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• W1988028386 abstract "Terminal differentiation of hematopoietic cells follows a precisely orchestrated program of transcriptional regulatory events at the promoters of both lineage-specific and ubiquitous genes. Here we show that the transcription factor ATF2 is associated with the induction of granulocytic differentiation, and the molecular interaction of ATF2 with a tissue-specific coactivator activating signal cointegator-2 (ASC-2) potentiates the differentiation procedure. All-trans retinoic acid (RA) induced the phosphorylation and expression of ATF2 in the early and middle phase of granulocyte differentiation, respectively. The activation of granulocyte-specific gene expression is increased with the concerted action of another basic regionleucine zipper factor, CCAAT/enhancer-binding protein (C/EBPα), and ASC-2, which function in a cooperative manner. The interaction between ATF2 and C/EBPα in RA-treated cells was enhanced by the ectopic expression of ASC-2. ATF2-mediated transactivation was also increased by co-transfection of ASC-2. This resulted from the direct protein interaction that the N-terminal transactivation domain of ATF2 interacts with the central region of ASC-2. Furthermore, the molecular interaction of ATF2 and ASC-2 was stimulated by RA treatment and inhibited by p38β kinase inhibitor. Taking these results together, these results suggest that the differentiation-dependent expression and phosphorylation of ATF2 protein physically and functionally interacts with C/EBPα and coativator ASC-2 and synergizes to induce target gene transcription during granulocytic differentiation. Terminal differentiation of hematopoietic cells follows a precisely orchestrated program of transcriptional regulatory events at the promoters of both lineage-specific and ubiquitous genes. Here we show that the transcription factor ATF2 is associated with the induction of granulocytic differentiation, and the molecular interaction of ATF2 with a tissue-specific coactivator activating signal cointegator-2 (ASC-2) potentiates the differentiation procedure. All-trans retinoic acid (RA) induced the phosphorylation and expression of ATF2 in the early and middle phase of granulocyte differentiation, respectively. The activation of granulocyte-specific gene expression is increased with the concerted action of another basic regionleucine zipper factor, CCAAT/enhancer-binding protein (C/EBPα), and ASC-2, which function in a cooperative manner. The interaction between ATF2 and C/EBPα in RA-treated cells was enhanced by the ectopic expression of ASC-2. ATF2-mediated transactivation was also increased by co-transfection of ASC-2. This resulted from the direct protein interaction that the N-terminal transactivation domain of ATF2 interacts with the central region of ASC-2. Furthermore, the molecular interaction of ATF2 and ASC-2 was stimulated by RA treatment and inhibited by p38β kinase inhibitor. Taking these results together, these results suggest that the differentiation-dependent expression and phosphorylation of ATF2 protein physically and functionally interacts with C/EBPα and coativator ASC-2 and synergizes to induce target gene transcription during granulocytic differentiation. The pluripotent blood stem cells mature by transcription factors that activate lineage-specific genes that are essential to the commitment and development of specific hematopoietic lineages, erythroid, myeloid, or lymphoid cells (1Shivdasani R.A. Orkin S.H. Blood. 1996; 87: 4025-4039Crossref PubMed Google Scholar). The vitamin A metabolite, all-trans retinoic acid, induces the granulocytic differentiation of the promyelocytic cell line U937, similar to HL60. C/EBPα,1 C/EBPϵ, PU.1, etc. have been known to contribute in granulopoiesis (2Yamanaka R. Lekstrom-Himes J. Barlow C. Wynshaw-Boris A. Xanthopoulos K.G. Int. J. Mol. Med. 1998; 1: 213-221PubMed Google Scholar). In particular C/EBPα is essential to granulocyte differentiation in an early stage, and in vitro it forms a heterodimeric DNA-binding complex with another transcription factor of the basic region-leucine zipper family, ATF2 in liver cells (3Shuman J.D. Cheong J. Coligan