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Stress-dependent Daxx-CHIP Interaction Suppresses the p53 Apoptotic Program

2009; Elsevier BV; Volume: 284; Issue: 31 Linguagem: Inglês

10.1074/jbc.m109.011767

1083-351X

Holly McDonough, Peter C. Charles, Eleanor Hilliard, Shu Bing Qian, Jin-Na Min, Andrea L. Portbury, Douglas Cyr, Cam Patterson,

Ubiquitin and proteasome pathways

Our previous studies have implicated CHIP (carboxyl terminus of Hsp70-interacting protein) as a co-chaperone/ubiquitin ligase whose activities yield protection against stress-induced apoptotic events. In this report, we demonstrate a stress-dependent interaction between CHIP and Daxx (death domain-associated protein). This interaction interferes with the stress-dependent association of HIPK2 with Daxx, blocking phosphorylation of serine 46 in p53 and inhibiting the p53-dependent apoptotic program. Microarray analysis confirmed suppression of the p53-dependent transcriptional portrait in CHIP+/+ but not in CHIP−/− heat shocked mouse embryonic fibroblasts. The interaction between CHIP and Daxx results in ubiquitination of Daxx, which is then partitioned to an insoluble compartment of the cell. In vitro ubiquitination of Daxx by CHIP revealed that ubiquitin chain formation utilizes non-canonical lysine linkages associated with resistance to proteasomal degradation. The ubiquitination of Daxx by CHIP utilizes lysines 630 and 631 and competes with the sumoylation machinery of the cell at these residues. These studies implicate CHIP as a stress-dependent regulator of Daxx that counters the pro-apoptotic influence of Daxx in the cell. By abrogating p53-dependent apoptotic pathways and by ubiquitination competitive with Daxx sumoylation, CHIP integrates the proteotoxic stress response of the cell with cell cycle pathways that influence cell survival. Our previous studies have implicated CHIP (carboxyl terminus of Hsp70-interacting protein) as a co-chaperone/ubiquitin ligase whose activities yield protection against stress-induced apoptotic events. In this report, we demonstrate a stress-dependent interaction between CHIP and Daxx (death domain-associated protein). This interaction interferes with the stress-dependent association of HIPK2 with Daxx, blocking phosphorylation of serine 46 in p53 and inhibiting the p53-dependent apoptotic program. Microarray analysis confirmed suppression of the p53-dependent transcriptional portrait in CHIP+/+ but not in CHIP−/− heat shocked mouse embryonic fibroblasts. The interaction between CHIP and Daxx results in ubiquitination of Daxx, which is then partitioned to an insoluble compartment of the cell. In vitro ubiquitination of Daxx by CHIP revealed that ubiquitin chain formation utilizes non-canonical lysine linkages associated with resistance to proteasomal degradation. The ubiquitination of Daxx by CHIP utilizes lysines 630 and 631 and competes with the sumoylation machinery of the cell at these residues. These studies implicate CHIP as a stress-dependent regulator of Daxx that counters the pro-apoptotic influence of Daxx in the cell. By abrogating p53-dependent apoptotic pathways and by ubiquitination competitive with Daxx sumoylation, CHIP integrates the proteotoxic stress response of the cell with cell cycle pathways that influence cell survival. Death domain-associated protein (Daxx) 3The abbreviations used are: Daxxdeath domain-associated proteinCHIPcarboxyl terminus of Hsc/Hsp 70-interacting proteinASK1apoptosis signal-regulating kinase 1HIPK2homeodomain-interacting protein kinase 2HSF1heat shock factor 1JNKc-Jun amino-terminal kinaseMDM2murine double minute 2MEFmouse embryonic fibroblastSUMO-1small ubiquitin-like modifier 1PBSphosphate-buffered salineHAhemagglutininTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labelingWTwild typeC/EBPCCAAT/enhancer-binding protein. 