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• W1990786539 abstract "The aberrant association of promyelocytic leukemia protein-retinoic acid receptor-α (PML-RARα) with corepressor complexes is generally thought to contribute to the ability of PML-RARα to regulate transcription. We report here that PML-RARα acquires aberrant association with coactivators. We show that endogenous PML-RARα interacts with the histone acetyltransferases CBP, p300, and SRC-1 in a hormoneindependent manner, an association not seen for RARα. This hormone-independent coactivator binding activity requires an intact ligand-binding domain and the NR box of the coactivators. Confocal microscopy studies demonstrate that exogenous PML-RARα sequesters and colocalizes with coactivators. These observations correlate with the ability of PML-RARα to attenuate the transcription activation of the Notch signaling downstream effector, CBF1, and of the glucocorticoid receptor. This includes attenuation of the glucocorticoid-induced leucine zipper (GILZ) and FLJ25390 target genes of the endogenous glucocorticoid receptor. Furthermore, treatment of NB4 cells with all-trans-retinoic acid, which promotes PML-RARα degradation, resulted in increased activation of GILZ. On the basis of these findings, we propose a model in which the hormone-independent association between PML-RARα and coactivators contributes to its ability to regulate gene expression. The aberrant association of promyelocytic leukemia protein-retinoic acid receptor-α (PML-RARα) with corepressor complexes is generally thought to contribute to the ability of PML-RARα to regulate transcription. We report here that PML-RARα acquires aberrant association with coactivators. We show that endogenous PML-RARα interacts with the histone acetyltransferases CBP, p300, and SRC-1 in a hormoneindependent manner, an association not seen for RARα. This hormone-independent coactivator binding activity requires an intact ligand-binding domain and the NR box of the coactivators. Confocal microscopy studies demonstrate that exogenous PML-RARα sequesters and colocalizes with coactivators. These observations correlate with the ability of PML-RARα to attenuate the transcription activation of the Notch signaling downstream effector, CBF1, and of the glucocorticoid receptor. This includes attenuation of the glucocorticoid-induced leucine zipper (GILZ) and FLJ25390 target genes of the endogenous glucocorticoid receptor. Furthermore, treatment of NB4 cells with all-trans-retinoic acid, which promotes PML-RARα degradation, resulted in increased activation of GILZ. On the basis of these findings, we propose a model in which the hormone-independent association between PML-RARα and coactivators contributes to its ability to regulate gene expression. Acute promyelocytic leukemia (APL) 3The abbreviations used are: APL, acute promyelocytic leukemia; RARα, retinoic acid receptor-α; PML, promyelocytic leukemia; PLZF, promyelocytic leukemia zinc finger; NPM, nucleophosmin; RXRs, retinoid X receptors; SMRT, silencing mediator for retinoic acid and thyroid hormone receptor; PCAF, p300/CBP-associated factor; ACTR, activator for thyroid hormone and retinoid receptor; CBP, cAMP response element-binding protein-binding protein; LBD, ligand-binding domain; GR, glucocorticoid receptor; ATRA, all-trans-retinoic acid; HA, hemagglutinin; PBS, phosphate-buffered saline; GST, glutathione S-transferase; RID, receptor interaction domain; RT, reverse transcription; GILZ, glucocorticoid-induced leucine zipper. 