J.E. J. Biol. Chem. 1997; 272: 12793-12800Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Among the ATF/CREB family, ATF2 (initially called CREBP1, Refs. 4Maekawa T. Sakura H. Kanei-Ishii C. Sudo T. Yoshimura T. Fujisawa J. Yoshida M. Ishii S. EMBO J. 1989; 8: 2023-2028Crossref PubMed Scopus (292) Google Scholar and 5Gaire M. Chatton B. Kedinger C. Nucleic Acids Res. 1990; 18: 3461-3473Crossref Scopus (75) Google Scholar) has been more extensively studied and shown to be ubiquitously expressed with the highest level of expression being observed in the brain. A common characteristic of these factors is the presence of a transcriptional activation domain containing the metal finger structure located in the N-terminal region and basic region-leucine zipper proteins in the C-terminal region (6Matsuda S. Maekawa T. Ishii S. J. Biol. Chem. 1991; 266: 18188-18193Abstract Full Text PDF PubMed Google Scholar). ATF2 is capable of forming homodimers or heterodimers with c-Jun for binding to cAMP-response element (CRE) (5′-TGACGTCA-3′) (6Matsuda S. Maekawa T. Ishii S. J. Biol. Chem. 1991; 266: 18188-18193Abstract Full Text PDF PubMed Google Scholar). In particular, ATF2 is known to play an important role in inducing cell differentiation including cardiomyocyte (7Monzen K. Hiroi Y. Kudoh S. Akazawa H. Oka T. Takimoto E. Hayashi D. Hosoda T. Kawabata M. Miyazono K. Ishii S. Yazaki Y. Nagai R. Komuro I. J. Cell Biol. 2001; 153: 687-698Crossref PubMed Scopus (127) Google Scholar), adipogenesis in early stage (8Lee M. Kong H.J. Cheong J. Biochem. Biophys. Res. Commun. 2001; 281: 1241-1247Crossref PubMed Scopus (20) Google Scholar), and central nervous system development (9Reimold A.M. Grusby M.J. Kosaras B. Fries J.W. Mori R. Maniwa S. Clauss I.M. Collins T. Sidman R.L. Glimcher M.J. Glimcher L.H. Nature. 1996; 379: 262-265Crossref PubMed Scopus (240) Google Scholar). Some cofactors, structural modification, and phosphorylation were known to influence the transcriptional activation of ATF2. For example, TIP49b was reported as a regulator of ATF2 response to stress and DNA damage (10Cho S.G. Bhoumik A. Broday L. Ivanov V. Rosenstein B. Ronai Z. Mol. Cell Biol. 2001; 21: 8398-8413Crossref PubMed Scopus (67) Google Scholar). ATF2 exhibits intramolecular inhibitory interaction between N-terminal transactivation domain and C-terminal DNA-binding domain under normal growth conditions (11Li X.Y. Green M.R. Genes Dev. 1996; 10: 517-527Crossref PubMed Scopus (106) Google Scholar). Stability, transcriptional activity, and histone acetyltransferase activity of the ATF2 transcription factor are regulated by phosphorylation and dephosphorylation (12Fuchs S.Y. Tappin I. Ronai Z. J. Biol. Chem. 2000; 275: 12560-12564Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 13Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (473) Google Scholar, 14Kawasaki H. Schiltz L. Chiu R. Itakura K. Taira K. Nakatani Y. Yokoyama K.K. Nature. 2002; 405: 195-200Crossref Scopus (223) Google Scholar). In response to various stressors, ATF2 has been shown to be phosphorylated on amino acid residues Thr-69, Thr-71 by stress-activated protein kinases, Jun-N-terminal kinase (JNK, Refs. 15van Dam H. Wilhelm D. Herr I. Steffen A. Herrlich P. Angel P. EMBO J. 1995; 14: 1798-1811Crossref PubMed Scopus (569) Google Scholar and 16Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1337) Google Scholar), p38 mitogen-activated protein kinase (17Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R.J. Mol. Cell Biol. 1996; 6: 1247-1255Crossref Scopus (1143) Google Scholar), and extracellular single-regulated kinases (ERK, Ref. 18Ouwens D.M. de Ruiter N.D. van der Zon G.C. Carter A.P. Schouten J. van der Burgt C. Kooistra K. Bos J.L. Maassen J.A. van Dam H. EMBO J. 2002; 21: 3782-3793Crossref PubMed Scopus (185) Google Scholar) and also by Thr-73 by Ca2+-/calmodulin-dependent protein kinase IV (CaMKIV) (19Ban N. Yamada Y. Someya Y. Ihara Y. Adachi T. Kubota A. Watanabe R. Kuroe A. Inada A. Miyawaki K. Sunaga Y. Shen Z.P. Iwakura T. Tsukiyama K. Toyokuni S. Tsuda K. Seino Y. Diabetes. 