HDAC5, a Key Component in Temporal Regulation of p53-Mediated Transactivation in Response to Genotoxic Stress  Nirmalya Sen, Rajni Kumari, Manika Indrajit Singh, Sanjeev Das  Molecular Cell  Volume 52, Issue 3, Pages 406-420 (November 2013) DOI: 10.1016/j.molcel.2013.09.003 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 52, 406-420DOI: (10.1016/j.molcel.2013.09.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 HDAC5 Interacts with p53 (A) H1299 cells were infected with adenovirus expressing GFP (Ad-GFP) or p53 tagged with hemagglutinin (HA) and Flag epitopes (Ad-p53 HF). Cells were harvested, and nuclear extracts were sequentially immunoprecipitated with Flag and HA antibody affinity resins. The p53-associated proteins were detected by SDS-PAGE and silver staining. (B) H1299 cells were infected with the indicated adenoviruses for 24 hr. The cells were then harvested and subjected to immunoprecipitations using anti-p53 antibody (left panel) or anti-HDAC5 antibody (right panel), and western blotting was performed for the indicated proteins. (C) GST, GST-p53, and His-HDAC5 were bacterially expressed and purified (left panel). GST pull-down assay was performed, followed by western blotting for the indicated proteins (right panel). (D) HCT116 cells were treated with etoposide (40 μM) for the indicated time points, and western blotting was performed for the indicated proteins. (E) HCT116 cells were treated with etoposide (40 μM) for the indicated time points, and western blotting was performed for the indicated proteins from nuclear (left) and cytoplasmic (right) fractions. (F) HCT116 cells were treated with etoposide (40 μM) for the indicated time points. The cells were then harvested, and nuclear extracts were subjected to immunoprecipitations using anti-p53 antibody (left panel) or anti-HDAC5 antibody (right panel), and western blotting was performed for the indicated proteins. (G) H1299 cells were transfected with constructs expressing full-length or different domains of p53 as GST fusion protein. At 6 hr after transfection, the cells were infected with Ad-GFP or Ad-HDAC5 Flag. At 24 hr after infection, cell lysates were subjected to GST pull-down followed by immunoblotting for the indicated proteins. (H) H1299 cells were transfected with constructs expressing full-length or different domains of HDAC5 as FLAG-tagged protein. At 6 hr after transfection, the cells were infected with Ad-GFP or Ad-p53. At 24 hr after infection, cell lysates were subjected to immunoprecipitation using anti-Flag antibody followed by immunoblotting for the indicated proteins. See also Figure S7. Molecular Cell 2013 52, 406-420DOI: (10.1016/j.molcel.2013.09.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Effect of Genotoxic Stress on HDAC5 Cellular Localization (A) HCT116 cells were treated with etoposide (40 μM) for the indicated time points. ROS levels in cells were measured by FACS analysis. Error bars are means ± SD of three independent experiments with duplicate samples. (B and C) HCT116 cells were treated with etoposide (40 μM) for the indicated time points, and KN-93 (10 μM) or KN-92 (10 μM) was added at 14 hr after drug addition, as indicated. The cells were then subjected to western blotting (B) and immunofluorescence staining (C) for the indicated proteins. (D) HCT116 cells were treated with etoposide (40 μM) for the indicated time points, and KN-93 (10 μM) or KN-92 (10 μM) was added at 14 hr after drug addition, as indicated. Western blotting was performed for the indicated proteins from nuclear (left) and cytoplasmic (right) fractions. See also Figures S1 and S2. Molecular Cell 2013 52, 406-420DOI: (10.1016/j.molcel.2013.09.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 HDAC5 Deacetylates p53 (A) HCT116 control (HCT116) and HDAC5 knockdown cells (HCT116 HDAC5kd) were treated with etoposide (40 μM) for the indicated time points, and KN-93 (10 μM) was added at 14 hr after drug addition, as indicated. The cells were then harvested, and western blotting was performed for the indicated proteins. The acetylated p53 blots were quantified, and normalized values (with respect to β-actin) are shown. (B) AcK120-p53 peptide was incubated either alone (control) or in the presence of recombinant HDAC5, and peptide molecular mass was determined by mass spectrometry. The relative positions of acetylated (1,246.36 Da) and deacetylated (1,204.36 Da) p53 peptides are shown. (C) Results of HDAC5 deacetylation reactions using acetylated p53 peptides. Peptide molecular mass was determined by mass spectrometry as in (B). See also Figures S1–S3. Molecular Cell 2013 52, 406-420DOI: (10.1016/j.molcel.2013.09.