3The abbreviations used are: APL, acute promyelocytic leukemia; RARα, retinoic acid receptor-α; PML, promyelocytic leukemia; PLZF, promyelocytic leukemia zinc finger; NPM, nucleophosmin; RXRs, retinoid X receptors; SMRT, silencing mediator for retinoic acid and thyroid hormone receptor; PCAF, p300/CBP-associated factor; ACTR, activator for thyroid hormone and retinoid receptor; CBP, cAMP response element-binding protein-binding protein; LBD, ligand-binding domain; GR, glucocorticoid receptor; ATRA, all-trans-retinoic acid; HA, hemagglutinin; PBS, phosphate-buffered saline; GST, glutathione S-transferase; RID, receptor interaction domain; RT, reverse transcription; GILZ, glucocorticoid-induced leucine zipper. is a disease in which a terminal differentiation block of myeloid precursors occurs at the promyelocytic stage of development (1Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2210) Google Scholar). APL pathogenesis has been attributed to aberrant signaling due to a common chromosomal translocation involving the retinoic acid receptor-α (RARα) gene on chromosome 17q21 (2de The H. Lavau C. Marchio A. Chomienne C. Degos L. Dejean A. Cell. 1991; 66: 675-684Abstract Full Text PDF PubMed Scopus (1188) Google Scholar). This chromosomal translocation results in two reciprocal fusion genes that are translated into reciprocal fusion proteins found to be oncogenic. There are five known proteins that create fusions with RARα: promyelocytic leukemia (PML), promyelocytic leukemia zinc finger (PLZF), nuclear matrix-associated (NuMA), nucleophosmin (NPM), and signal transducer and activator of transcription 5β (Stat5β) (2de The H. Lavau C. Marchio A. Chomienne C. Degos L. Dejean A. Cell. 1991; 66: 675-684Abstract Full Text PDF PubMed Scopus (1188) Google Scholar, 3Wells R.A. Catzavelos C. Kamel-Reid S. Nat. Genet. 1997; 17: 109-113Crossref PubMed Scopus (239) Google Scholar, 4Redner R.L. Rush E.A. Faas S. Rudert W.A. Corey S.J. Blood. 1996; 87: 882-886Crossref PubMed Google Scholar, 5Kakizuka A. Miller Jr., W.H. Umesono K. Warrell Jr., R.P. Frankel S.R. Murty V.V. Dmitrovsky E. Evans R.M. Cell. 1991; 66: 663-674Abstract Full Text PDF PubMed Scopus (1282) Google Scholar, 6Arnould C. Philippe C. Bourdon V. Grégoire M.J. Berger R. Jonveaux P. Hum. Mol. Genet. 1999; 8: 1741-1749Crossref PubMed Scopus (230) Google Scholar, 7Goddard A.D. Borrow J. Freemont P.S. Solomon E. Science. 1991; 254: 1371-1374Crossref PubMed Scopus (434) Google Scholar). Among them, >90% of APL patients express a fusion with PML to generate RARα-PML and PML-RARα. Disruption of the retinoid signaling pathway is a key pathogenic feature of APL (8Melnick A. Licht J.D. Blood. 1999; 93: 3167-3215Crossref PubMed Google Scholar, 9Rousselot P. Hardas B. Patel A. Guidez F. Gaken J. Castaigne S. Dejean A. de The H. Degos L. Farzaneh F. et al.Oncogene. 1994; 9: 545-551PubMed Google Scholar, 10Grignani F. Ferrucci P.F. Testa U. Talamo G. Fagioli M. Alcalay M. Mencarelli A. Peschle C. Nicoletti I. et al.Cell. 1993; 74: 423-431Abstract Full Text PDF PubMed Scopus (535) Google Scholar). RARs are members of the nuclear receptor family that controls processes such as development, differentiation, and homeostasis through regulation of complex gene networks. RARs form heterodimers with retinoid X receptors (RXRs) and bind to DNA sequences harboring direct repeats, (A/G)G(G/T)TCA, separated by 5 bp. Transcription regulation by RXR/RAR heterodimers involves the exchange of corepressor and coactivator complexes, which are controlled by the hormone binding status of the receptor (11Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2805) Google Scholar, 12Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. et al.Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6000) Google Scholar). Unliganded RXR/RAR heterodimers bind corepressor complexes to inhibit transcription, whereas hormone-bound RXR/RAR heterodimers dissociate from the corepressors and concomitantly recruit the coactivator complexes, leading to transcription activation. The best known corepressors include SMRT and the nuclear receptor corepressor, which form complexes with mSin3A and histone deacetylases (13Li J. Wang J. Nawaz Z. Liu J.M. Qin J. Wong J. EMBO J. 2000; 19: 4342-4350Crossref PubMed Scopus (498) Google Scholar, 14Jones P.L. Sachs L.M. Rouse N. Wade P.A. Shi Y.B. J. Biol. Chem. 2001; 276: 8807-8811Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), leading to deacetylation of histone tails to generate compact chromatin, thus inhibiting the general transcription machinery from reaching the promoter. The coactivators include members of the p160 family, SRC-1, ACTR/RACK1/pCIP/SRC-3, and TIF2/GRIP1, as well as the potent histone acetyltransferases PCAF and p300/CBP (15Yao T.P. Ku G. Zhou N. Scully R. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10626-10631Crossref PubMed Scopus (392) Google Scholar, 16Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1915) Google Scholar, 17Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1250) Google Scholar, 18Hong H. Kohli K. Trivedi A. Johnson D.L. Stallcup M.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4948-4952Crossref PubMed Scopus (610) Google Scholar, 19Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2039) Google Scholar, 20Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1101) Google Scholar, 21Chakravarti D. LaMorte V.J. Nelson M.C. Nakajima T. Schulman I.G. Juguilon H. Montminy M. Evans R.M. Nature. 1996; 383: 99-103Crossref PubMed Scopus (839) Google Scholar, 22Blanco J.C. Minucci S. Lu J. Yang X.J. Walker K.K. Chen H. Evans R.M. Nakatani Y. Ozato K. Genes Dev. 1998; 12: 1638-1651Crossref PubMed Scopus (333) Google Scholar). In contrast to the action of histone deacetylases, histone acetyltransferases acetylate histone tails, resulting in weaker associations of histones with chromatin and creating a local environment conducive for binding of the general transcription machinery to the target promoter. These notions are consistent with the observations that histone acetylation is linked to transcription activation and that deacetylation is associated with transcription repression. Molecular and structural studies of the nuclear receptor ligand-binding domain (LBD) of RXRα indicate that, in the absence of hormone, helix 12 of the LBD is held in a position extended away from the rest of the LBD (23Egea P.F. Mitschler A. Rochel N. Ruff M. Chambon P. Moras D. EMBO J. 2000; 19: 2592-2601Crossref PubMed Google Scholar). In this conformation, corepressors bind receptors via a common motif, (I/L)XX(I/V)I (where X is any amino acid), which is known as the CoRNR box (24Hu X. Lazar M.A. Nature. 1999; 402: 93-96Crossref PubMed Scopus (514) Google Scholar, 25Nagy L. Kao H.-Y. Love J.D. Li C. Banayo E. Gooch J.T. Krishna V. Chatterjee K. Evans R.M. Schwabe J.W. Genes Dev. 1999; 13: 3209-3216Crossref PubMed Scopus (341) Google Scholar, 26Perissi V. Staszewski L.M. McInerney E.M. Kurokawa R. Krones A. Rose D.W. Lambert M.H. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1999; 13: 3198-3208Crossref PubMed Scopus (418) Google Scholar). Hormone binding induces a conformational change in which helix 12 folds back to contact helixes 3-5 of the LBD (23Egea P.F. Mitschler A. Rochel N. Ruff M. Chambon P. Moras D. EMBO J. 2000; 19: 2592-2601Crossref PubMed Google Scholar, 27Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 375: 377-382Crossref PubMed Scopus (1042) Google Scholar). Consequently, such conformational changes result in dissociation of the corepressors and recruitment