3The abbreviations used are: Daxxdeath domain-associated proteinCHIPcarboxyl terminus of Hsc/Hsp 70-interacting proteinASK1apoptosis signal-regulating kinase 1HIPK2homeodomain-interacting protein kinase 2HSF1heat shock factor 1JNKc-Jun amino-terminal kinaseMDM2murine double minute 2MEFmouse embryonic fibroblastSUMO-1small ubiquitin-like modifier 1PBSphosphate-buffered salineHAhemagglutininTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labelingWTwild typeC/EBPCCAAT/enhancer-binding protein. is a nuclear protein active in a number of apoptotic pathways (1Salomoni P. Khelifi A.F. Trends Cell Biol. 2006; 16: 97-104Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In the nucleus, Daxx is found in promyelocytic leukemia oncogenic domains and in heterochromatic domains of the chromatin (2Ishov A.M. Vladimirova O.V. Maul G.G. J. Cell Sci. 2004; 117: 3807-3820Crossref PubMed Scopus (123) Google Scholar). The interaction of Daxx with a number of transcription factors generally has a repressive influence on transcriptional activity, tipping cells toward suppressed growth and enhanced apoptosis (3Lin D.Y. Huang Y.S. Jeng J.C. Kuo H.Y. Chang C.C. Chao T.T. Ho C.C. Chen Y.C. Lin T.P. Fang H.I. Hung C.C. Suen C.S. Hwang M.J. Chang K.S. Maul G.G. Shih H.M. Mol. Cell. 2006; 24: 341-354Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). Under certain stress conditions, Daxx translocates to the cytoplasm, promoting the c-Jun amino-terminal kinase (JNK)-dependent pathway of apoptosis through activation of the mitogen-activated protein (MAP) kinase kinase kinase, apoptosis signal-regulating kinase (ASK1) (4Chang H.Y. Nishitoh H. Yang X. Ichijo H. Baltimore D. Science. 1998; 281: 1860-1863Crossref PubMed Scopus (531) Google Scholar, 5Hwang J.R. Zhang C. Patterson C. Cell Stress Chaperones. 2005; 10: 147-156Crossref PubMed Scopus (77) Google Scholar). death domain-associated protein carboxyl terminus of Hsc/Hsp 70-interacting protein apoptosis signal-regulating kinase 1 homeodomain-interacting protein kinase 2 heat shock factor 1 c-Jun amino-terminal kinase murine double minute 2 mouse embryonic fibroblast small ubiquitin-like modifier 1 phosphate-buffered saline hemagglutinin terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling wild type CCAAT/enhancer-binding protein. death domain-associated protein carboxyl terminus of Hsc/Hsp 70-interacting protein apoptosis signal-regulating kinase 1 homeodomain-interacting protein kinase 2 heat shock factor 1 c-Jun amino-terminal kinase murine double minute 2 mouse embryonic fibroblast small ubiquitin-like modifier 1 phosphate-buffered saline hemagglutinin terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling wild type CCAAT/enhancer-binding protein. However, other studies have indicated that Daxx has anti-apoptotic functions. The increased apoptosis found after depletion of endogenous Daxx (6Chen L.Y. Chen J.D. Mol. Cell. Biol. 2003; 23: 7108-7121Crossref PubMed Scopus (97) Google Scholar) and the ability of overexpressed Daxx to enhance the heat shock transcription factor-1 (HSF1)-dependent transcriptional program (7Boellmann F. Guettouche T. Guo Y. Fenna M. Mnayer L. Voellmy R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 4100-4105Crossref PubMed Scopus (72) Google Scholar) both support an anti-apoptotic role for this protein. That Daxx knockouts are embryonic lethals attests to its importance in normal cellular growth and development (8Michaelson J.S. Bader D. Kuo F. Kozak C. Leder P. Genes Dev. 1999; 13: 1918-1923Crossref PubMed Scopus (196) Google Scholar). Because the physiological state of the cell affects Daxx localization and Daxx binding partners, the exact role and the regulation of this molecule has been difficult to discern. Carboxyl terminus of Hsp70-interacting protein (CHIP) is a ubiquitin ligase that is a critical regulator of proteotoxic stress through its ability to mediate degradation of misfolded proteins (9Ballinger C.A. Connell P. Wu Y. Hu Z. Thompson L.J. Yin L.Y. Patterson C. Mol. Cell. Biol. 1999; 19: 4535-4545Crossref PubMed Scopus (750) Google Scholar, 10Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L.J. Höhfeld J. Patterson C. Nat Cell Biol. 2001; 3: 93-96Crossref PubMed Scopus (0) Google Scholar, 11Jiang J. Ballinger C.A. Wu Y. Dai Q. Cyr D.M. Höhfeld J. Patterson C. J. Biol. Chem. 2001; 276: 42938-42944Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). CHIP provides protection against apoptosis under some circumstances (12Dai Q. Zhang C. Wu Y. McDonough H. Whaley R.A. Godfrey V. Li H.H. Madamanchi N. Xu W. Neckers L. Cyr D. Patterson C. EMBO J. 2003; 22: 5446-5458Crossref PubMed Scopus (249) Google Scholar, 13Zhang C. Xu Z. He X.R. Michael L.H. Patterson C. Am. J. Physiol. Heart Circ Physiol. 