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 HDAC5 Modulates p53 Transactivation Function (A) HCT116 control (HCT116) and HDAC5 knockdown cells (HCT116 HDAC5kd) were treated with etoposide (40 μM) for the indicated time points, and KN-93 (10 μM) was added at 14 hr after drug addition, as indicated. Relative mRNA levels were analyzed by RT-qPCR for the indicated genes. Error bars are means ± SD of three independent experiments with triplicate samples. (B) HCT116 control (HCT116) and HDAC5 knockdown cells (HCT116 HDAC5kd) were treated with etoposide (40 μM) for the indicated time points, and KN-93 (10 μM) was added at 14 hr after drug addition, as indicated. The cells were then harvested, and western blotting was performed for the indicated proteins. (C and D) HCT116 control (HCT116) and HDAC5 knockdown cells (HCT116 HDAC5kd) were treated with etoposide (40 μM) for the indicated time points, and KN-93 (10 μM) was added at 14 hr after drug addition, as indicated. ChIP assay was then performed with control immunoglobulin G (IgG) antibody or p53 antibody (C) and control IgG antibody or AcK120-p53 antibody (D). Error bars are means ± SD of three independent experiments with triplicate samples. (E) Part of the chromatin immunoprecipitated with p53 antibody (4C above) was again subjected to ChIP using control IgG or HDAC5 antibody. Error bars are means ± SD of three independent experiments with triplicate samples. See also Figures S1–S4 and S7. Molecular Cell 2013 52, 406-420DOI: (10.1016/j.molcel.2013.09.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Effect of HDAC5 on p53-Mediated Cell Fate Decisions (A) HCT116 control (HCT116) and HDAC5 knockdown cells (HCT116 HDAC5kd) were treated with etoposide (40 μM) for the indicated time points, and KN-93 (10 μM) was added at 14 hr after drug addition, as indicated. Cells were harvested and stained with propidium iodide and analyzed by FACS. The data shown are representative of three independent experiments. (B) HCT116 control (HCT116) and HDAC5 knockdown cells (HCT116 HDAC5kd) were treated with etoposide (40 μM) for the indicated time points, and KN-93 (10 μM) was added at 14 hr after drug addition, as indicated. Cells were harvested, and TUNEL assay was performed. Error bars are means ± SD of three independent experiments with duplicate samples. (C and D) HCT116 control (empty vector) cells (HCT116) and HCT116 cells stably expressing phospho-dead HDAC5 double mutant (HCT116 HDAC5 mut) were treated with etoposide (40 μM) for the indicated time points, and KN-93 (10 μM) was added at the same time, as indicated. The cells were then subjected to western blotting for the indicated proteins (C) and senescence-associated β-galactosidase staining (D). (E and F) HCT116 and HCT116 HDAC5 mut cells were treated with etoposide (40 μM) for the indicated time points, and KN-93 (10 μM) was added at the same time, as indicated. The cells were harvested and then subjected to RT-qPCR for the indicated genes (E) and ChIP assay with either control IgG antibody or H3K9me3 antibody for the indicated promoters (F). Error bars are means ± SD of three independent experiments with triplicate samples. See also Figures S1–S3 and S5. Molecular Cell 2013 52, 406-420DOI: (10.1016/j.molcel.2013.09.