2000; 49: 1142-1148Crossref PubMed Scopus (56) Google Scholar). To stimulate transcriptional activity of specific transcription factors, the cooperative association of transcriptional coactivator CBP/p300 was necessary. Previously, we determined that differentiation-dependent expressed ASC-2 protein physically and functionally interacts with C/EBPα and increases its transactivation activity in granulocyte differentiation (21Hong S. Lee M.Y. Cheong J. Biochem. Biophys. Res. Commun. 2001; 282: 1257-1262Crossref PubMed Scopus (15) Google Scholar). Lee et al. (22Lee S-K. Anzick S.L. Choi J-E. Bubendorf L. Guan X-Y. Jung Y-K. kallioniemi O.P. Kononen J. Trent J.M. Azorsa D. Jhun B-H. Cheong J. Lee Y.C. Meltzer P.S. Lee J.W. J. Biol. Chem. 1999; 274: 34283-34293Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) isolated a novel coactivator ASC-2 by using retinoid X receptor as a bait. ASC-2 was also subsequently identified from several groups named RAP250, PRIP, and TRBP (23Caira F. Antonson P. Pelto-Huikko M. Treuter E. Gustafsson J.A. J. Biol. Chem. 2000; 275: 5308-5317Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 24Ko L. Cardona G.R. Chin W.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6212-6217Crossref PubMed Scopus (128) Google Scholar, 25Zhu Y. Kan L. Qi C. Kanwar Y.S. Yeldandi A.V. Rao M.S. Reddy J.K. J. Biol. Chem. 2000; 275: 13510-13516Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) and was also identical to AIB-3, which was amplified in breast and other human cancers (26Anzick S.L. Kononen J. Walker R.L. Azorsa D.O. Tanner M.M. Guan X.Y. Sauter G. Kallioniemi O.P. Trent J.M. Meltzer P.S. Science. 1997; 277: 965-968Crossref PubMed Scopus (1430) Google Scholar). ASC-2, a typical ligand- and AF2-dependent interactor of nuclear receptors, enhances the receptor transactivation, either alone or in conjunction with SRC-1 and CBP/p300, and functionally interacts with specific transcription factors including peroxisome proliferator-activated receptor-γ, thyroid receptor, NF-κB, AP-1, and serum response factor (27Lee S-K. Na S-Y. Jung S-Y. Jhun B.H. Cheong J. Meltzer P.S. Lee Y.C. Lee J.W. Mol. Endocrinol. 2000; 14: 915-925Crossref PubMed Scopus (60) Google Scholar). To understand the process of normal myeloid differentiation, it is important to identify and characterize the transcription factors that specifically activate important genes in the myeloid lineage. Until now, evidence is wholly lacking that ATF2 is involved in hematopietic differentiation. However, we already reported that retinoic acid activates the p38β kinase pathway leading to phosphorylation and activation of ATF2, thereby enhancing PEPCK gene transcription and glucose production (20Lee M.Y. Jung C.H. Lee K. Choi Y.H. Hong S. Cheong J. Diabetes. 2002; 51: 3400-3407Crossref PubMed Scopus (142) Google Scholar). Therefore, this reflects the indirect evidence that ATF2 may be related in granulocyte differentiation. In this study, we show two novel points that ATF2 is required for the induction of granulocytic differentiation and associates with granulocyte-specific transcription factor C/EBPα by retinoic acid (RA) treatment and that the ASC-2 functions as a coactivator for ATF2 in the differentiation process. These results support that the granulocytic differentiation requires a specific transcription factor-regulated cascade action including a variety of specific transcription factors and coactivators. U937 Cell Culture and Induction of Differentiation—U937 promyelocytic leukemia cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin 100 units/ml, and streptomycin 100 μg/ml (Invitrogen). To induce granulocyte differentiation, the cells in logarithmic growth were seeded at 2 × 105/ml and grown in the presence of 1 μm all-trans RA for up to 4 days. At the end of differentiation experiments, differentiated cells were confirmed with nitro blue tetrazolium (NBT) assay, harvested, and resuspended in an appropriate buffer for each experiments. Reduction of nitro blue tetrazolium by respiratory burst products was assayed with nitro blue tetrazolium tablets (Sigma) in accordance with the manufacturer's protocols. Cells were cytospun and counterstained with safranin. Western Blot Analysis—Cells were harvested on ice-cold lysis buffer (150 mm NaCl, 50 mm Tris-Cl, pH 7.5, 1% Nonidet