Volume 44, Issue 5, Pages 797-810 (December 2011) DNA Damage-Induced RORα Is Crucial for p53 Stabilization and Increased Apoptosis  Hyunkyung Kim, Ji Min Lee, Gina Lee, Jinhyuk Bhin, Se Kyu Oh, Kyeongkyu Kim, Ki Eun Pyo, Jason S. Lee, Hwa Young Yim, Keun Il Kim, Daehee Hwang, Jongkyeong Chung, Sung Hee Baek  Molecular Cell  Volume 44, Issue 5, Pages 797-810 (December 2011) DOI: 10.1016/j.molcel.2011.09.023 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Identification of RORα as a p53 Target Gene Induced by DNA Damage (A) HCT116 p53+/+ cells were treated with either Dox (1 μg/ml) or IR (10 Gy). After 24 hr, cells were harvested and total RNAs were extracted. RORα and p21 mRNA levels were analyzed by real-time RT-PCR. Error bars represent mean ± SD (n = 3); ∗p < 0.05. (B) HCT116 p53+/+ cells treated with either Dox (1 μg/ml) or IR (10 Gy) were analyzed by immunoblot for RORα, p21, and p53. (C) HCT116 p53−/− cells were reconstituted with p53. RORα transcripts were analyzed by real-time RT-PCR with or without restored p53 after Dox treatment for 24 hr. Error bars represent mean ± SD (n = 3); ∗p < 0.05. (D) RORα and p21 transcripts were measured by real-time RT-PCR in WT or p53−/− MEFs after Dox treatment for 24 hr. Error bars represent mean ± SD (n = 3); ∗p < 0.05. (E) WT or p53−/− MEFs treated with Dox (1 μg/ml) for 24 hr were analyzed by immunoblot for RORα, p21, and p53. (F) A schematic representation of the RORα promoter region. The triangle depicts locations of p53RE1 (−688 to −664) and p53RE2 (−760 to −740), and PCR primer pairs used for ChIP assay are shown. (G) RORα promoter-luciferase reporter plasmid was transfected into HCT116 p53+/+ or p53−/− cells. Luciferase activity was measured after Dox treatment at the indicated times and normalized by β-galactosidase activity. Values are expressed as mean ± SD for three independent experiments. Error bars represent mean ± SD. ∗p < 0.05. (H) Introduction of p53 plasmids into HCT116 p53−/− cells increased the WT RORα promoter-luciferase activity. RORα promoter-luciferase activity with deleted p53REs was not affected by p53. Luciferase activity was measured after Dox treatment for 12 hr and normalized by β-galactosidase activity. Values are expressed as mean ± SD for three independent experiments. Error bars represent mean ± SD. ∗p < 0.05. (I) Shown is a schematic representation of the WT RORα promoter-luciferase (WT; from −2,000 to +100) and the RORα promoter-luciferase with deleted p53RE (Δp53RE; from −660 to +100). (J) ChIP assay using anti-p53 and anti-p300 antibodies to the p53RE in the RORα promoter after Dox treatment in HCT116 p53+/+ or p53−/− cells. (K) ChIP assay to the p53RE in the RORα promoter in the p53-reconstituted HCT116 p53−/− cells. Occupancies of p53 and RNA polymerase II are indicated. (L) ChIP assay was performed on the WT RORα promoter or Δp53RE RORα promoter in HCT116 cells. Promoter occupancy by p53 was analyzed. Error bars represent mean ± SD (n = 3); ∗p < 0.05. (M) Reaction mixtures for EMSA containing 32P-labeled oligonucleotide probes 1 and 2 and p53 were incubated and analyzed by electrophoresis. Unlabeled probes were added for the competition. (N and O) Lysates from HCT116 p53+/+ and p53−/− cells (N) or WT and p53−/− MEFs (O) treated with Dox for the indicated times were analyzed by immunoblot for RORα and p53. See also Figure S1. Molecular Cell 2011 44, 797-810DOI: (10.1016/j.molcel.2011.09.023) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 RORα Regulates p53 Stability by Inhibiting p53 Ubiquitination (A) Coimmunoprecipitation of RORα and p53 at endogenous levels in HCT116 cells with Dox treatment for 12 hr. (B) HCT116 cells were transfected with shRNA against RORα, and samples were collected to determine the p53 protein levels for immunoblot after Dox treatment at the indicated times. The quantification of the immunoblot results is also represented. (C) Overexpression of RORα in HCT116 p53−/− cells increased p53 