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DAZAP2 acts as specifier of the p53 response to DNA damage

Liebl, Magdalena C, Moehlenbrink, Jutta, Becker, Huong, Raddatz, Günter, Abdeen, Suhaib K, Aqeilan, Rami I, Lyko, Frank, Hofmann, Thomas G·2021·Nucleic Acids Research
DOI 10.1093/nar/gkab084PMC PMC7969023
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Abstract

The DNA damage-responsive tumor suppressors p53 and HIPK2 are well established regulators of cell fate decision-making and regulate the cellular sensitivity to DNA-damaging drugs. Here, we identify Deleted in Azoospermia-associated protein 2 (DAZAP2), a small adaptor protein, as a novel regulator of HIPK2 and specifier of the DNA damage-induced p53 response. Knock-down or genetic deletion of DAZAP2 strongly potentiates cancer cell chemosensitivity both in cells and in vivo using a mouse tumour xenograft model. In unstressed cells, DAZAP2 stimulates HIPK2 polyubiquitination and degradation through interplay with the ubiquitin ligase SIAH1. Upon DNA damage, HIPK2 site-specifically phosphorylates DAZAP2, which terminates its HIPK2-degrading function and triggers its re-localization to the cell nucleus. Interestingly, nuclear DAZAP2 interacts with p53 and specifies target gene expression through modulating a defined subset of p53 target genes. Furthermore, our results suggest that DAZAP2 co-occupies p53 response elements to specify target gene expression. Collectively, our findings propose DAZAP2 as novel regulator of the DNA damage-induced p53 response that controls cancer cell chemosensitivity.

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Figures

Figure 1. DAZAP2 binds to HIPK2 in vitro and in cells. (A) Schematic representation of the domain structure of HIPK2 and the HIPK2 truncation used as bait in the yeast two-hybrid screening that identified DAZAP2 as a binding partner of HIPK2. AID = autoinhibitory domain. (B) DAZAP2 associates with HIPK2 in vitro. GST pulldown assays were performed with bacterially expressed GST or GST-DAZAP2 and 35S-labeled HIPK2K221A and analyzed by SDS-PAGE, Coomassie Brilliant Blue (CBB) staining and autoradiography. n = 3. (C) Interaction of ectopically expressed DAZAP2 and HIPK2. FLAG-HIPK2 and Myc-DAZAP2 were expressed in H1299 cells as indicated in the presence of 20 μM MG-132. FLAG-HIPK2 was precipitated from the lysates and binding of Myc-DAZAP2 to FLAG-HIPK2 was analyzed by immunoblotting. n = 2. (D) Interaction of endogenous DAZAP2 and HIPK2. Lysates from HCT116 cells treated with MG-132 were subjected to immunoprecipitation with control or HIPK2 antibodies and analyzed by immunoblotting (n = 2). A non-specific band is marked with an asterisk. IgGL = Immunoglobulin G Light Chain. (E) DAZAP2 and HIPK2 colocalize. Co-localization of EGFP-DAZAP2 and mCherry-HIPK2 in U2OS cells was assessed by confocal microscopy (n = 2). DNA is stained by Hoechst (blue). Two representative cells showing nuclear (upper panel) or cytoplasmic (lower panel) HIPK2 and DAZAP2 expression are shown.