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Effect of ROS on HDAC5-Mediated Regulation of p53 Functions (A) HCT116 control (HCT116) and HDAC5 knockdown cells (HCT116 HDAC5kd) were treated with etoposide (40 μM) for the indicated time points, and tiron (5 mM) was added at 14 hr after drug addition, as indicated. ROS levels in cells were measured by FACS analysis. Error bars are means ± SD of three independent experiments with duplicate samples. (B) HCT116 control (HCT116) and HDAC5 knockdown cells (HCT116 HDAC5kd) were treated with etoposide (40 μM) for the indicated time points, and tiron (5 mM) was added at 14 hr after drug addition, as indicated. Cells were harvested, and western blotting was performed for the indicated proteins. (C) HCT116 control cells were treated with etoposide (40 μM) for the indicated time points, and tiron (5 mM) was added at 14 hr after drug addition, as indicated. Immunofluorescence staining was performed for the indicated proteins. (D) HCT116 control (HCT116) and HDAC5 knockdown cells (HCT116 HDAC5kd) were treated with etoposide (40 μM) for the indicated time points, and tiron (5 mM) was added at 14 hr after drug addition, as indicated. Relative mRNA levels were analyzed by RT-qPCR for the indicated genes. Error bars are means ± SD of three independent experiments with triplicate samples. (E and F) HCT116 control (HCT116) and HDAC5 knockdown cells (HCT116 HDAC5kd) were treated with etoposide (40 μM) for the indicated time points, and tiron (5 mM) was added at 14 hr after drug addition, as indicated. Cells were harvested and analyzed by propidium iodide staining and FACS (E) and TUNEL assay (F). Error bars are means ± SD of three independent experiments with duplicate samples. See also Figure S4. Molecular Cell 2013 52, 406-420DOI: (10.1016/j.molcel.2013.09.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 HDAC5 Modulates p53-Mediated Stress Response In Vivo (A) Mice were injected with etoposide (10 mg/kg) for the indicated days. KN-93 (6 mg/kg) was injected 1 day after etoposide injection, as indicated. Representative TUNEL staining performed on sections of intestine and spleen from these mice is shown. (B) Mice were injected with etoposide (10 mg/kg) for the indicated days. KN-93 (6 mg/kg) was injected 1 day after etoposide injection, as indicated. ROS levels in intestinal cells and splenocytes isolated from these mice were measured by FACS. The data shown are representative of three independent experiments using samples from five individual mice. Error bars are means ± SD from five individual mice (n = 5). (C) Mice were injected with etoposide (10 mg/kg) for the indicated days. KN-93 (6 mg/kg) was injected 1 day after etoposide injection, as indicated. Tissue from intestine and spleen was harvested and processed for western blotting of the indicated proteins. The data shown are representative of three independent experiments. (D) Mice were injected with etoposide (10 mg/kg) for the indicated days. KN-93 (6 mg/kg) was injected 1 day after etoposide injection, as indicated. Total RNA was isolated from intestine and spleen, and RT-qPCR was performed for the indicated genes. The data shown are representative of three independent experiments using samples from five individual mice. Error bars are means ± SD from five individual mice (n = 5). (E) Mice were injected with adenovirus expressing control (scrambled) or HDAC5 shRNA and subsequently injected with etoposide (10 mg/kg) for the indicated days. Tissue from intestine and spleen was isolated and processed for western blotting of the indicated proteins. The data shown are representative of three independent experiments. (F) Mice were injected with adenovirus expressing control (scrambled) or HDAC5 shRNA and subsequently treated with etoposide (10 mg/kg) for the indicated days. Representative TUNEL staining performed on sections of intestine and spleen from these mice is shown. (G) Mice were injected with adenovirus expressing control (scrambled) or HDAC5 shRNA and subsequently injected with etoposide (10 mg/kg) for the indicated days. ROS levels in intestinal cells and splenocytes isolated from these mice were measured by FACS analysis. The data shown are representative of three independent experiments using samples from five individual mice. Error bars are means ± SD from five individual mice (n = 5). (H) Mice were injected with adenovirus expressing control (scrambled) or HDAC5 shRNA and subsequently injected with etoposide (10 mg/kg) for the indicated days. Total RNA was isolated from intestine and spleen, and RT-qPCR was performed for the indicated genes. The data shown are representative of three independent experiments using samples from five individual mice. Error bars are means ± SD from five individual mice (n = 5). See also Figure S6. Molecular Cell 2013 52, 406-420DOI: (10.1016/j.molcel.2013.09.003) Copyright © 2013 Elsevier Inc. Terms and Conditions