2005; 288: H2836-H2842Crossref PubMed Scopus (99) Google Scholar). Primarily a cytoplasmic molecule, CHIP inhibits the JNK-dependent apoptotic pathway by ubiquitinating and targeting ASK1 for proteasomal degradation (5Hwang J.R. Zhang C. Patterson C. Cell Stress Chaperones. 2005; 10: 147-156Crossref PubMed Scopus (77) Google Scholar). In the nuclear compartment, CHIP is part of the HSF1 transcriptional complex that up-regulates the production of heat shock proteins (12Dai Q. Zhang C. Wu Y. McDonough H. Whaley R.A. Godfrey V. Li H.H. Madamanchi N. Xu W. Neckers L. Cyr D. Patterson C. EMBO J. 2003; 22: 5446-5458Crossref PubMed Scopus (249) Google Scholar), which in turn interact with and suppress many apoptotic signaling molecules (14Beere H.M. J. Clin. Invest. 2005; 115: 2633-2639Crossref PubMed Scopus (355) Google Scholar). The counter-regulation of ASK1 by both Daxx and CHIP, the effect of CHIP on soluble Daxx distribution and steady-state levels (5Hwang J.R. Zhang C. Patterson C. Cell Stress Chaperones. 2005; 10: 147-156Crossref PubMed Scopus (77) Google Scholar), and the transcriptional regulation of HSF1 by both CHIP and Daxx (7Boellmann F. Guettouche T. Guo Y. Fenna M. Mnayer L. Voellmy R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 4100-4105Crossref PubMed Scopus (72) Google Scholar) suggest intersections between Daxx signaling and CHIP-governed proteotoxic stress responses. These observations led us to examine more carefully the molecular relationship between CHIP and Daxx in the setting of cell stress. In this study, we describe a stress-induced interaction between Daxx and CHIP that results in reduced homeodomain-interacting protein kinase 2 (HIPK2) binding to Daxx, ubiquitination and reduced steady-state levels of cytoplasmic Daxx, and reduced stress-dependent sumoylation of Daxx. These stress-induced effects of CHIP on Daxx shift the cellular stress response toward survival pathways and away from apoptotic pathways. COS-7 or HEK-293 cells were grown at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen). CHIP+/+ and CHIP−/− primary mouse embryonic fibroblasts (MEFs) isolated from day 13.5 embryos of C57BL6 CHIP+/− crosses were grown under similar conditions with the addition of β-mercaptoethanol. Cells were transfected with FuGENE 6 reagent (Roche Applied Science) using pcDNA3 to equalize DNA. Transfection efficiencies were ≥95% as estimated by cells transfected with green fluorescent protein. Cells were treated and harvested 24 h after transfection. To create CHIP deletion mutants, site-directed mutagenesis was performed with the QuikChange site-directed mutagenesis protocol (Stratagene) using appropriate oligonucleotides. The sequences of the mutated deletions were verified by DNA sequencing. For whole cell lysates, cells were harvested, washed twice in PBS, and then lysed in lysis buffer (150 mm NaCl, 50 mm Tris, pH 7.5, 2 mm EGTA, 1 mm Na3VO4, 1× protease inhibitor mixture, 20 mm NaH2PO4, pH 7.2, 25 mm NaF, 10% glycerol, 0.50% Triton X-100, 1 mm phenylmethylsulfonyl fluoride). Cell lysates were centrifuged at 12,000 rpm for 10 min, and the supernatants were removed as the soluble fraction. The pellets were solubilized by adding lysis buffer and 10% DNase and incubating for 30 min at room temperature with mechanical trituration with a pipette. Pellet samples were then boiled for 30 min in SDS sample buffer (0.5 m Tris-HCl, pH 6.8, 30% glycerol, 10% SDS, 0.035% bromphenol blue, 7.5% β-mercaptoethanol). For cytoplasmic nuclear separation, the NER-PERTM nuclear and cytoplasmic extraction kit from Pierce was utilized. After the cytoplasmic and nuclear fractions had been removed, the remaining insoluble pellet was solubilized with radioimmune precipitation buffer and 10% DNase with mechanical trituration. 