P-40, 1 mm EDTA, 10% glycerol) containing 1× protease inhibitor at 4 °C. The protein content of cell lysates was determined with Bradford reagent (Bio-Rad) using bovine serum albumin as standard. After heating at 100 °C for 5 min in 1× Laemmli sample buffer, the samples were separated by 10% SDS-PAGE. The resulting gels were either stained with Coomassie Blue or transferred to polyvinylidene difluoride (Immobilon-P) membrane (Millipore). For Western blotting, the membrane was incubated with anti-phospho-ATF2 (New England Biolabs), anti-ATF2, anti-TATA-binding protein, anti-C/EBPα (Santa Cruz Biotechnology), Mac-1 (Caltag) in TBS containing 1% non-fat dried milk for 1 h at room temperature. After washing three times with cold TBS-T (TBS containing 0.04% Tween 20), the blotted membranes incubated with peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 30 min at room temperature. After washing three times with cold TBS-T, the protein bands were visualized by the enhanced chemiluminescence detection system according to the recommended procedure (Amersham Biosciences). Transient and Stable Transfection—HeLa and U937 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) and 1% antibiotics. The cells were seeded in 24-well plates with growth medium and cotransfected with pRSV/β-galactosidase vector and expression vectors for ASC-2 and/or ATF2 by using Superfect (Qiagen) or electroporation. Total amounts of expression vectors were kept constant by adding pcDNA3.1/His C. Relative luciferase and β-galactosidase activities were determined as described (28Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Short Protocols in Molecular Biology. 5th Ed. John Wiley & Sons, Inc., 2002: 9-32Google Scholar). All the transfection results represent the mean of three independent experiments. For establishment of the N-terminal domain ATF2 (ATF2-N)-expressing stable cell line, HeLa cells were transfected with 3 μg of ATF2-N expression plasmid (pcDNA3/hemagglutinin) using calcium phosphate co-precipitation method with BES. At 48 h after transfection, cells were cultured in the presence of 500 μg/ml G418 (Invitrogen). After 21 days in selective medium, individual G418-resistant colonies were isolated. Glutathione S-Transferase (GST) Pull-down Assay between ASC-2 and ATF2—GST fusion proteins were purified as described previously. Equal amounts (∼1 mg) of GST and several GST-ATF2 proteins (1–323, 1–352, 323–352, 323–492, 1–492) immobilized on 20 μl of glutathione-Sepharose 4B beads were incubated with in vitro translated [35S]ASC-2 in the reaction buffer (25 mm HEPES, pH 7.6, 20% glycerol, 100 mm NaCl, 0.2 mm EDTA, 1 mm dithiothreitol, 300 μm phenylmethylsulfonyl fluoride, 1.5% bovine serum albumin) for 4 h at 4 °C. After washing three times with phosphate-buffered saline, the bound proteins were eluted with 10 mm reduced glutathione and boiled with an equal volume of SDS-PAGE sample buffer at 100 °C for 3 min prior to electrophoresis. After electrophoresis, the gel was dried and analyzed with the Molecular Imager Fx (Bio-Rad). Mammalian Two-hybrid Assay between ASC-2 and C/EBP α and ATF2—CV-1 and HeLa Cells were seeded in 24-well plates with growth medium supplemented with 10% fetal bovine serum and 1% antibiotics and co-transfected with expression vectors encoding Gal4-DNA-binding domain fusions (pCMX/Gal4N/-, pCMX/Gal4N-ASC-2 series) and VP16-activation domain fusions (pCMX/VP16/-, pCMX/VP16-C/EBP α, or pCMX/VP16-ATF2) as well as the previously described Gal4-tk-luc reporter plasmid. After 48 h, cells were harvested, and the luciferase activity was normalized to the β-galactosidase expression. All the results represent the average of at least three independent experiments. Co-immunoprecipitation Assay—Cell lysates (500 μg) were incubated with 1 μg of anti-ATF2 antibody at 4 °C for 2 h with gentle agitation. Immune complexes were collected on protein G-Sepharose beads (Invitrogen). After washing three times with radioimmune precipitation buffer (–) buffer (1% Triton X-100, 1% deoxycholate in phosphate-buffered saline), the precipitates were boiled with an equal volume of 2× Laemmli sample buffer at 100 °C for 3 min and analyzed by SDS-PAGE. Chromatin