Figure 1. DAZAP2 binds to HIPK2 in vitro and in cells. (A) Schematic representation of the domain structure of HIPK2 and the HIPK2 truncation used as bait in the yeast two-hybrid screening that identified DAZAP2 as a binding partner of HIPK2. AID = autoinhibitory domain. (B) DAZAP2 associates with HIPK2 in vitro. GST pulldown assays were performed with bacterially expressed GST or GST-DAZAP2 and 35S-labeled HIPK2K221A and analyzed by SDS-PAGE, Coomassie Brilliant Blue (CBB) staining and autoradiography. n = 3. (C) Interaction of ectopically expressed DAZAP2 and HIPK2. FLAG-HIPK2 and Myc-DAZAP2 were expressed in H1299 cells as indicated in the presence of 20 μM MG-132. FLAG-HIPK2 was precipitated from the lysates and binding of Myc-DAZAP2 to FLAG-HIPK2 was analyzed by immunoblotting. n = 2. (D) Interaction of endogenous DAZAP2 and HIPK2. Lysates from HCT116 cells treated with MG-132 were subjected to immunoprecipitation with control or HIPK2 antibodies and analyzed by immunoblotting (n = 2). A non-specific band is marked with an asterisk. IgGL = Immunoglobulin G Light Chain. (E) DAZAP2 and HIPK2 colocalize. Co-localization of EGFP-DAZAP2 and mCherry-HIPK2 in U2OS cells was assessed by confocal microscopy (n = 2). DNA is stained by Hoechst (blue). Two representative cells showing nuclear (upper panel) or cytoplasmic (lower panel) HIPK2 and DAZAP2 expression are shown.CC BY-NC · PubMed Central
Figure 2. DAZAP2 enhances HIPK2 poly-ubiquitination and degradation through the ubiquitin ligase SIAH1. (A) DAZAP2 depletion increases HIPK2 protein levels. HepG2 cells were transduced with vectors encoding control (Luciferase) or DAZAP2 shRNAs. Total cell lysates were analyzed by immunoblotting using the indicated antibodies. A non-specific band is marked with an asterisk. n = 3. (B) Ectopic expression of DAZAP2 decreases HIPK2 levels. H1299 cells were transfected with the indicated expression vectors and total protein lysates were analyzed by immunoblotting. n = 2. (C) DAZAP2 potentiates proteasome-dependent HIPK2 degradation. H1299 cells were transfected with the indicated expression vectors and exposed to MG-132 or z-VAD-FMK as indicated. Total protein lysates were analyzed by immunoblotting. n = 3. (D) SIAH1 is essential for DAZAP2-mediated HIPK2 degradation. H1299 cells were transduced with the indicated shRNAs and transfected with the indicated expression vectors. Total protein lysates were analyzed by Immunoblotting. Depletion of SIAH1 was investigated by RT-PCR. n = 2. (E) DAZAP2 interacts with SIAH1. The proteins indicated were expressed in H1299 cells in the presence of MG-132. FLAG-SIAH1 was precipitated from the lysates and immunoprecipitates were analyzed by immunoblotting. n = 2. (F) DAZAP2 interacts with SIAH1 in vitro. Bacterially expressed GST or GST-SIAH1 were incubated with in vitro translated 35S-labeled DAZAP2. GST pulldown assays were performed and analyzed by SDS-PAGE, Coomassie Brilliant Blue (CBB) staining and autoradiography. n = 3. (G) DAZAP2 potentiates HIPK2-SIAH1 complex formation. The indicated proteins were expressed in H1299 cells in the presence of MG-132. FLAG-HIPK2 was precipitated from the lysates and immunoprecipitates were analyzed by immunoblotting. n = 3. (H) DAZAP2 stimulates SIAH1-dependent HIPK2 ubiquitination. Recombinant proteins were expressed in E. coli and purified by affinity chromatography. The isolated proteins were incubated with UBE1 (= E1 ubiquitin-activating enzyme), UBE2D2 ( = E2 ubiquitin-conjugating enzyme) and HA-Ubiquitin and in vitro ubiquitination assays were performed. Subsequently, His-HIPK2 was purified from the reactions using Ni-NTA-beads followed by immunoblotting with a Ubiquitin- and HIPK2-specific antibody. Total levels of GST-DAZAP2 and GST-SIAH1 were analyzed by Ponceau S staining and immunoblotting, respectively. n = 3. Representative experiments are shown.
