key genes in SOD1 knockdown and LD100 treated HeLa cells (INA, UNC13A, PTK6 and NRG1 in (C); PBX2 and FGFR4 in (E); TUSC2 and CRTC3 in (F). (D) An enriched KEGG pathway scatterplot of the DEGs in (A) shows the most affected 20 signaling pathways with high statistical significance. (G) A heat map of the genes regulates by SOD1 at transcriptional levels, drawn using normalized log10(FPKM+1) of the genes. (H and K) Genome browser views of the SOD1 binding sites near TSS of all genes in (G) and in (J) (red for the ChIP-Seq under normal conditions; blue for the Input). (I and J) RT-pPCR tests for the SOD1-regulated genes in knockdown HeLa cells (TUSC2, CRTC3, KCNAB2, SPEG, QRFP, MAF1, DNM2, NFIC, PTK6, TNFRSF25 and FGFR4 in (I); INA, UNC13A and NRG1 in (J)). Data are mean of triplicate samples ± SD (*P < 0.05, **P < 0.01, ***P < 0.001; unpaired Student's t test), and all error bars are SD.

Fig 3: Binding model of DNA–SOD1 complexes. (A) The SL bases directly involved in the DNA–SOD1 interactions fixed by 1% formaldehyde cross-linking were determined by capillary electropherograms following DNase I digestion of 5'-FAM-labelled pentameric SL. Stars represent the sites protected by SOD1. (C) Capillary electropherograms were generated from the SL-SOD1 complexes treated without 1% formaldehyde following DNase I digestion of 5'-FAM-labelled SL. (B and D) Cartoon representation of SL highlights the SOD1 binding DNA sites resulted from DNase I footprinting tests in (A) and (C). (E) The structural docking model for S1-DNA complex. The purple regions in DNA represent the potential SOD1 binding sites in S1 motif. a-helix, red; ß-sheet, yellow; random coil and DNA, green; SOD1 binding sites, purple. (F) A final concentration of 100 µM SOD1 and 100 µM of S1 was incubated in 20 mM Tris–HCl (pH 7.4) at 37°C for 24 h. Before data collection, the sample was centrifuged at 10,000 rpm at 4°C for 10 min to remove the potential aggregates. Based on SAXS results, space-filling models of the S1-SOD1 complexes were constructed. Space-filling models of the S1-SOD1 complexes derived from SAXS data are depicted in grey, with HADDOCK model of this complexes docked into mesh envelope.

Fig 4: SOD1 binds to DNA with different sequence preferences under varied redox conditions. (A) Person correlations between parallel ChIP-Seq samples in control (designed as C1, C2 and C3), H2O2 (H1, H2 and H3), and LD100 (L1, L2 and L3) treated groups. Unless otherwise specified, the labels for all ChIP-Seq samples were the same as here. All the ChIP-Seq data were representative of three independent experiments. (B) Typical SOD1 binding motifs under varied redox conditions. (C) Motif logo matching between motif A (bottom) and four consensus motifs (top), determined by TOMTOM (MEME). (D and E) Hierarchical clustering analyses of fold enrichment values in control, H2O2 and LD100 treated groups. (F–H) Enriched GO terms of all ChIP-Seq samples in control (F), H2O2 (G), and LD100 treated groups (H).

Fig 5: SOD1 binding to DNA depends on redox environments and its conformation. (A and B) The treatment with H2O2 for 4 h reduced not only the activity of SOD1 (A) but also the formation of S1-SOD1 complexes (B). (C and D) The treatment with GSH for 4 h slightly reduced the activity of SOD1 (C) but significantly elevated the formation of S1-SOD1 complexes (D). (E and H) The specific inhibitor of SOD1 (LD100, E) and the chelator of Zn2+ (TPEN, H) both prevented SOD1 from interactions with DNA under tested conditions. (F and G) The formation of S1-A4V (F) and S1-H46R/H48Q (G) complexes were dependent on concentrations of SOD1 in 10 mM pH 7.4 PBS. Data are mean of triplicate samples ± SD (*P < 0.05, **P < 0.01, ***P < 0.001; unpaired Student's t test), and all error bars are SD.