protein levels in a dose-dependent manner. (D) The effects of RORα overexpression on the half-life of endogenous p53 in HCT116 cells. The transfected cells were treated with CHX (20 μg/ml), collected at the indicated times, and analyzed by immunoblot to determine p53 protein levels. The quantification of the immunoblot results is also represented. (E) HCT116 p53−/− cells were transfected with HisMax-p53, siRORα, or negative control siRNA (siNS), and ubiquitination assay was performed followed by treatment of 20 μM of MG132. Whole-cell extracts and Ni-NTA+ affinity-purified precipitates (Ni-NTA+ pull-down) were analyzed by immunoblot with anti-Ub antibody to detect ubiquitinated p53. (F) HCT116 p53−/− cells were cotransfected with p53, MDM2, RORα, and HisMax-ubiquitin, and protein extracts were pulled down with Ni-NTA+ beads followed by treatment of cells with 20 μM of MG132. Ubiquitination of p53 was measured with anti-p53 antibody. (G) HCT116 p53−/− cells were transfected with p53 and MDM2 with increasing amounts of RORα, and immunoprecipitation assay was performed with anti-Mdm2 antibody followed by immunoblot with anti-p53 antibody. (H) RORα-interacting proteins were obtained from HEK293 cells stably expressing Flag-RORα. The bound proteins were resolved by SDS-PAGE and prepared for LC-MS/MS analysis, and obtained peptide sequence of HAUSP was indicated. (I) Coimmunoprecpitation assay between HAUSP and RORα in HCT116 cells. (J) HCT116 cells were transfected with Flag-RORα, and two-step immunoprecipitation assay was performed first with anti-Flag antibody followed by anti-p53 antibody. The precipitates were analyzed by immunoblot with anti-p53, anti-HAUSP, and anti-Flag antibodies to visualize three factors present in the same complex. (K) Coimmunoprecipitation assays among RORα, p53, and HAUSP at endogenous expression level in HCT116 cells. (L) Direct interaction between in vitro-translated 35S-RORα and GST-HAUSP NTD or GST-p53 was analyzed. See also Figure S1. Molecular Cell 2011 44, 797-810DOI: (10.1016/j.molcel.2011.09.023) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 RORα Enhances p53 Deubiquitination in a HAUSP-Dependent Manner (A and B) Coimmunoprecipitation of p53 and HAUSP in HCT116 cells treated with 20 μM of MG132 for 4 hr. Overexpression of RORα increased the binding between p53 and HAUSP (A), whereas knockdown of RORα decreased their binding (B). (C and D) HCT116 p53−/− cells were cotransfected with p53, MDM2, HAUSP, HisMax-ubiquitin, and RORα (C) or siRORα (D), and protein extracts were pulled down with Ni-NTA+ beads. Ubiquitination of p53 was measured with an anti-p53 antibody. (E) Shown is coimmunoprecipitation of p53 and RORα in HCT116 cells treated with 20 μM of MG132 for 4 hr. Knockdown of HAUSP diminished the binding between p53 and RORα. (F and G) HCT116 p53−/− cells were cotransfected with p53, MDM2, siHAUSP, HisMax-ubiquitin, and HA-RORα (F) or siRORα (G). Ubiquitination of p53 was assessed as in (C). (H) H1299 (p53 null) cells were cotransfected with p53, PG13-luciferase reporter, and increasing amounts of RORα expression plasmids as indicated. Luciferase activities were measured and normalized by β-galactosidase activity. Values are expressed as mean ± SD for three independent experiments. ∗p < 0.05, ∗∗∗p < 0.001. (I and J) Binding between p53 and p300 (I) or CBP (J) in HCT116 p53−/− cells treated with 20 μM of MG132 for 4 hr. Coimmunoprecipitation was performed using either anti-p300 or anti-CBP antibody followed by immunoblot analysis. See also Figure S2. Molecular Cell 2011 44, 797-810DOI: (10.1016/j.molcel.2011.09.023) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Genome-wide Analysis Reveals that RORα Regulates a Subset of p53-Responsive Genes Involved in Apoptosis (A) Flow chart showing the strategy for gene expression analysis of WT and Sg MEFs. (B and C) RORα-dependent up- and downregulated genes (patterns 1 and 2, respectively) (B), and RORα-independent up- and