Figure 2. DAZAP2 enhances HIPK2 poly-ubiquitination and degradation through the ubiquitin ligase SIAH1. (A) DAZAP2 depletion increases HIPK2 protein levels. HepG2 cells were transduced with vectors encoding control (Luciferase) or DAZAP2 shRNAs. Total cell lysates were analyzed by immunoblotting using the indicated antibodies. A non-specific band is marked with an asterisk. n = 3. (B) Ectopic expression of DAZAP2 decreases HIPK2 levels. H1299 cells were transfected with the indicated expression vectors and total protein lysates were analyzed by immunoblotting. n = 2. (C) DAZAP2 potentiates proteasome-dependent HIPK2 degradation. H1299 cells were transfected with the indicated expression vectors and exposed to MG-132 or z-VAD-FMK as indicated. Total protein lysates were analyzed by immunoblotting. n = 3. (D) SIAH1 is essential for DAZAP2-mediated HIPK2 degradation. H1299 cells were transduced with the indicated shRNAs and transfected with the indicated expression vectors. Total protein lysates were analyzed by Immunoblotting. Depletion of SIAH1 was investigated by RT-PCR. n = 2. (E) DAZAP2 interacts with SIAH1. The proteins indicated were expressed in H1299 cells in the presence of MG-132. FLAG-SIAH1 was precipitated from the lysates and immunoprecipitates were analyzed by immunoblotting. n = 2. (F) DAZAP2 interacts with SIAH1 in vitro. Bacterially expressed GST or GST-SIAH1 were incubated with in vitro translated 35S-labeled DAZAP2. GST pulldown assays were performed and analyzed by SDS-PAGE, Coomassie Brilliant Blue (CBB) staining and autoradiography. n = 3. (G) DAZAP2 potentiates HIPK2-SIAH1 complex formation. The indicated proteins were expressed in H1299 cells in the presence of MG-132. FLAG-HIPK2 was precipitated from the lysates and immunoprecipitates were analyzed by immunoblotting. n = 3. (H) DAZAP2 stimulates SIAH1-dependent HIPK2 ubiquitination. Recombinant proteins were expressed in E. coli and purified by affinity chromatography. The isolated proteins were incubated with UBE1 (= E1 ubiquitin-activating enzyme), UBE2D2 ( = E2 ubiquitin-conjugating enzyme) and HA-Ubiquitin and in vitro ubiquitination assays were performed. Subsequently, His-HIPK2 was purified from the reactions using Ni-NTA-beads followed by immunoblotting with a Ubiquitin- and HIPK2-specific antibody. Total levels of GST-DAZAP2 and GST-SIAH1 were analyzed by Ponceau S staining and immunoblotting, respectively. n = 3. Representative experiments are shown.CC BY-NC · PubMed Central
Figure 3. Loss of DAZAP2 sensitizes cancer cells to chemotherapeutic drug treatment. (A, B) DAZAP2 depletion sensitizes HCT116 cells to 5-FU treatment. HCT116 cells were transfected with DAZAP2-specific or control siRNAs and DNA damage was induced using 50 μM 5-FU. (A) Cells were harvested 24 h post-treatment and lysates were analyzed by immunoblotting. n = 3. (B) Cells were harvested 48 h post-treatment and co-stained with Annexin V and PI. Stained cells were analyzed by flow cytometry. Bar graph showing quantification of % Annexin V-positive cells + SD from three biological replicates. ** P-value ≤ 0.01, ns non-significant (un-paired t-test). n = 3. (C) DAZAP2 depletion sensitizes HCT116 cells to Adriamycin and Oxaliplatin. HCT116 cells were transfected with DAZAP2-specific or control siRNAs and DNA damage was induced for 24 h using 0.75 μg/ml Adriamycin or 50 μM Oxaliplatin. Cell lysates were analyzed by immunoblotting. n = 3. (D) DAZAP2 deletion chemosensitizes cells. HCT116 control CRISPR/Cas9 and DAZAP2 knockout CRISPR/Cas9 clonal cell lines were treated with 50 μM 5-FU for 24 h and total protein lysates were subjected to immunoblot analysis for the expression of the indicated proteins. n = 3. (E, F) DAZAP2 deletion increases Caspase-3/7 activity upon DNA damage. HCT116 CRISPR/Cas9 control and CRISPR/Cas9 DAZAP2 knockout clonal cell lines were treated with 50 μM 5-FU and Caspase-3/7 activity was assessed 24 h post-treatment by fluorescence microscopy. n = 3. (E) Representative image of cells with active Caspase-3/7 (green) are shown. (F) Bar graph represents mean and SD of % NucView-positive, green cells from three images. (G) DAZAP2 deletion sensitizes tumors to chemotherapeutic drug treatment in vivo. CRISPR/Cas9 DAZAP2 KO or control CRISPR/Cas9 HCT116 cells were subcutaneously injected into NOD/SCID mice. When the tumor size reached 100 mm3, mice were treated with DMSO or 50 mg/kg 5-FU for 21 days. Analysis of weight of tumor size after 21 days of treatment is shown. Data are displayed as sample mean ± standard error of the mean. * P-value ≤0.05, ns non-significant (unpaired t-test).