Immunoprecipitation Analysis—Cells were lysed for 5 min in L1 buffer (50 mm Tris, pH 8.0, 2 mm EDTA, 0.1% Nonidet P-40, and 10% glycerol) supplemented with protease inhibitors. Nuclei were pelleted at 3000 rpm and resuspended in L2 buffer (50 mm Tris, pH 8.0, 0.1% SDS, and 5 mm EDTA). Chromatin was sheared by sonication, centrifuged, and diluted 10 times in dilution buffer (50 mm Tris, pH 8.0, 0.5% Nonidet P-40, 0.2 m NaCl, and 0.5 mm EDTA). Extracts were precleared for 3 h with 60 μl of a 50% suspension of salmon sperm-saturated protein A-agarose. Immunoprecipitations were carried out overnight at 4 °C. Immunocomplexes were collected with salmon sperm-saturated protein A for 30 min and washed three times (5 min each) with high-salt buffer (20 mm Tris, pH 8.0, 0.1% SDS, 1% Nonidet P-40, 2 mm EDTA, and 0.5 m NaCl) followed by three washes in no salt buffer (1× Tris/EDTA buffer). Immunocomplexes were extracted in 1× Tris/EDTA buffer containing 2% SDS, and protein-DNA cross-links were reverted by heating at 65 °C overnight. After proteinase K digestion, DNA was extracted with phenol-chloroform and precipitated in ethanol. About one-twentieth of the immunoprecipitated DNA was used in each PCR. Quantitative duplex PCR assay was performed to analyze the amount of DNA precipitated by specified antibodies in proportion to input DNA. Two pairs of primers were used: forward (5′-TTGGGCGGGTTGCAGCAGGCA-3′) and reverse (5′-GTCTGTATTCATGATTCTTC-3′) for the G-CSF promoter. The PCR conditions were as follows: 1.25 units of TaqDNA polymerase (Amersham Biosciences), 100 ng of each primer, 200 μm dNTP, 2.5 μl of 10× Taq buffer, and double-distilled water to a final volume of 25 μl. The cycles were as follows: 94 °C for 180 s; 34 cycles at 94 °C for 45 s, 60 °C for 60 s and 72 °C for 60 s; final elongation at 72 °C for 10 min. Protein Expression of ATF2 Is Induced by Exposure to RA in U937 Cells—Human U937 cells undergo differentiation in response to RA and have a commitment of mature granulocytic cells, suggesting that granulocytic differentiation of U937 cells may also require responsivenesses against distinct transcription factors by RA signaling. It is known that C/EBPα is expressed during early granulocytic differentiation induced by RA exposure. In addition to this, our previous two different results showed that C/EBPα physically and functionally interacts with ATF2 in vitro and in vivo in liver cells (3Shuman J.D. Cheong J. Coligan J.E. J. Biol. Chem. 1997; 272: 12793-12800Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The other finding is that RA increased ATF2-driven transactivation by inducing the phosphorylation and inhibiting the intramolecular interaction of ATF2 itself, although HepG2 cells were used (20Lee M.Y. Jung C.H. Lee K. Choi Y.H. Hong S. Cheong J. Diabetes. 2002; 51: 3400-3407Crossref PubMed Scopus (142) Google Scholar). These results prompted us to examine whether ATF2 acts on RA-induced granulocytic differentiation of U937 cells. From the result of Fig. 1, the protein expression level of ATF2 was examined with Western blot analysis by using antibody against the full-length ATF2 during the granulocytic differentiation of U937 cells by exposure to RA. The protein expression of ATF2 was very low in untreated U937 cells, almost at a background level, but its expression was gradually induced after RA treatment. The time course study showed that the induction of ATF2 was detectable on day 2, and the level was slightly increased until day 4. Therefore, it can be considered that ATF2 is one of the markers for granulocytic differentiation. The level of C/EBPα protein was increased at early times after RA treatment, although C/EBPα is highly expressed and transcriptionally active in untreated U937 cells. Consistent with the finding of ASC-2 in blood cells (25Zhu Y. Kan L. Qi C. Kanwar Y.S. Yeldandi A.V. Rao M.S. Reddy J.K. J. Biol. Chem. 2000; 275: 13510-13516Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), the protein expression of ASC-2 here may provide a clue for a functional role in granulocytic differentiation. ASC-2 Functions as a Coactivator for ATF2-dependent Transactivation of Granulocyte-specific Gene Expression—Previously, we presented