downregulated genes (patterns 3 and 4, respectively) (C) in either WT or Sg MEFs upon DNA damage. Upregulated and downregulated clusters are represented as the log2 fold changes in red and green, respectively (color bar). The RORα dependency is denoted by the relative differences between the log2 fold changes in WT and Sg in the third column (red and green, negative and positive dependency, respectively). (D) RORα-dependent genes represent approximately 76.3% of all DNA damage-responsive genes. (E) The p53-responsive genes were collected (see text in detail), and the enrichment of these genes was analyzed for each cluster (shown as the percentage of the total number of genes within each cluster). (F) Functional classification of RORα-dependent p53 target genes (186 genes in the C1 cluster). (G) The RORα-dependent p53 target genes (43 out of the 186 genes in the C1 cluster), shown as described above. Asterisk, genes with previously reported p53 target genes involved in apoptosis. See also Table S1 and Table S2. Molecular Cell 2011 44, 797-810DOI: (10.1016/j.molcel.2011.09.023) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Recruitment of RORα to a Subset of p53 Target Promoters along with HAUSP (A and B) Quantitative RT-PCR analyses of RORα-dependent (A) and RORα-independent (B) p53 target gene expressions, identified by cDNA microarray from WT and Sg MEFs in the absence or presence of Dox. Error bars represent mean ± SD (n = 3); ∗p < 0.05. (C and D) The ChIP assays on the RORα-dependent SIVA and Fas promoters (C) and RORα-independent Rrm2B and Flit-1 promoters (D) in HCT116 p53+/+ or p53−/− cells with or without Dox treatment for 12 hr. (E and F) The ChIP assay was performed in the absence or presence of MG132 to block p53 ubiquitination. ChIP assays were performed on the HRAS, CASP1, and SIVA promoters (E) and the Fas and Pmap1 promoters (F) in HCT116 cells with or without Dox treatment for 12 hr. Promoter occupancy by RORα, p53, and HAUSP was analyzed. (G) ChIP assay was performed on the Fas and Mdm2 promoters in HCT116 cells with or without Dox treatment for 12 hr. Promoter occupancy by RORα, p53, HAUSP, CBP, and RNA Pol II was analyzed. See also Figure S3. Molecular Cell 2011 44, 797-810DOI: (10.1016/j.molcel.2011.09.023) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 RORα Increases Apoptosis via p53 in Response to DNA Damage (A and B) HCT116 p53+/+ (A) or HCT116 p53−/− cells (B) were cotransfected with siRORα or nonspecific scramble siRNA. After 24 hr of Dox treatment, cells were fixed and stained with propidium iodide followed by flow cytometric analysis. Values are expressed as mean ± SD for three independent experiments. ∗∗∗p < 0.001. (C and D) HCT116 p53+/+ (C) or HCT116 p53−/− cells (D) transfected with the mock vector or RORα were stained using the TUNEL assay system after Dox treatment for 24 hr. Fragmented apoptotic cell nuclei were shown by TUNEL (TdT, green), and the nucleus was stained with DAPI (blue). Error bars represent mean ± SD (n = 3); ∗∗∗p < 0.001. See also Figure S4. Molecular Cell 2011 44, 797-810DOI: (10.1016/j.molcel.2011.09.023) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 RORα Regulates IR- and p53-Induced Apoptosis in Drosophila (A) Shown are RT-PCR analyses for DmRORα (DHR3-1, DHR3-2) and Dmp53 in third-instar larvae in the absence or presence of IR. Rp49 was used as a loading control. (B) Shown are microscopic images of the adult fly eyes from the indicated genotypes. DmRORα-RA and DmRORα-RB are splicing variants (as described in the Experimental Procedures). (C–E) Larval eye discs from the indicated genotypes in the absence or presence of IR were stained with TUNEL (TdT, green) and immunostained with anti-cleaved caspase-3 antibody (red). The nucleus was stained with Hoechst 33258 (blue). Posterior is at the right. See also Figure S5. Molecular Cell 2011 44, 797-810DOI: (10.1016/j.molcel.2011.09.023) Copyright © 2011 Elsevier Inc. Terms and Conditions