Figure 1.
Methodologies and genome distribution of the sequences.
(A) Scheme of the Methodologies followed: Streptavidin chromatin precipitations and ChIPs followed by streptavidin precipitations were performed in Ba/F3 cells expressing BirA or BirA/bioSTAT5a (left). Single and double ChIPs with anti-STAT5a antibody were performed in Ba/F3 cells (right). All cells were deprived of IL-3 for 6 h and subsequently stimulated with IL-3 for 30 min. (B) Genome distribution of the sequences: Genome distribution of the sequences from the ChIP followed by streptavidin precipitation library is shown on the left and from the double ChIP library on the right. The pie charts show the genome distribution of the sequences relative to the nearest neighbor gene, located within <3 kb and 3–5 kb relative to TSS (transcription start site), introns, exons and intergenic regions.
Figure 2.
The novel STAT5 target gene Dpf3: Expression levels (in wild type and knock-down cells) and STAT5a, STAT5b binding.
(A) Expression levels of Dpf3: Expression levels (mRNA) were measured by real time PCR in Ba/F3 cells deprived of IL-3 for 6 h (starved) and stimulated with IL-3 for 30 min and 3 h. The deprived of IL-3 cells were set as 1. Bars demonstrate mean and standard deviation (SD) values obtained at least in three independent experiments. The statistical significance of the difference in expression between 30 min stimulated and deprived of IL-3 cells is indicated with asterisks (**P = 0.005). (B) STAT5a and STAT5b binding to Dpf3: Cross-linked chromatin from Ba/F3 cells deprived of IL-3 for 6 h and stimulated with IL-3 for 30 min was used in ChIPs with anti-STAT5a or anti-STAT5b antibody. Three sets of primers were used for each gene. One set specific for the amplification of the isolated genomic region/cloned sequence (seq) containing at least one TTCN3/4GAA motif, one set specific for the promoter of each gene (pr) containing at least one TTCN3GAA motif and one set of negative control primers (neg) for amplification of a region lacking TTCN3/4GAA motifs. IgG was utilized in parallel with anti-STAT5a or anti-STAT5b antibodies, as control. Bars demonstrate mean and SD values of specific enrichments (fold differences) versus input obtained at least in three independent experiments. The statistical significance of enrichment versus the negative control region is indicated with asterisks (ns: not significant, *P = 0.01, **P = 0.002, ***P<0.001). (C) Expression levels of Dpf3 in cells with knock-down of STAT5a, STAT5b or both: Expression levels (mRNA) of Dpf3 were measured in Ba/F3 cells with knock-down of STAT5a, STAT5b or both. The cells were deprived of IL-3 for 6 h (starved) and stimulated with IL-3 for 30 min. Expression levels were measured by real time PCR and compared with Ba/F3 cells in the deprived of IL-3 state, transduced with scrambled shRNA and set as 1. Bars demonstrate mean and SD values obtained at least in three independent experiments. The statistical significance of the difference in expression between knock-downs and the respective scrambled control is indicated with asterisks (ns: not significant, *P = 0.02, **P = 0.005, ***P<0.001).
Figure 3.
Expression levels of DPF3 in hematologic malignancies.
DPF3 expression levels (mRNA) in patients with hematologic malignancies [AML (PB and/or BM), ALL (PB and/or BM), CML (PB and/or BM), MPN-JAK2-positive (+) (PB) and CLL (PB)] are shown and compared with the expression in PB of healthy controls. The boxes represent the interquartile range that contains the 50% of values. The whiskers are lines that extend from the box to the highest and lowest values, excluding outliers. A line across the box indicates the median value for each patient cohort. In this graph outliers and extremes have been omitted. P-values versus healthy controls were calculated by the Mann-Whitney U test and are shown in Table 1 (ns: not significant, *0.01≤P<0.05, **P = 0.009).
Table 1.
Expression of DPF3 gene in patients with hematologic malignancies.
Figure 4.
Detection of activated STAT5 (p-STAT5), DPF3 staining and mRNA levels in CLL.
(A) Flow cytometry analysis:Indicative plots of peripheral blood cells analysis are shown (healthy individual with low DPF3 expression levels (3.6) in upper panels and CLL patient with high DPF3 expression levels (19.69) in lower panels). Dot plots on the left indicate percentages of gated cells used on the analysis (A: lymphocytes in pink, B: monocytes in green and C: granulocytes in red). Histograms on the right indicate p-STAT5 levels in lymphocytes (pink), monocytes (green) and granulocytes (red). Isotype control (IgG1) is depicted in grey. (B) Levels of p-STAT5 in the peripheral blood cell subpopulations analyzed by flow cytometry: The boxes represent the interquartile range containing 50% of values in lymphocytes, monocytes and granulocytes of healthy individuals (n = 4) and CLL patients (n = 10). The whiskers are lines that extend from the box to the highest and lowest values, excluding outliers. A line across the box indicates the median value for each group. The statistical significance of difference is noted on the charts. (C) DPF3 expression levels (mRNA) in granulocytes and monocytes: DPF3 expression levels were measured in granulocytes and monocytes of CLL patients (n = 5) and healthy individuals (n = 4) by real time PCR. Bars demonstrate mean and standard error of the mean (SEM) values of expression. The statistical significance of the differences in expression is indicated with an asterisk (*0.01≤P<0.05). (D) Immunofluorescence detection of nuclear p-STAT5: Granulocytes with low to high intensity nuclear p-STAT5 staining were scored as positive for the nucleus. Granulocytes with absent nuclear staining and low to high intensity cytoplasmic staining were considered positive for the cytoplasm (***P<0.001). (E) Immunofluorescence detection of nuclear DPF3: Scoring of nuclear DPF3 staining (detected as puncta in the nucleus) was performed on granulocytes positive for p-STAT5 staining in the cytoplasm and/or the nucleus. Granulocytes with low to high intensity puncta in the nucleus were considered positive (***P<0.001). (F) Immunofluorescence detection of both nuclear p-STAT5 and DPF3: For co-localization of DPF3 (red) and p-STAT5 (green) nuclear staining, granulocytes with green and red, and/or yellow staining puncta (representing p-STAT5 and DPF3 co-localization) were scored (*P = 0.05). In D, E and F: Scoring was performed in granulocytes showing p-STAT5 staining in the nucleus, the cytoplasm or both; bars demonstrate mean and SD values obtained at least in three different slides or regions of the same slide; the statistical significance of the difference between samples with low (healthy individuals) and high (CLL patients) DPF3 expression is shown; DPF3 expression levels (mRNA) are shown below the samples; ND: not determined.
Figure 5.
STAT5 binding to human DPF3 promoter.
(A) STAT5a binding to human DPF3 promoter in granulocytes: Cross-linked chromatin from granulocytes of a CLL patient and a healthy individual was used in ChIPs with anti-STAT5a antibody. (B) STAT5a binding to human DPF3 promoter in CLL cell lines: Cross-linked chromatin from EHEB and JVM-2 cells was used in ChIPs with anti-STAT5a antibody. In A and B, IgG was utilized in parallel with anti-STAT5a antibody, as control. Two sets of primers were used: One set specific for the amplification of the DPF3 promoter containing a STAT5 motif and one set of negative control primers (neg) for amplification of a region lacking STAT5 motifs. Bars represent mean and SD values of specific enrichments (fold differences) versus input obtained at least in three independent experiments. The statistical significance of enrichment for promoter sequences versus the negative control region (*0.01≤P<0.03) and for patient versus healthy individual (**P = 0.007) is indicated; a-STAT5a: anti-STAT5a.