the conclusion that the coactivator ASC-2 specifically interacts with C/EBPα during granulocytic differentiation and was also known to induce the protein expression during the granulocytic differentiation. In addition to these results, the quantitative changes of ATF2 protein were observed in Fig. 1. Taken together, these results prompted us to examine whether ASC-2 functionally helps the transactivation activity with ATF2. First, the transactivation of ASC-2 on ATF2-mediated transactivation was examined. Second, ASC-2 increased the transactivation of ATF2 synergistically in a dose-dependent manner (Fig. 2A). As the stimulatory effects of RA are specific to the onset of the differentiation program, we examined the effect of RA treatment and expression of the granulocyte colony-stimulating factor receptor (G-CSFR) in the differentiation cascade, and the ability of the ATF2 to influence transcription from extended regions of G-CSFR promoters was assessed directly in transient transfection assays (Fig. 2B). In U937 cells, ATF2 and a combination of ATF2- and ASC-2 induced gene expression from the G-CSFR promoter between 3- and 11-fold (Fig. 2B). By contrast, c-Jun failed completely to activate transcription from the G-CSFR promoter. RA treatment had only a modest stimulatory effect on basal transcription from the G-CSFR promoter but a significant additional effect on expression induced by ATF2. In the presence of ATF2 and a combination of ATF2 and ASC-2, RA treatment enhanced expression from the G-CSFR promoter 22-fold while enhancing expression dependent on ATF2 alone a more modest 10-fold. Identification of the Protein-Protein Interaction of ATF2 and ASC-2 and Each Interacting Region—The association between ATF2 and ASC-2 was characterized by co-immunoprecipitation analysis. ATF2 was immunoprecipitated from freshly prepared RA-treated U937 cells, gel-fractionated, and analyzed for ASC-2 coprecipitation by Western blotting with anti-ASC-2 IgG. As shown in Fig. 3A, endogenous ASC-2 was detected as a coprecipitant in ATF2 immunoprecipitates. A parallel immunoprecipitated formed with preimmune serum failed to sow ASC-2 immunoreactivity. This result suggests that ASC-2 can stimulate the ATF2 transactivation through direct protein-protein interaction. To further characterize the interacting region of ATF2 against ASC-2 in vitro, GST pull-down assay was performed. GST fusion proteins encoding the full-length, 1–323 amino acids, and 323–492 amino acids of ATF2 were expressed in Escherichia coli, immobilized on glutathione-Sepharose 4B beads, and incubated with 35S-labeled full-length ASC-2 produced by an in vitro translation system. The N-terminal-containing transactivation domain of ATF2 interacts with ASC-2 protein (Fig. 3B). The reciprocal strategy was used to delineate the region of ASC-2 required for interaction with ATF2 using the mammalian two-hybrid assay. For this study, Gal4-ASC-2 and VP16-ATF2 expression vectors were transfected into U937 cells. The 849–1057 region of ASC-2 including LXXLL the motif was interacted with ATF2 (Fig. 3C), similarly to the region bound by another transcription factor. As shown by a mammalian two-hybrid assay and GST pull-down assay, the central domain of ASC-2 specifically interacts with the N-terminal transactivation domain of ATF2. The Coactivator ASC-2 Enforces the Interaction between ATF2 and C/EBP a—From the previous results, ATF2 and C/EBPα associate with ASC-2 in the process of granulocytic differentiation and interact with each other. These results prompted us to examine whether the temporal expression of ASC-2 affects the intermolecular interaction of ATF2 and C/EBPα in U937 cells. For this, in the absence or presence of ASC-2 transfection, the mammalian expression plasmids for ATF2 and C/EBPα were ectopically expressed in U937 cells. As shown in Fig. 4A, ASC-2 expression significantly increased the protein interaction of ATF2 and C/EBPα. In addition to these, we already observed that the 849–1057 region of ASC-2 interacts with ATF2 in Fig. 3C and that the 392–930 region of ASC-2 interacts with C/EBPα (data not shown). Next, we examined the dominant negative function of the 849–1057 domain or the 392–930 