30–50 μg of protein were loaded and separated by SDS-PAGE gel electrophoresis. To preserve sumoylated species, cells were lysed in an "anti-desumoylation" buffer containing 1% SDS, 1% Nonidet P-40, 5 mm EDTA, and 1× protease inhibitor mixture in PBS heated to 95 °C and followed by sonication. Blots were probed with the appropriate dilutions of the primary antibodies: anti-FLAG (Sigma), anti-Myc (Santa Cruz Biotechnology), anti-Daxx (Santa Cruz Biotechnology), anti-CHIP (9Ballinger C.A. Connell P. Wu Y. Hu Z. Thompson L.J. Yin L.Y. Patterson C. Mol. Cell. Biol. 1999; 19: 4535-4545Crossref PubMed Scopus (750) Google Scholar), anti-β-actin (Sigma), anti-SUMO-1 (Upstate Cell Signaling), anti-p53 (Santa Cruz Biotechnology), anti-phospho p53 Ser-46 (Cell Signaling), anti-HIPK2 (Santa Cruz Biotechnology), anti-HA (Roche Applied Science), and anti-ubiquitin (Chemicon). All Western blots included in FIGURE 1, FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5, FIGURE 6, FIGURE 7 are representative of at least three blots from three independent experiments with β-actin as the standard loading control.FIGURE 2Co-expression of CHIP reduces Daxx under stress of heat shock. A, COS-7 cells were transiently transfected with FLAG-Daxx or FLAG-Daxx and Myc-CHIP. 24 h after transfection, cells were left untreated or heat shocked at 43 °C for 30 min. The cell lysates were immunoprecipitated (IP) with anti-FLAG resin for immunoprecipitating Daxx, or conversely, immunoprecipitated with anti-Myc resin for immunoprecipitating CHIP. Lysates were analyzed on Western blot (IB) with β-actin as a loading control and probed for Daxx and CHIP with anti-FLAG and anti-Myc respectively. B, COS-7 cells were transiently transfected as in A and then separated into cytoplasmic (cy) and nuclear (n) fractions. These fractions were then immunoprecipitated with anti-FLAG resin for Daxx and immunoblotted with anti-FLAG for Daxx and anti-Myc for CHIP. Immunoblotting for lamin B1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as controls for the nuclear and cytoplasmic compartments respectively. HS, heat shocked.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3The interaction between CHIP and Daxx requires specific domains. A, HEK-293 cells were transiently transfected with FLAG-Daxx WT and Myc-CHIP WT or one of the indicated Myc-CHIP deletion mutants. 24 h after transfection, cells were left untreated or heat shocked at 43 °C for 30 min, lysed, and immunoprecipitated (IP) with anti-FLAG resin for Daxx and then immunoblotted (IB) with anti-Myc for CHIP and anti-FLAG for Daxx. TPR, tetratricopeptide. B, COS-7 cells were transiently transfected with Myc-CHIP WT and FLAG-Daxx WT or the indicated FLAG-Daxx deletion mutants. 24 h after transfection, cells were treated as in A. PML, promyelocytic leukemia protein; FAS, transforming growth factor-β receptor. C, COS-7 cells were transiently transfected with FLAG-CHIP WT and the indicated Myc-Daxx deletion mutants. 24 h after transfection, the cells were treated as in A except for immunoprecipitating with anti-Myc agarose conjugate for Daxx, immunoblotting with anti-Myc for Daxx, and immunoblotting with anti-FLAG for CHIP.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4CHIP ubiquitinates Daxx under the stress of heat shock. A, FLAG-Daxx and increasing amounts of Myc-CHIP were co-expressed in COS-7 cells. 24 h after transfection, cells were left untreated or heat shocked at 43 °C for 30 min and then lysed and separated into soluble (s) and insoluble (p) fractions. IB, immunoblot. B, left panel, in vitro ubiquitination was performed with immunopurified FLAG-Daxx as substrate and the indicated components of the ubiquitination reaction system. E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; Ub, ubiquitin; 3KTR, K29R,K48R,K63R. B, right panel, in vitro ubiquitination was performed using the indicated WT and mutant ubiquitin proteins with immunopurified FLAG-Daxx as the substrate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Endogenous CHIP and Daxx co-immunoprecipitate with the stress of heat shock (HS). A, CHIP+/+ MEFs were left untreated or heat shocked at 43 °C for 30 min and then lysed. The lysates were