Figure 3. Loss of DAZAP2 sensitizes cancer cells to chemotherapeutic drug treatment. (A, B) DAZAP2 depletion sensitizes HCT116 cells to 5-FU treatment. HCT116 cells were transfected with DAZAP2-specific or control siRNAs and DNA damage was induced using 50 μM 5-FU. (A) Cells were harvested 24 h post-treatment and lysates were analyzed by immunoblotting. n = 3. (B) Cells were harvested 48 h post-treatment and co-stained with Annexin V and PI. Stained cells were analyzed by flow cytometry. Bar graph showing quantification of % Annexin V-positive cells + SD from three biological replicates. ** P-value ≤ 0.01, ns non-significant (un-paired t-test). n = 3. (C) DAZAP2 depletion sensitizes HCT116 cells to Adriamycin and Oxaliplatin. HCT116 cells were transfected with DAZAP2-specific or control siRNAs and DNA damage was induced for 24 h using 0.75 μg/ml Adriamycin or 50 μM Oxaliplatin. Cell lysates were analyzed by immunoblotting. n = 3. (D) DAZAP2 deletion chemosensitizes cells. HCT116 control CRISPR/Cas9 and DAZAP2 knockout CRISPR/Cas9 clonal cell lines were treated with 50 μM 5-FU for 24 h and total protein lysates were subjected to immunoblot analysis for the expression of the indicated proteins. n = 3. (E, F) DAZAP2 deletion increases Caspase-3/7 activity upon DNA damage. HCT116 CRISPR/Cas9 control and CRISPR/Cas9 DAZAP2 knockout clonal cell lines were treated with 50 μM 5-FU and Caspase-3/7 activity was assessed 24 h post-treatment by fluorescence microscopy. n = 3. (E) Representative image of cells with active Caspase-3/7 (green) are shown. (F) Bar graph represents mean and SD of % NucView-positive, green cells from three images. (G) DAZAP2 deletion sensitizes tumors to chemotherapeutic drug treatment in vivo. CRISPR/Cas9 DAZAP2 KO or control CRISPR/Cas9 HCT116 cells were subcutaneously injected into NOD/SCID mice. When the tumor size reached 100 mm3, mice were treated with DMSO or 50 mg/kg 5-FU for 21 days. Analysis of weight of tumor size after 21 days of treatment is shown. Data are displayed as sample mean ± standard error of the mean. * P-value ≤0.05, ns non-significant (unpaired t-test).CC BY-NC · PubMed Central
Figure 4. DAZAP2 interacts with p53. (A) A fraction of DAZAP2 accumulates in the nucleus upon DNA damage. HCT116 cells were treated with DMSO or 50 μM 5-FU for 24 h. Cells were fractionated into nuclear and cytoplasmic fractions, and equal total protein amounts of all fractions were analyzed by immunoblotting. WCL = whole cell lysate. Cyto = Cytoplasm. n = 3. (B) Interaction of endogenous DAZAP2 and p53. Lysates from untreated or Adriamycin-treated HepG2 cells in the presence of MG-132 were subjected to immunoprecipitation with control or p53 antibodies. Lysates and immunoprecipitates were analyzed by immunoblotting. n = 2. (C, D) In vitro interaction of DAZAP2 and p53. (C) GST pull-down assays were performed using in vitro translated 35S-labeled p53 and bacterially expressed GST-DAZAP2 or GST as a control. n = 3. (D) GST pull-down assays were performed using in vitro translated 35S-labeled DAZAP2 and bacterially expressed GST-p53 or GST as a control. n = 3. GST pulldown assays were analyzed by SDS-PAGE, Coomassie Brilliant Blue (CBB) staining and autoradiography. Representative experiments are shown.
