Fig 2: Carboxy-terminal EVI1 phosphorylation. Mass spectrometry analysis of the EVI1 peptide Asn849-Arg862 from SB1690CB AML cells, demonstrating the presence of non-phosphorylated (A) and single Ser860 phosphorylated peptides (B) in untreated SB1690CB cells. y- and b-type ions illustrating their position within the peptide sequence indicated. The mass/charge ratio (m/z) of the precursor ion is also shown. (C) Schematic representation of the EVI1 protein with sequence alignment of EVI1 from other species as indicated, showing DNA binding zinc finger domains (ZF), CtBP1 binding motifs (CtBP) and acidic domain (AC). Carboxy-terminal phosphorylation sites are shown in red circles and annotated in relation to the EVI1-SQS containing epitope alignment. (D) Confirmation of induction of DNA damage by γH2AX foci formation after H2O2 or radiation treatment of SB1690CB cells (left panel). White arrowheads denote cells with increased number of foci (green signal). DAPI co-stain for the nucleus (blue signal). Western blot (right panel) of γH2AX and p-p53 (Ser15) as markers of DNA damage response activation and ATM activity after H2O2-treatment of SB1690CB cells. (E) EVI1 MS spectrum obtained for peptide Asn849-Arg862 from irradiated SB1690CB cells, inferring double phosphorylation of the carboxy-terminal S858/S860 SQS motif.

Fig 3: Effect of EVI1-WT and EVI1-AQA on transcription. (A) Luciferase assays on promoters of EVI1-repressed PLZF (left panel) and FOS (right panel) transcripts (n = 4, t-test, ns: not significant). (B) Heat map illustration of significantly differentially expressed transcripts (P < 0.01) after transfection with EVI1-WT and EVI1-AQA of untreated cells (left half of the heatmap), and after treatment with H2O2 (150 μM, right half of the heat map, indicated by black line) (n = 1306). The heatmap was generated applying a group ANOVA analysis to the entire dataset. Clusters of EVI1 regulated transcripts were identified by dendrogram delineation, and boxed for repressed (1A and 1B), and upregulated transcripts (2A and 2B). Patterns of sub-clusters in H2O2 treated cells with different changes comparing EVI-WT with EVI1-AQA indicated by arrow in 1B and dashed box in 2B.

Fig 4: Nuclear association of EVI1 with CtBP1. (A) EVI1 (green) and CtBP1 (red) immunofluorescence signal of HEK293T cells transfected with EVI1-WT (upper panel) and EVI1-AQA mutant (middle panel), or SB1690CB AML cells (lower panel) after genotoxic stress induced either with H2O2 treatment or irradiation (2 Gy). Left top corner inserts represent 4 μm2 regions of interests (ROI) in HEK293T cells or 3 μm2 ROIs in SB1690CB cells (eight ROIs/cell analyzed). (B) Quantification of EVI1 and CtBP1 co-localization in EVI1-WT or EVI1-AQA transfected HEK293T cells after genotoxic stress. Degree of co-localization is expressed as Pearson's Coefficient (P’sC). Each bar represents the average P’sC of independent experiments (n = 3) for H2O2 (left panel) and irradiated cells (right panel). (C) Distribution of P’sC for endogenously expressed EVI1 and CtBP1 co-localization in SB1690CB leukemia cells after H2O2 treatment or irradiation. Each dot represents a single cell (left panel). Average P’sC of independent experiments (n = 3) plotted as bar graphs (right panel). Statistical analysis: one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant).

Fig 5: Induction of EVI1 double phosphorylation by DNA damage. (A) Immunoprecipitated EVI1 from SB1690CB cells probed with anti-pS860-EVI1 (upper panel), and anti-pS858/pS860-EVI1 antibody, untreated (lane 1), and after 1 and 2 h (lane 2 and 3) post radiation. (B) Western blot (WB) of EVI1-immunoprecipitates from untreated (lanes 1 and 2) and irradiated (lanes 3 and 4) SB1690CB cells pre-treated (1 h) with 10 μM ATM-kinase inhibitor KU55933 (lanes 2 and 4), and not pre-treated (lanes 1 and 3). Membranes were probed for doubly phosphorylated EVI1 with anti-pS858/pS860-EVI1 antibody. Middle panel: membrane as upper panel re-probed with pan-EVI1 antibody. Lower panel: WB of p-p53 (Ser15) carried out on input samples as positive control for ATM-activity and ATM-inhibition.