of the coactivators via the LXXLL motif present in the coactivators (28Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1742) Google Scholar, 29Darimont B.D. Wagner R.L. Apriletti J.W. Stallcup M.R. Kushner P.J. Baxter J.D. Fletterick R.J. Yamamoto K.R. Genes Dev. 1998; 12: 3343-3356Crossref PubMed Scopus (816) Google Scholar). It has been proposed that these consensus motifs signify a common mechanism of transcription regulation throughout the nuclear receptor family members. The mechanisms underlying transcription regulation by PML-RARα appear to be more complex than those of RXRα/RARα heterodimers. In vitro studies have shown that, in addition to its ability to form heterodimers with RXRα, PML-RARα is capable of forming homodimers (30Jansen J.H. Mahfoudi A. Rambaud S. Lavau C. Wahli W. Dejean A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7401-7405Crossref PubMed Scopus (73) Google Scholar, 31Perez A. Kastner P. Sethi S. Lutz Y. Reibel C. Chambon P. EMBO J. 1993; 12: 3171-3182Crossref PubMed Scopus (286) Google Scholar) that bind corepressors more tightly than does RXRα/RARα and that require retinoic acid concentrations higher than physiological levels to dissociate from corepressors (≥1 μm) (2de The H. Lavau C. Marchio A. Chomienne C. Degos L. Dejean A. Cell. 1991; 66: 675-684Abstract Full Text PDF PubMed Scopus (1188) Google Scholar, 5Kakizuka A. Miller Jr., W.H. Umesono K. Warrell Jr., R.P. Frankel S.R. Murty V.V. Dmitrovsky E. Evans R.M. Cell. 1991; 66: 663-674Abstract Full Text PDF PubMed Scopus (1282) Google Scholar, 31Perez A. Kastner P. Sethi S. Lutz Y. Reibel C. Chambon P. EMBO J. 1993; 12: 3171-3182Crossref PubMed Scopus (286) Google Scholar). Therefore, at physiological concentrations of retinoic acid (10-100 nm), PML-RARα acts as a dominant-negative inhibitor of RARs, leading to a constitutive repression of RAR target genes. This model is supported by transient transfection assays, in which PML-RARα can potently repress transcription when it is tethered to the yeast Gal4 DNA-binding domain (32Lin R.J. Evans R.M. Mol. Cell. 2000; 5: 821-830Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). There is also evidence suggesting that PML-RARα can promote formation of a heterochromatin-like structure to keep target genes repressed through recruitment of histone methyltransferases such as SUV39H1 (33Carbone R. Botrugno O.A. Ronzoni S. Insinga A. Di Croce L. Pelicci P.G. Minucci S. Mol. Cell. Biol. 2006; 26: 1288-1296Crossref PubMed Scopus (92) Google Scholar). However, there is no in vivo evidence to suggest that PML-RARα homodimers compete with RXRα/RARα for DNA binding. Furthermore, recent reports suggested that overexpression of PML-RARα leads to a reduction of nuclear receptor corepressor protein levels (34Khan M.M. Nomura T. Chiba T. Tanaka K. Yoshida H. Mori K. Ishii S. J. Biol. Chem. 2004; 279: 11814-11824Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 35Ng A.P. Howe Fong J. Sijin Nin D. Hirpara J.L. Asou N. Chen C.S. Pervaiz S. Khan M. Cancer Res. 2006; 66: 9903-9912Crossref PubMed Scopus (19) Google Scholar). Likewise, other studies have indicated that, in the absence of hormone, PML-RARα activates retinoic acid response element-mediated reporter activity in the absence of retinoic acid (36Kogan S.C. Hong S.H. Shultz D.B. Privalsky M.L. Bishop J.M. Blood. 2000; 95: 1541-1550Crossref PubMed Google Scholar, 37Hauksdottir H. Privalsky M.L. Cell Growth & Differ. 