Figure 4. DAZAP2 interacts with p53. (A) A fraction of DAZAP2 accumulates in the nucleus upon DNA damage. HCT116 cells were treated with DMSO or 50 μM 5-FU for 24 h. Cells were fractionated into nuclear and cytoplasmic fractions, and equal total protein amounts of all fractions were analyzed by immunoblotting. WCL = whole cell lysate. Cyto = Cytoplasm. n = 3. (B) Interaction of endogenous DAZAP2 and p53. Lysates from untreated or Adriamycin-treated HepG2 cells in the presence of MG-132 were subjected to immunoprecipitation with control or p53 antibodies. Lysates and immunoprecipitates were analyzed by immunoblotting. n = 2. (C, D) In vitro interaction of DAZAP2 and p53. (C) GST pull-down assays were performed using in vitro translated 35S-labeled p53 and bacterially expressed GST-DAZAP2 or GST as a control. n = 3. (D) GST pull-down assays were performed using in vitro translated 35S-labeled DAZAP2 and bacterially expressed GST-p53 or GST as a control. n = 3. GST pulldown assays were analyzed by SDS-PAGE, Coomassie Brilliant Blue (CBB) staining and autoradiography. Representative experiments are shown.CC BY-NC · PubMed Central
Figure 5. Phosphorylation of DAZAP2 by HIPK2 regulates DAZAP2-mediated HIPK2 degradation and DAZAP2 localization. (A) FLAG-HIPK2 and kinase-dead FLAG-HIPK2 K221A were purified from 293T cells, recombinant GST-DAZAP2 was purified from E. coli. In vitro kinase assays were performed and DAZAP2 phosphorylation as well as HIPK2 autophosphorylation were examined by autoradiography. Protein levels were analyzed by Coomassie Brilliant Blue (CBB) staining. n = 2. (B) HIPK2 phosphorylates DAZAP2 on five Ser/Thr residues. In vitro kinase assays were performed using bacterially expressed His-HIPK2 and GST-DAZAP2 mutants and analyzed by SDS–PAGE and autoradiography. Protein levels were analyzed by CBB staining. n = 3. (C) Validation of the DAZAP2 phospho-Ser77 antibody. In vitro kinase assays were performed with His-HIPK2 and GST-DAZAP2. Non-phosphorylated GST-DAZAP2 and in vitro phosphorylated GST-DAZAP2 phosphorylation were examined by SDS–PAGE and immunoblotting. n = 3. (D) DAZAP2 is phosphorylated at Ser77 upon DNA damage in cells. Proteins were expressed in H1299 cells as indicated in the presence of the indicated compounds. Myc-DAZAP2 was precipitated from the lysates and analyzed by immunoblotting as indicated. n = 2. (E) Effect of DAZAP2 phosphorylation on its HIPK2-degrading capacity. H1299 cells were transfected with the indicated expression vectors and total protein lysates were analyzed by immunoblotting. n = 3. (F, G) DAZAP2 phosphorylation regulates its localization. The indicated EGFP-DAZAP2 fusion proteins were expressed in U2OS cells and their subcellular localization was assessed by confocal microscopy. DNA is stained by Hoechst (blue). n = 3. DAZAP23A: S77A, S85A, T86A; DAZAP25A: S62A, S69A, S77A, S85A, T86A; DAZAP25D: S62D, S69D, S77D, S85D, T86D. Representative experiments are shown.
Figure 5. Phosphorylation of DAZAP2 by HIPK2 regulates DAZAP2-mediated HIPK2 degradation and DAZAP2 localization. (A) FLAG-HIPK2 and kinase-dead FLAG-HIPK2 K221A were purified from 293T cells, recombinant GST-DAZAP2 was purified from E. coli. In vitro kinase assays were performed and DAZAP2 phosphorylation as well as HIPK2 autophosphorylation were examined by autoradiography. Protein levels were analyzed by Coomassie Brilliant Blue (CBB) staining. n = 2. (B) HIPK2 phosphorylates DAZAP2 on five Ser/Thr residues. In vitro kinase assays were performed using bacterially expressed His-HIPK2 and GST-DAZAP2 mutants and analyzed by SDS–PAGE and autoradiography. Protein levels were analyzed by CBB staining. n = 3. (C) Validation