immunoprecipitated with anti-Daxx, and the co-immunoprecipitating (IP) proteins were then subjected to Western blot (IB) analysis and immunoblotting with anti-Daxx, anti-CHIP, and anti-β-actin as loading control. B, CHIP+/+ MEFs were heat shocked at 43 °C, and samples were collected at 0, 10, 20, 30, 40, and 150 min from the start of heat shock. The nuclear fraction of the lysates was immunoprecipitated with agarose-conjugated anti-Daxx (Santa Cruz Biotechnology). A Western blot of the immunoprecipitated proteins was probed with anti-Daxx (Santa Cruz Biotechnology) and anti-CHIP (Chemicon). C, CHIP+/+ and CHIP−/− MEFs were treated as in A and separated into soluble (SOL) and insoluble (INSOL) fractions. The fractions were immunoblotted with anti-Daxx, anti-CHIP, and anti-β-actin as loading control. The * indicates possible sumoylated Daxx. D, CHIP+/+ and CHIP−/− MEFs treated as in A. The lysates were separated into soluble and insoluble fractions and immunoprecipitated with agarose conjugated anti-Daxx (Santa Cruz Biotechnology). A Western blot of the two fractions was probed with anti-Daxx and anti-ubiquitin (Chemicon). E, CHIP+/+ and CHIP−/− MEFs were untreated or heat shocked at 43 °C and allowed to recover for the indicated times before lysis. The lysates were immunoprecipitated with anti-Daxx resin (Santa Cruz Biotechnology), and the co-immunoprecipitating proteins were analyzed by Western blot and immunoblotted with anti-Sumo-1 (Cell Signaling), anti-Daxx and anti-β-actin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6CHIP interferes with the stress-induced sumoylation of Daxx. A, COS-7 cells were transiently transfected with combinations of Myc-CHIP, FLAG-Daxx, and HA-SUMO-1. 24 h after transfection, the cells were left untreated or heat shocked at 43 °C for 30 min and then lysed and immunoprecipitated (IP) for 3 h with anti-FLAG-conjugated resin (Santa Cruz Biotechnology). The co-immunoprecipitating proteins were analyzed by Western blot (IB) with the indicated probes. B, COS-7 cells were transfected with FLAG-Daxx WT or FLAG-Daxx-K630A/K631A (K630/1A, a sumoylation mutant) and with Myc-CHIP. 24 h after transfection, the cells were left untreated or heat shocked at 43 °C for 30 min and then lysed. Both the soluble (s) and the insoluble (p) fractions were run on an 8% SDS gel and immunoblotted for Daxx with anti-FLAG, for CHIP with anti-Myc, and for β-actin as loading control. C, COS-7 cells treated as in B, and the soluble and insoluble fractions were immunoprecipitated with anti-FLAG for Daxx. The recovered proteins were run on an 8% SDS gel and immunoblotted for ubiquitin (Ub) and CHIP (anti-Myc).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Under conditions of heat shock stress, CHIP interferes with HIPK2 phosphorylation of Ser-46 of p53. A, COS-7 cells were transiently transfected with FLAG-Daxx alone or with Myc-CHIP. 24 h after transfection, the cells were left untreated or heat shocked at 43 °C for 30 min. The cells were then lysed, separated into soluble and insoluble fractions, and immunoprecipitated (IP) for Daxx with anti-FLAG resin. The immunoprecipitated proteins were then run on an 8% SDS gel and probed with anti-FLAG for FLAG-Daxx, anti-HIPK2, anti-phospho p53 Ser-46, and anti-Myc for Myc-CHIP. IB, immunoblot. B, COS-7 cells were plated on slides and then transiently transfected with WT FLAG-Daxx alone or with Myc-CHIP and with the HIPK2 binding mutant FLAG-Daxx 154–740, alone or with Myc-CHIP. The cells were heat shocked for 30 min at 45 °C and then allowed to recover at 37 °C for 16 h. They were then stained, and 10 random fields were analyzed for TUNEL-positive apoptotic cells. Student's t test assuming equal variance gave p = 0.0002 for * and p = 0.003 for **. C, CHIP+/+ and CHIP−/− MEFs were heat shocked at 43 °C for 30 min and allowed to recover for the indicated times. The cell lysates were then run on a 12% SDS gel and immunoblotted for the indicated proteins.