2001; 12: 85-98PubMed Google Scholar). This differential regulation of transcription is likely promoter- and cell type-specific; however, it highlights the fact that a simple model of constitutive repression of RARα target genes by PML-RARα is not sufficient to explain all of its possible roles in transcription regulation. Although the aberrant association of PML-RARα with corepressors is well characterized, interactions with coactivators have not been well explored, although one study with NPM-RARα suggests that this fusion protein may interact with coactivators in an aberrant manner compared with wild-type RXR/RARα (38Dong S. Qiu J. Stenoien D.L. Brinkley W.R. Mancini M.A. Tweardy D.J. Oncogene. 2003; 22: 858-868Crossref PubMed Scopus (24) Google Scholar). In this study, we show that PML-RARα homodimers acquire the ability to interact with coactivators in a hormone-independent manner. Consistent with these data, we found that ectopic expression of PML-RARα in the absence of hormone is able to induce mislocalization of some coactivators to a non-nuclear compartment. We also found that overexpression of PML-RARα decreases transcription activation by the glucocorticoid receptor (GR) and CBF1. In light of these findings, we propose a distinct mechanism by which PML-RARα regulates transcription not only as a repressor of RARα target genes, but as a factor capable of repressing the activation of a subset of genes whose activation relies on coactivator recruitment. Plasmid Construction—The plasmids pCMX, pCMX-PML(S)-RARα, pCMX-TAN-1, pCBF1-TK-Luc, pCMX-GRα, pGRE-TK-Luc, pCMX-ERα, pERE-TK-Luc, pCMX-MEF2C, and pMEF2-TK-Luc have been described previously (39Hollenberg S.M. Giguere V. Evans R.M. Cancer Res. 1989; 49: 2292s-2294sPubMed Google Scholar, 40Kao H.-Y. Ordentlich P. Koyano-Nakagawa N. Tang Z. Downes M. Kintner C.R. Evans R.M. Kadesch T. Genes Dev. 1998; 12: 2269-2277Crossref PubMed Scopus (486) Google Scholar, 41Schulman I.G. Juguilon H. Evans R.M. Mol. Cell. Biol. 1996; 16: 3807-3813Crossref PubMed Scopus (117) Google Scholar, 42Kao H.-Y. Verdel A. Tsai C.C. Simon C. Juguilon H. Khochbin S. J. Biol. Chem. 2001; 276: 47496-47507Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). pCMX-FLAG-ACTR, pGEX4T-1-ACTR(RID), pBGT9-CBP, pCMX-HA-PML, pCMX-HA-RARα, pCMX-HA-PML(S)-RARα, pCMX-HA-PLZF, pCMX-HA-PLZF-RARα, pCMX-FLAG-PCAF, pGAD-RARα(LBD), and pGAD-PML(S)-RARα were generated by PCR of the corresponding cDNA and subcloning into the indicated vector. Yeast Methods—Yeast two-hybrid assays were carried out according to the protocol of Clontech. Where indicated, yeast cells were grown overnight (16-24 h) in medium containing all-trans-retinoic acid (ATRA). Liquid β-galactosidase activity was assayed, and the values were derived from duplicate experiments with two independent clones. Luciferase Reporter Assays—Transient transfection assays were carried out according to our published protocol (43Kao H.-Y. Han C.C. Korma A.A. Evans R.M. J. Biol. Chem. 2003; 278: 7366-7373Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). CV-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin G, and 50 μg/ml streptomycin sulfate at 37 °C in 7% CO2. For reporter assays, cells (85-95% confluence, 48-well plates) were cotransfected with equal amounts of the corresponding pCMX constructs, not exceeding 1 μg, as well as 100 ng of pCBF1-TK-Luc, pGRE-TK-Luc, pERE-TK-Luc, or pMEF2-TK-Luc and 100 ng of pCMX-LacZ in 200 μl of Opti-MEM I using the transfection reagent Lipofectamine 2000 following a procedure adapted from Invitrogen. CV-1 cells were transfected, washed, and placed in Dulbecco's modified Eagle's medium containing 10% charcoal-stripped fetal bovine serum, 50 units/ml penicillin G, and 50 μg/ml streptomycin sulfate. After 24 h, the medium was replaced or not with 1 μm dexamethasone for GR assays or with 1 μm estradiol for estrogen receptor-α assays or grown in regular Dulbecco's modified Eagle's medium and non-stripped