of the DAZAP2 phospho-Ser77 antibody. In vitro kinase assays were performed with His-HIPK2 and GST-DAZAP2. Non-phosphorylated GST-DAZAP2 and in vitro phosphorylated GST-DAZAP2 phosphorylation were examined by SDS–PAGE and immunoblotting. n = 3. (D) DAZAP2 is phosphorylated at Ser77 upon DNA damage in cells. Proteins were expressed in H1299 cells as indicated in the presence of the indicated compounds. Myc-DAZAP2 was precipitated from the lysates and analyzed by immunoblotting as indicated. n = 2. (E) Effect of DAZAP2 phosphorylation on its HIPK2-degrading capacity. H1299 cells were transfected with the indicated expression vectors and total protein lysates were analyzed by immunoblotting. n = 3. (F, G) DAZAP2 phosphorylation regulates its localization. The indicated EGFP-DAZAP2 fusion proteins were expressed in U2OS cells and their subcellular localization was assessed by confocal microscopy. DNA is stained by Hoechst (blue). n = 3. DAZAP23A: S77A, S85A, T86A; DAZAP25A: S62A, S69A, S77A, S85A, T86A; DAZAP25D: S62D, S69D, S77D, S85D, T86D. Representative experiments are shown.CC BY-NC · PubMed Central
Figure 6. DAZAP2 regulates a subset of p53 target genes and restricts cell death in a p53-mediated manner. (A) Results of RNA-sequencing analyses are visualized by a Volcano plot showing differentially expressed genes between DAZAP2-depleted and control HCT116 cells upon 5-FU treatment. Each dot represents one gene. Red dots indicate genes with higher expression in DAZAP2-depleted cells and a q-value ≤ 0.05, while blue dots represent genes with lower expression in DAZAP2-depleted cells and a q-value ≤ 0.05. Grey dots show the genes with an q-value ≥ 0.05. Highlighted in green is DAZAP2 expression. (B, C) Ingenuity pathway analysis of genes differentially expressed in RNA-seq data from DAZAP2-depleted versus control HCT116 cells upon 5-FU treatment. (B) Top predicted biological functions are ranked by -LOG(P-value). (C) Top predicted upstream regulators are ranked by -LOG(P-value). (D) Identification of DAZAP2-regulated p53 target genes. Venn diagram displaying the overlap between DAZAP2-regulated genes upon 5-FU treatment and p53 target genes(52). (E) Heatmap of the p53 target genes regulated by DAZAP2 upon genotoxic stress. RNA-Seq analysis was performed as described in (A). Relative expression of DAZAP2 is shown in the last row of the heatmap. R1–3 indicate the three biological replicates. (F) DAZAP2 depletion enhances the expression of a subset of cell death-associated p53 target genes upon 5-FU exposure. HCT116 or HCT116 p53−/− were transfected with DAZAP2-specific or control siRNAs and DNA damage was induced using 50 μM 5-FU. mRNA expression was analyzed by qRT-PCR 48 h post-treatment for the indicated genes. Gene expression was normalized to GAPDH/B2M expression and the fold change relative to the untreated cells was calculated. Data are shown as mean + SD from three replicates. *** P-value ≤0.001 (two-way ANOVA for left panel, un-paired t-test for right panel). (G, H) DAZAP2 depletion sensitizes HCT116 cells to cell death in a p53-regulated manner. HCT116 wt and p53−/− cells were transfected with DAZAP2-specific or control siRNAs and DNA damage was induced using 50 μM 5-FU. n = 3. (G) Total protein extracts were prepared 24 h post-treatment and analyzed by immunoblotting for the indicated proteins. A representative experiment is shown. (H) Cells were harvested 48 h post-treatment and co-stained with Annexin V and PI. Stained cells were analyzed by flow cytometry. Bar graph showing quantification of % Annexin V-positive cells + SD from three biological replicates. ** P-value ≤0.01, ns non-significant (un-paired t-test).
