Active Regulator of SIRT1 Cooperates with SIRT1 and Facilitates Suppression of p53 Activity  Eun-Joo Kim, Jeong-Hoon Kho, Moo-Rim Kang, Soo-Jong Um  Molecular Cell  Volume 28, Issue 2, Pages 277-290 (October 2007) DOI: 10.1016/j.molcel.2007.08.030 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Identification of AROS and Its Expression (A) The nucleotide sequence and deduced amino acid sequence of human AROS. (B) Yeast two-hybrid assays were performed using LexA DBD-fused SIRT1 (in pBTM116 vector) and Gal4 AD-fused AROS (in pGAD10 vector). The interaction was quantified using a β-gal assay. Data are presented as mean ± standard deviation (SD) of triplicate experiments. (C) Analysis of AORS mRNA expression in human tissue. Northern blotting was performed using the open reading frame of AROS as a probe and human multiple tissue northern blot membrane. β-actin was used as an internal control. (D) The expression levels of AROS and SIRT1 proteins in various cell lines were analyzed by western blotting with AROS and SIRT1 antibodies. (E) Localization of endogenous AROS (left) and overexpressed AROS (right). The location of endogenous AROS in H1299 cells was determined using anti-AROS antibody and Texas Red-conjugated anti-rabbit IgG. Cells were transfected with GFP-AROS to locate overexpressed AROS. Hoechst staining was used to locate the nucleus. Molecular Cell 2007 28, 277-290DOI: (10.1016/j.molcel.2007.08.030) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Interaction between SIRT1 and AROS In Vitro and In Vivo (A) AROS specifically interacts with SIRT1 from the SIRT family. Yeast two-hybrid assays were performed using LexA DBD-fused SIRT family (SIRT1–7) and Gal4 AD-fused AROS. The interaction was evaluated by β-gal assays. Folding activity indicates the relative value of the Gal4 AD empty control. Data are presented as mean ± standard deviation (SD) of triplicate experiments. (B) Mapping of the SIRT1 domain responsible for the interaction with AROS. SIRT1 deletions were fused to the LexA DBD vector and introduced into yeast L40 together with Gal4 AD-fused AROS. Folding activity indicates the relative β-gal value compared to LexA DBD-SIRT1 plus Gal4 AD empty control. Data are presented as mean ± standard deviation (SD) of triplicate experiments. (C) Direct interaction between SIRT1 and AROS. In vitro-translated Flag-tagged AROS was incubated with GST or GST-SRIT1 (114–217). The bound proteins were visualized by SDS-PAGE and subsequent western blotting (WB) using anti-Flag antibody. (D) Interaction between SIRT1 and AROS under transfection conditions. H1299 cells were transfected with either Flag or Flag-tagged AROS alone, and lysates were subjected to immunoprecipitation (IP) followed by WB with anti-Flag and anti-SIRT1 antibodies, or vice versa. (E) Endogenous interaction between SIRT1 and AROS. HCT116 cells were treated with 20 μM etoposide plus 0.5 μM TSA for 6 hr. Cell lysates were prepared and immunoprecipitated with preimmune serum or anti-AROS antibody. Precipitated proteins were revealed by WB using anti-SIRT1 antibody. (F) Endogenous location of SIRT1 and AROS. SIRT1 was stained with mouse monoclonal anti-SIRT1 antibody and Texas Red-conjugated anti-mouse IgG. AROS was stained with rabbit polyclonal anti-AROS antibody and FITC-conjugated anti-rabbit IgG in H1299 cells. Images were visualized by immunofluorescence microscopy. Molecular Cell 2007 28, 277-290DOI: (10.1016/j.molcel.2007.08.030) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 AROS Enhances SIRT1-Promoted p53 Deacetylation (A) AROS stimulates p53 deacetylation of SIRT1 in vitro. The fluorescent substrate (Lys379/382 residues of p53) was incubated with recombinant SIRT1 (1 U; 0.17 μg) in the presence of 100 μM NAD+. Purified GST-AROS was added in increasing amounts (0, 0.5, 1, and 1.5 μg). Amounts of GST used were 0, 0.3, 0.6, and 0.9 μg to keep the same molar ratio as GST-AROS. In the absence of SIRT1, 0.9 μg GST or 1.5 μg GST-AROS was added. Data are presented