fetal bovine serum for TAN-1 assays. Cells were harvested and assayed for luciferase and β-galactosidase activities 36-48 h after transfection. Luciferase activity was normalized to the level of β-galactosidase activity. Each transfection was performed in triplicate and repeated at least three times. Electroporation of HL-60 cells—HL-60 cells were grown in RPMI 1640 medium supplemented with 10% charcoal-stripped bovine calf serum, 50 units/ml penicillin G, and 50 μg/ml streptomycin sulfate at 37 °C in 7% CO2. For transfection, 7-10 × 106 cells were spun down and resuspended in 200 μl of Opti-MEM I. 60 μg of hemagglutinin (HA)-PML-RARα expression plasmid was added, and the mixture was placed into a 0.2-mm gap electroporation cuvette. The cells were electroporated with a 140-V square-wave pulse for 0.25 ms using the Bio-Rad Gene Pulser system. Following this, the cells were grown 24 h in RPMI 1640 medium and then harvested for immunofluorescence microscopy. Immunoprecipitation—To detect endogenous interactions, NB4 cells were grown in RPMI 1640 medium supplemented with 10% charcoal-stripped bovine calf serum, 50 units/ml penicillin G, and 50 μg/ml streptomycin sulfate at 37 °C in 7% CO2. Lysates were made using NETN buffer (100 mm NaCl, 1 mm EDTA, 20 mm Tris-HCl (pH 8.0), 0.1% Nonidet P-40, 10% glycerol, and 1 mm dithiothreitol) with mixture of protease inhibitors (Sigma) and lysed by sonication. The resulting lysates were immunoprecipitated in NETN buffer for 4 h with 4 μg of antibody (anti-RARα (C-20), anti-SRC-1 (M-341), anti-p300 (N-15), or anti-CBP (A-22); Santa Cruz Biotechnology, Inc.). The immunoprecipitated complexes were resolved by SDSPAGE and immunoblotted overnight with the antibodies indicated in Fig. 4 (anti-RARα, anti-SRC-1, anti-p300, anti-CBP, or anti-PML (H-238)) at a 1:500 dilution in 1× phosphate-buffered saline (PBS)/Tween 20 at 4 °C. Human embryonic kidney 293 cells were transfected with 10 μg of DNA composed of pCMX-FLAG-PCAF, pCMX-FLAG-ACTR, pCMX-HA-PML-RARα, pCMX-RARα, or pCMX alone using Lipofectamine 2000 following the published protocol from Invitrogen. 48 h transfection, whole cell lysates were made using radioimmune precipitation assay buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) plus protease inhibitors and then immunoprecipitated in radioimmune precipitation assay buffer using red anti-FLAG antibody M2 affinity gel (Sigma) for 4 h at 4°C.The immunoprecipitates were analyzed by Western blotting using anti-FLAG (Sigma) and HA-conjugated anti-horseradish peroxidase (Roche Applied Science) antibodies (1:1000 dilution in 1× PBS/Tween 20 for 1 h at room temperature). The corresponding secondary antibodies were used, and visualization of the products was done using an ECL detection kit (Pierce). For immunoprecipitations done in the presence of hormone, cells were grown for 12 h in medium with charcoal-stripped serum supplemented or not with 1 μm ATRA. ATRA was also included in the immunoprecipitation reactions and wash buffer at the same concentration. Immunofluorescence Microscopy—CV-1 and HL-60 cells were transfected with an expression plasmid of HA-PML-RARα. Transfected cells were grown in medium containing stripped serum for an additional 24-48 h. For immunofluorescence microscopy, transfected cells were fixed in 3.7% paraformaldehyde in 1× PBS for 30 min at room temperature and permeabilized in 1× PBS with the addition of 0.1% Triton X-100 and 10% goat serum for 10 min. The cells were washed three times with 1× PBS and incubated in a solution of PBS, 10% goat serum, and 0.1% Tween 20 (buffer A) for 60 min. Incubation with primary antibodies was carried out for 120 min in buffer A. The cells were washed three