Figure 6. DAZAP2 regulates a subset of p53 target genes and restricts cell death in a p53-mediated manner. (A) Results of RNA-sequencing analyses are visualized by a Volcano plot showing differentially expressed genes between DAZAP2-depleted and control HCT116 cells upon 5-FU treatment. Each dot represents one gene. Red dots indicate genes with higher expression in DAZAP2-depleted cells and a q-value ≤ 0.05, while blue dots represent genes with lower expression in DAZAP2-depleted cells and a q-value ≤ 0.05. Grey dots show the genes with an q-value ≥ 0.05. Highlighted in green is DAZAP2 expression. (B, C) Ingenuity pathway analysis of genes differentially expressed in RNA-seq data from DAZAP2-depleted versus control HCT116 cells upon 5-FU treatment. (B) Top predicted biological functions are ranked by -LOG(P-value). (C) Top predicted upstream regulators are ranked by -LOG(P-value). (D) Identification of DAZAP2-regulated p53 target genes. Venn diagram displaying the overlap between DAZAP2-regulated genes upon 5-FU treatment and p53 target genes(52). (E) Heatmap of the p53 target genes regulated by DAZAP2 upon genotoxic stress. RNA-Seq analysis was performed as described in (A). Relative expression of DAZAP2 is shown in the last row of the heatmap. R1–3 indicate the three biological replicates. (F) DAZAP2 depletion enhances the expression of a subset of cell death-associated p53 target genes upon 5-FU exposure. HCT116 or HCT116 p53−/− were transfected with DAZAP2-specific or control siRNAs and DNA damage was induced using 50 μM 5-FU. mRNA expression was analyzed by qRT-PCR 48 h post-treatment for the indicated genes. Gene expression was normalized to GAPDH/B2M expression and the fold change relative to the untreated cells was calculated. Data are shown as mean + SD from three replicates. *** P-value ≤0.001 (two-way ANOVA for left panel, un-paired t-test for right panel). (G, H) DAZAP2 depletion sensitizes HCT116 cells to cell death in a p53-regulated manner. HCT116 wt and p53−/− cells were transfected with DAZAP2-specific or control siRNAs and DNA damage was induced using 50 μM 5-FU. n = 3. (G) Total protein extracts were prepared 24 h post-treatment and analyzed by immunoblotting for the indicated proteins. A representative experiment is shown. (H) Cells were harvested 48 h post-treatment and co-stained with Annexin V and PI. Stained cells were analyzed by flow cytometry. Bar graph showing quantification of % Annexin V-positive cells + SD from three biological replicates. ** P-value ≤0.01, ns non-significant (un-paired t-test).CC BY-NC · PubMed Central
Figure 7. DAZAP2 co-occupies p53 response elements. (A) DAZAP2 binds to p53 response elements. ChIP analyses were performed with anti-DAZAP2 antibody in cross-linked lysates from HCT116 cells treated with 50 μM 5-FU for 24 h. A non-specific IgG was used as a negative binding control. Quantitative PCR was carried out on the immunoprecipitated DNA with primers specific for regions encompassing the p53 RE. Results are represented as fold enrichment + SD from three replicates. * P-value < 0.05, *** P-value < 0.001 (unpaired t-test). (B) Schematic model summarizing the regulation of HIPK2 and the p53 pathway by the adaptor protein DAZAP2 in undamaged cells and in response to DNA damage.
Figure 7. DAZAP2 co-occupies p53 response elements. (A) DAZAP2 binds to p53 response elements. ChIP analyses were performed with anti-DAZAP2 antibody in cross-linked lysates from HCT116 cells treated with 50 μM 5-FU for 24 h. A non-specific IgG was used as a negative binding control. Quantitative PCR was carried out on the immunoprecipitated DNA with primers specific for regions encompassing the p53 RE. Results are represented as fold enrichment + SD from three replicates. * P-value < 0.05, *** P-value < 0.001 (unpaired t-test). (B) Schematic model summarizing the regulation of HIPK2 and the p53 pathway by the adaptor protein DAZAP2 in undamaged cells and in response to DNA damage.CC BY-NC · PubMed Central

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Authors
Liebl, M. C., Moehlenbrink, J., Becker, H., Raddatz, G., Abdeen, S. K., Aqeilan, R. I., Lyko, F., & Hofmann, T. G.
Affiliations
  • Institute of Toxicology, University Medical Center Mainz, Johannes Gutenberg University, Mainz, Germany
  • Division of Epigenetics, German Cancer Research Center (dkfz), Heidelberg, Germany
  • The Concern Foundation Laboratories, The Lautenberg Center for Immunology and Cancer Research, Department of Immunology and Cancer Research-IMRIC, Hebrew University-Hadassah Medical School, Jerusalem, Israel
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2021-02-16
Volume
49
Issue
5
Pages
2759-2776
ISSN
  • 0305-1048
  • 1362-4962
DOI
10.1093/nar/gkab084
PMC
PMC7969023
Cited by
20
References
61
Language
en
Licence
creativecommons.org/licenses/by-nc/4.0/ (vor)
Funders
Deutsche Forschungsgemeinschaft (SFB 1036, SFB 1361)

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