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cell extracts were incubated for 3 h at 4 °C with the appropriate agarose-conjugated antibody: EZ view anti-FLAG-AG (Sigma), anti-Myc-AG (Santa Cruz Biotechnology), or anti-Daxx-AG (Santa Cruz Biotechnology). After three washes, the immunoprecipitated proteins were recovered by boiling in SDS sample buffer and analyzed by Western blot using the appropriate antibodies. MEFs were grown on coverslips, fixed with 3.7% formaldehyde, rinsed with PBS, blocked, and permeabilized with 1% bovine serum albumin, 0.5% Triton X-100 in PBS. They were then incubated with anti-Daxx (Santa Cruz Biotechnology) for 1.5 h, washed twice with PBS, 1% bovine serum albumin, 0.05% Triton X-100, and then incubated in rhodamine-conjugated goat anti-rabbit (Molecular Probes) for 45 min, washed three times in PBS and then washed for 5 min in 4′,6-diamidino-2-phenylindole (100 ng/ml) to stain the nuclei. The cells were then dehydrated, mounted in Prolong Gold antifade mounting medium (Invitrogen), and viewed using a Nikon Eclipse E800 upright fluorescent microscope utilizing Qcapture (QImaging Corp.) and IPLab (Scanalytics Inc.) software. Nuclei from cells in 10 random fields were scored for the number of Daxx speckles. COS-7 cells were transiently transfected with FLAG-Daxx. Cells were lysed (50 mm Tris, pH 7.4 with 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, and protease inhibitor mixture) and then incubated with anti-FLAG resin for 1.5 h at 4 °C. After three lysis buffer washes, the resin was incubated with FLAG peptide (100 μg/ml) to chase off FLAG-Daxx. Excess FLAG peptide was cleared from the supernatant by centrifugation through a Centricon YM-30 (Millipore). In vitro ubiquitination assays were performed as described previously (15Qian S.B. McDonough H. Boellmann F. Cyr D.M. Patterson C. Nature. 2006; 440: 551-555Crossref PubMed Scopus (287) Google Scholar). Briefly, the immunopurified FLAG-Daxx was incubated for 2 h at 30 °C in the presence of 4 μm CHIP, 0.1 μm purified human ubiquitin-activating enzyme (E1), 2 μm UbcH5a, 50 μm ubiquitin, 2 mm Mg2+, and 5 mm ATP. The reactions were quenched by the addition of 1× loading buffer. COS-7 cells that had been plated on slides and then transiently transfected were analyzed for TUNEL-positive cells using the Promega DeadEnd fluorometric TUNEL system following the manufacturer's instructions. Ten randomly chosen fields were analyzed for each transfection condition. Total RNA was extracted from MEFs using the Qiagen RNeasy Plus mini kit. RNA integrity was verified by assay on an Agilent Bioanalyzer 2100. 500 ng of MEF total RNA were labeled with cyanine-5 CTP in a T7 transcription reaction using the Agilent low input linear RNA amplification/labeling system. Labeled cRNA from test samples was hybridized to Agilent G4122F mouse 4 × 44,000 microarray slides in the presence of equimolar concentrations of cyanine-3 CTP-labeled mouse reference RNA prepared from pools of 1-day-old mouse pups (16He X.R. Zhang C. Patterson C. BioTechniques. 2004; 37: 464-468Crossref PubMed Google Scholar). Microarray data (n = 24 arrays) were loess-normalized (17Riva A. Carpentier A.S. Torrésani B. Hénaut A. Comput Biol. Chem. 2005; 29: 319-336Crossref PubMed Scopus (22) Google Scholar, 18Smyth G.K. Speed T. Methods. 2003; 31: 265-273Crossref PubMed Scopus (1482) Google Scholar), and probes were filtered for features having a normalized intensity of <30 arbitrary fluorescence units in either channel. A probe was removed if <70% of the data were present across all samples. Missing data points were imputed using the k nearest neighbors algorithm (k = 3). 41,174 probes passed these filters and were subsequently used for analysis. Scripts written in the R Statistical Language and Environment (R; version 2.2.1, build r36812, release date 12/20/2005) and Perl (ActiveState Perl 5.8.1, build 807, release date 11/6/2003) were used to standardize (μ = 0, σ = 1) the data set. Lists of differentially expressed genes were identified using the statistical analysis of microarray algorithm (17Riva A. Carpentier A.S. Torrésani B. Hénaut A. Comput Biol. Chem. 2005; 29: 319-336Crossref PubMed Scopus (22) Google Scholar, 19Storey J.D. Tibshirani R. Methods Mol. Biol. 2003; 224: 149-157PubMed Google Scholar, 20Storey J.D. Tibshirani R. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 9440-9445Crossref PubMed Scopus (7083) Google Scholar, 21Tusher V.G. Tibshirani R. Chu G. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 5116-5121Crossref PubMed Scopus (9751) Google Scholar, 22Yu H. Gao L. Tu K. Guo Z. Gene. 2005; 352: 75-81Crossref PubMed Scopus (56) Google Scholar) with a typical false discovery rate of 10% and custom R scripts written in our laboratory. Unsupervised, semisupervised, and supervised clustering analysis was performed on gene lists essentially as described (23Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 14863-14868Crossref PubMed Scopus (13204) Google Scholar) using Cluster (version 2.11, available from the Eisen laboratory). Heat maps of cluster analyses were visualized with Java TreeView (version 1.0.12, release date 3/14/2005). High level pathway analysis and mapping to gene ontology (Gene Ontology (GO) Home) categories and identification of predicted transcription factor binding sites were performed on gene lists using GATHER (Gene Annotation Tool to Help Explain Relationships) (24Chang J.T. Nevins J.R. Bioinformatics. 2006; 22: 2926-2933Crossref PubMed Scopus (277) Google Scholar). The Chilibot (25Chen H. Sharp B.M. BMC Bioinformatics. 2004; 5: 147Crossref PubMed Scopus (263) Google Scholar) contextual data mining algorithm was used for text mining of the PubMed data base with selected genes and keywords. Differential expression values as a function of time were calculated, and the significance analysis of microarray (10% false discovery rate threshold) algorithm was used to identify genes that were significantly differentially expressed between 30 and 240 min in the heat shocked CHIP−/− when compared with the CHIP+/+ cells. The 30 and 240 min after heat shock treatments were then normalized by their respective unshocked controls. This gene list was subsequently subjected to higher order analysis using the GATHER algorithm to associate the differentially expressed genes with significantly over-represented predicted transcription factor binding sites (26Al'Qteishat A. Gaffney J. Krupinski J. Rubio F. West D. Kumar S. Kumar P. Mitsios N. Slevin M. Brain. 2006; 129: 2158-2176Crossref PubMed Scopus (109) Google Scholar). The complete data set is available online through the Gene Expression Omnibus (record GSE14339) at www.ncbi.nlm.nih.gov/geo. Having observed changes in Daxx localization and steady-state levels coincident with the ectopic expression of CHIP (5Hwang J.R. Zhang C. Patterson C. Cell Stress Chaperones. 2005; 10: 147-156Crossref PubMed Scopus (77) Google Scholar), we compared Daxx levels in CHIP+/+ MEFs and CHIP−/− MEFs to examine the relationship of these proteins in a more physiological setting. MEFs were grown on coverslips and left untreated or exposed to heat shock for 30 min at 43 °C and then fixed and stained for Daxx. Although the well documented speckle appearance of Daxx (27Nefkens I. Negorev D.G. Ishov A.M. Michaelson J.S. Yeh E.T. Tanguay R.M. Müller W.E. Maul G.G. J. Cell Sci. 2003; 116: 513-524Crossref PubMed Scopus (66) Google Scholar) was observed in both cell strains, there were significantly more (Student's t test p < 0.0001) and larger speckles in the CHIP−/− MEFs when compared with the CHIP+/+ MEFs (Fig. 1, A and B). Western blotting demonstrated similar differences in endogenous soluble Daxx levels between CHIP+/+ MEFs and CHIP−/− MEFs (Fig. 1C). Following heat shock, the CHIP−/− MEFs displayed much brighter Daxx fluorescence in both the nuclei and the cytoplasm when compared with CHIP+/+ MEFs. Collectively, these data suggest a CHIP-dependent regulation of distribution and abundance of endogenous Daxx under both steady-state and stress conditions. Having observed a decrease in soluble Daxx coincident with CHIP expression, we addressed whether a direct stress-dependent interaction occurred between Daxx and CHIP. COS-7 cells were transiently transfected either with FLAG-tagged Daxx (FLAG-Daxx) alone or with FLAG-Daxx in combination with Myc-tagged CHIP (Myc-CHIP). 24 h after transfection, the cells were left untreated or exposed to heat shock. Western blot ana