times with 1× PBS, and the secondary antibodies were added for 30-60 min in the dark at room temperature in buffer A. Coverslips were mounted on slides using VECTASHIELD mounting medium with 4′,6-diamidino-2-phenylindole (H-1200, Vector Laboratories). Imaging was performed on a Leica Model DMLB microscope, and pictures were taken with a SPOT camera using SPOT Advanced software (Diagnostic Instruments, Inc.). The primary antibodies used were as follows: mouse anti-HA monoclonal, rabbit anti-p300 polyclonal (N-15), rabbit anti-CBP polyclonal (A-22), and rabbit anti-SRC-1 polyclonal (M-341) (all from Santa Cruz Biotechnology, Inc.). The secondary antibodies used were Alexa Fluor 594-conjugated anti-mouse and Alexa Fluor 488-conjugated anti-rabbit antibodies (Molecular Probes). Confocal Microscopy—All confocal images were acquired using a Zeiss LSM 510 inverted laser-scanning confocal microscope. A ×63 numerical aperture of a 1.4 oil immersion Plan Apochromat objective was used for all experiments. To investigate the localization of transiently transfected HA-PML-RARα, images of Alexa Fluor 488 were collected using a 488-nm excitation light from an argon laser, a 488-nm dichroic mirror, and 500-550-nm band pass barrier filter. For endogenous coregulator anti-SRC-1, anti-CBP, and anti-p300 antibodies, images of Alexa Fluor 594 were collected using a 633-nm excitation light from a He/Ne2 laser, a 633-nm dichroic mirror, and 650-nm long pass filter. All 4′,6-diamidino-2-phenylindole-stained nuclear images were collected using a Coherent Mira-F-V5-XW-220 (Verdi 5W) Ti-sapphire laser tuned at 750 nm, a 700-nm dichroic mirror, and a 390-465-nm band pass barrier filter. Glutathione S-Transferase (GST) Pulldown Assays—For GST pulldown assays, in vitro translated HA-RARα, HA-PML, HA-PML-RARα, HA-PLZF-RARα, or HA-PLZF was incubated for 30 min on a nutator at room temperature with GST-tagged ACTR receptor interaction domain (RID)-conjugated glutathione-Sepharose beads in NETN buffer with a mixture of protease inhibitors (Sigma) in the presence or absence of ATRA (10-1000 nm). After incubation, the beads were washed three times with NETN buffer and collected by centrifugation. The proteins were eluted and denatured by placing the samples at 100 °C for 5 min and then run on 10% SDS-polyacrylamide gels. The products were visualized by Western blot analysis with HA-conjugated anti-horseradish peroxidase antibody. Reverse Transcription (RT)-PCR—A549 cells were transfected with HA, HA-PML-RARα, or HA-PML-RARα(F584A) using Lipofectamine 2000. Where indicated, samples were treated for 16-20 h with 1 μm dexamethasone before harvesting. One-third of the cells were used to make whole cell lysates using radioimmune precipitation assay buffer plus protease inhibitors. The other two-thirds of the cells were used for RNA isolation. NB4 cells were grown in RPMI 1640 medium supplemented with 10% charcoal-stripped fetal bovine serum, 50 units/ml penicillin G, and 50 μg/ml streptomycin sulfate at 37 °C in 7% CO2. They were either left untreated or treated alone or in combination with 1 μm ATRA for 72 h and 1 μm dexamethasone for 16-18 h. RNA isolation was performed using an RNeasy mini RNA isolation kit (Qiagen Inc.). All procedures were performed according to the manufacturer's protocol. DNase I digestion was performed on an RNeasy column following the manufacturer's protocol using RNase- and DNase-free DNase I (Qiagen Inc.). The isolated RNA was used in a semiquantitative RT-PCR using a one-step RT-PCR kit (Invitrogen) according to the manufacturer's protocol. 