as mean ± standard deviation (SD) of triplicate experiments. Analysis of variance was employed to calculate the p value. A p value <0.05 was considered to be significant. (B) AROS deacetylates CBP-induced p53 in vivo. H1299 cells were cotransfected with p53 and HA-tagged CBP expression vector in combination with SIRT1, AROS, or SIRT1 and AROS. Cell lysates were analyzed by WB with antibodies against HA, Ac-p53 (K-382), and p53. (C) AROS cooperates with SIRT1 to reduce DNA-damage-induced deacetylation of endogenous p53. HCT116 cells were transfected with AROS, SIRT1, or AROS/SIRT1 expression vectors and treated with 20 μM etoposide and 0.5 μM TSA. The cell lysates were then analyzed by WB using antibodies against Flag, Ac-p53, and p53. (D) The effect of AROS downregulation on p53 acetylation. HCT116 cells were transfected with either control siRNA (50, 100 nM) or AROS siRNA (50, 100 nM). After 1 day, cell were treated with 20 μM etoposide and 0.5 μM TSA for 6 hr. The expression of AROS and acetylation of p53 were monitored by WB using the antibodies indicated. The expression of β-actin was monitored as a loading control. Molecular Cell 2007 28, 277-290DOI: (10.1016/j.molcel.2007.08.030) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 AROS Cooperates with SIRT1 and Suppresses p53-Driven Transcriptional Activation (A) The deacetylase activity of SIRT1 is required for AROS cooperation. H1299 cells were transfected with increasing amounts (0, 0.1, 0.3, and 0.5 μg) of Flag-AROS, Flag-SIRT1 WT, or the Flag-SIRT1 HY mutant (deacetylase-defective) in combination with the p53 expression vector and p53-responsive Bax-luciferase reporter. Luciferase activity was determined using the luciferase assay after normalizing to the observed β-gal activity. Fold-luciferase activity indicates the relative ratio obtained in the presence and absence of the p53 vector. Data are presented as mean ± standard deviation (SD) of triplicate experiments. Analysis of variance was employed to calculate the p value. A p value <0.05 was considered to be significant. (B) The effect of nicotinamide (NAM) on AROS repression. H1299 cells were transfected with combinations of p53, SIRT1, and AROS expression vector. Cells were treated with increasing concentrations (1, 5, and 10 mM) of NAM for 12 hr. Cell lysates were then subjected to luciferase assays. Data are presented as mean ± standard deviation (SD) of triplicate experiments. Analysis of variance was employed to calculate the p value. A p value <0.05 was considered to be significant. (C) AROS knockdown increases p53 transactivation. H1299 cells were transfected with p53 and the AROS antisense expression vector, and cell lysates were subjected to luciferase assays. Levels of acetylated p53, AROS, and p53 were determined by WB. (D) AROS overexpression inhibits p21WAF1 expression under DNA-damaging conditions. HCT116 cells were transfected with Flag or Flag-AROS and treated with 20 μM etoposide and 0.5 μM TSA. Levels of acetylated p53, p21WAF1, AROS, and p53 were determined by WB. Molecular Cell 2007 28, 277-290DOI: (10.1016/j.molcel.2007.08.030) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 SIRT1 Is Required for AROS-Driven p53 Inactivation (A) SIRT1, not AROS, interacts with p53. In vitro-translated Flag-SIRT1 or Flag-AROS was incubated with GST or GST-p53 (full-length). The bound proteins were visualized by SDS-PAGE and subsequent WB using anti-Flag antibody. (B) The interaction of AROS with p53 is SIRT1 dependent. HCT116 cells were transfected with the Flag-AROS vector in the absence and presence of SIRT1 siRNA treatment. Lysates were subjected to immunoprecipitation with preimmune serum or p53 antibody, followed by WB using the anti-Flag antibody. The expression levels of SIRT1, p53, and Flag-AROS were shown by WB. (C) The transcription activity of p53 is unaltered by expression of AROS in HeLa cells, with minutely detectable levels of SIRT1. HeLa cells were transfected with increasing amounts of Flag-AROS (0.1, 0.3, and 0.5 μg) with p53 and/or SIRT1 vectors. Data are presented as mean ± standard deviation (SD) of triplicate experiments. Analysis of variance was employed to calculate the p value. A p value <0.05 was considered to be significant. (D) SIRT1 knockdown abolishes AROS-driven inactivation of p53 transcriptional activity. HCT116 cells were transfected with the Flag-AROS vector and treated with 100 nM of SIRT1 siRNA or control siRNA. Fold-luciferase activity indicates the relative ratio obtained in the presence and absence of Bax-luciferase reporter. Levels of SIRT1 and AROS were shown by WB. Data are presented as mean ± standard deviation (SD) of triplicate experiments. Analysis of variance was employed to calculate the p value. A p value <0.05 was considered to be significant. (E) Depletion of SIRT1 abrogates AROS-mediated p53 deacetylation. HCT116 cells were transfected with Flag-AROS, SIRT1 siRNA, or control siRNA (shown by “C”). After 1 day, cells were either untreated or treated with 20 μM etoposide and 0.5 μM TSA for 6 hr. Protein levels were analyzed by WB with antibodies against Flag (for AROS), Ac-p53, SIRT1, and β-actin. (F) AROS-mediated p53 deacetylation is blocked by SIRT1 inhibitor splitomicin. H1299 cells were cotransfected with combinations of p53, CBP, and AROS vectors and treated with 240 μM splitomicin for 9 hr. Cell lysates were subjected to WB blotting using the antibodies indicated. Molecular Cell 2007 28, 277-290DOI: (10.1016/j.molcel.2007.08.030) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 AROS Binding Is Required for the Promotion of SIRT1 Activity (A) The N-terminal deletion of SIRT1 abolishes AROS binding. HCT116 cells were cotransfected with Flag-SIRT1 wild-type (WT) or SIRT1Δ (deletion of amino acids 1–217) and GFP-AROS constructs. Lysates were subjected to immunoprecipitation (IP) with an anti-Flag antibody and subsequent WB with an anti-GFP antibody. (B) AROS-binding-defective SIRT1Δ does not cooperate with ARSO in p53 inactivation. H1299 cells were transfected with combinations of p53, AROS, SIRT1 WT, and SIRT1Δ together with Bax-luciferase reporter. Luciferase assays were performed as described. Data are presented as mean ± standard deviation (SD) of triplicate experiments. Analysis of variance was employed to calculate the p value. A p value <0.05 was considered to be significant. (C) The effect of SIRT1Δ on p53 deacetylation. HCT116 was transfected with combinations of Flag-tagged AROS, SIRT1 WT, and SIRT1Δ and treated with 20 μM etoposide and 0.5 μM TSA. Cell lysates were subjected to WB using Ac-p53, Flag, and β-actin antibodies. Molecular Cell 2007 28, 277-290DOI: (10.1016/j.molcel.2007.08.030) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 AROS Modulates Cell Growth in Response to DNA Damage (A) The effect of AROS on cell proliferation. HCT116 cells stably expressed Flag-empty, Flag-AROS sense (S), or AROS antisense (AS) treated with 1 μM etoposide and 0.5 μM TSA for the times indicated. AROS expression was analyzed by WB with the anti-AROS antibody (left). Cell viability was determined by MTT assay (right). Data are presented as mean ± SD from triplicate experiments. (B) The effect of AROS on p21WAF1 expression. HCT116 stable cells were treated as described above. After 2 days of treatment, cells were harvested and lysates subjected to WB. (C) The effect of AROS on cell-cycle progression. Cells were treated as described in Figure 7B and subjected to FACS analysis. (D) The effect of AROS on apoptosis. Cells were treated as described in Figure 7B and subjected to Hoechst staining to monitor chromatin fragmentation, an indicator of apoptosis. (E) Diagram for AROS function. AROS cooperates with SIRT1 and inactivates p53 in mediating cell-cycle arrest and apoptosis under conditions that cause DNA damage. Combined, AROS and SIRT1 may function as a cell survival factor. Molecular Cell 2007 28, 277-290DOI: (10.1016/j.molcel.2007.08.030) Copyright © 2007 Elsevier Inc. Terms and Conditions