500 ng of RNA template was used in each reaction, and PCR amplification was repeated for 40 cycles. The final primer concentration in each reaction was 0.2 μm. Each assay was performed at least three times. The actin primers used as a control were 5′-ggtctcaaacatgatctgggtc-3′ (forward) and 5′-aaatctggcaccacaccttc-3′ (reverse). The GR target gene primers used were as follows: glucocorticoid-induced leucine zipper (GILZ), 5′-agatcgaacaggccatggat-3′ (forward) and 5′-ttacaccgcagaaccaccag-3′ (reverse); and FLJ25390, 5′-ggctcatgctggatgacaa-3′ (forward) and 5′-cccagatggtggagatcagt-3′ (reverse). 4K. R. Yamamoto and A. So, personal communication. The observations that PML-RARα requires higher concentrations of hormone to dissociate from corepressors and that both corepressor- and coactivator-binding domains overlap in the modeled nuclear receptor LBD suggested that PML-RARα may interact with both sets of coregulators differently from wild-type RARα (23Egea P.F. Mitschler A. Rochel N. Ruff M. Chambon P. Moras D. EMBO J. 2000; 19: 2592-2601Crossref PubMed Google Scholar). To investigate this possibility, we characterized the association of PML-RARα with coactivators in solution by GST pulldown experiments. A fusion protein composed of GST attached to the RID of SMRT or ACTR was expressed and purified from bacteria. Purified immobilized GST-SMRT-(RID) or GST-ACTR(RID) fusion protein was incubated with in vitro transcribed and translated RARα, PML, or PML-RARα in the presence and absence of hormone. To our surprise, PML-RARα could be pulled down by GST-ACTR(RID) in both the presence and absence of hormone (Fig. 1A, lanes 4 and 5). No binding was observed with any of the proteins to GST alone. In contrast and as expected, RARα was able to bind to ACTR in a DNA-independent manner, but only after the addition of hormone (Fig. 1B, lane 4 versus lane 5). Also, PML was unable to bind to GST-ACTR(RID) regardless of the presence of hormone (Fig. 1C, lanes 4 and 5). As a control, Fig. 1D shows that the association of PML-RARα with SMRT in this assay is consistent with the well documented association, which requires 1 μm ATRA to induce complete dissociation (lanes 3-5). These results suggest that PML-RARα binds coactivators in solution in both the absence and presence of hormone. To investigate the molecular basis of the hormone-independent association between PML-RARα and coactivators, we performed yeast-two hybrid assays. The yeast Gal4 activation domain (pGAD) was fused to the LBD of RARα, PML, or PML-RARα, whereas ACTR(RID) was fused to the Gal4 DNA-binding domain (pGBT9). The constructs were cotransformed into yeast. The interaction of ACTR(RID) and PML-RARα was assayed by measuring β-galactosidase expression. As shown in Fig. 2A, as controls, coexpression of the pGBT9 vector alone and pGAD-PML-RARα or pGBT9-ACTR(RID) and the pGAD vector alone resulted in only basal reporter activity. Moreover, coexpression of pGBT9-ACTR(RID) and pGAD-PML did not activate reporter activity in the presence or absence of hormone. As expected, coexpression of pGBT9-ACTR(RID) and pGAD-RARα(LBD) resulted in activation of the reporter only in the presence of hormone. However, consistent with our in vitro protein-protein interaction data (Fig. 1), coexpression of pGBT9-ACTR(RID) and pGAD-PML-RARα led to a potent activation of reporter activity in the absence of hormone, indicating a strong a" @default.
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• W1990786539 title "Aberrant Association of Promyelocytic Leukemia Protein-Retinoic Acid Receptor-α with Coactivators Contributes to Its Ability to Regulate Gene Expression" @default.
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• W1990786539 doi "https://doi.org/10.1074/jbc.m700330200" @default.
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