domain of ASC-2 in the interaction between ATF2 and C/EBPα. As shown by a mammalian two-hybrid assay, ASC-2 increased the interaction of ATF2 and C/EBPα (Fig. 4A, lane 5) and the 849–1057 region of ASC-2, the binding region to ATF2, or the 392–930 region of ASC-2, the binding region of C/EBPα, inhibited the interaction as a dominant negative mutant (Fig. 4A, lanes 6 and 7). These results support that the coactivator ASC-2 specifically enforces the interaction between ATF2 and C/EBPα. To further confirm that ASC-2 induces a functional transcriptional protein complex with ATF2 and C/EBPα at the granulocytic target gene promoter site, it was addressed whether these factors interact and are assembled on promoters in cells by chromatin immunoprecipitation assays with endogenous G-CSFR promoters as well as endogenous transcription factor proteins. After RA treatment, cells were lysed, and solubilized chromatin was immunoprecipitated, initially with antibodies against Myo-D ATF2, C/EBPα, or ASC-2, and recovered DNAs were amplified by PCR using promoter-specific primers. It is clear from the data in Fig. 4B that ASC-2 recruitment to the -ATF2 and C/EBPα-recognized promoter was confirmed in cells but not by Myo-D. Collectively, these findings support the notion that RA-induced differentiation controls the recruitment of essential components of the transcriptional activation machinery and consequently the efficiency of ATF2-dependent transcriptional activation of G-CSFR, which is one of the granulopoietic genes. RA Potentiates the Physical Association of ATF2 and C/EBPα in Granulocytic Differentiation—The results shown in Fig. 4 indicate that the temporally increased coactivator increases the association of ATF2 and C/EBPα. The ASC-2 expression was induced in the process of RA-dependent granulocytic differentiation (Fig. 1). To investigate the possibility that the differentiation inducer, RA, regulates the similar increased protein association of ATF2 and C/EBPα as shown in ASC-2 expression, the same experimental strategy was applied with RA treatment. The synergistic transactivation effect of RA on ATF2 and C/EBPα transactivation prompted us to examine the direct effect on the protein-protein interaction of ATF2 and C/EBPα by RA. To identify the possibility, we applied the mammalian two-hybrid assay with the cognate ATF2 and C/EBPα constructs (Fig. 5). The physical interaction of ATF2 and C/EBPα was increased considerably by the treatment of 1 μm RA, but not Me2SO, as a vehicle. ATF2 and p38β Kinase Are Phosphorylated by RA during Granulocytic Differentiation—p38β kinase activity is high during the initial stages of differentiation but drastically lower as the U937 leukemic cells undergo terminal differentiation into granulocytes. To identify the phosphorylation control of ATF2 in differentiation, we applied the appearance of phosphorylated ATF2 protein after induction of granulocyte differentiation. The ability to stimulate ATF2 phosphorylation with differentiation-inducing agents clearly points to a role for ATF2 in granulopoiesis. The degree of phosphorylation of ATF2 by p38β kinase, which displayed earlier than the protein expression of ATF2, increased up to 4-fold (Fig. 6). To confirm these results further, the effect of the specific inhibitor of p38β, the pyridinyl imidazole derivative SB203580, on the granulocyte differentiation-induced phosphorylation of ATF2 was examined. Since the SB203580 treatment almost blocked the differentiation procedure, it was not detected in the phosphorylated ATF2 protein dependent on the differentiation (data not shown). To further confirm that p38β kinase activation is involved in the RA response, we assayed for the activation of p38β kinase itself by phosphorylation of the kinase following RA treatment. Total cell extracts were prepared at the times indicated and assayed for expression and phosphorylation of p38β kinase. As shown in Fig. 6, although p38β kinase expression levels do not change, its phosphorylation state increased from 1 day after RA treatment. This time course correlates with the phosphorylation of ATF2 as shown in Fig. 6. Hence, these data provide direct evidence that RA stimulation leads to p38β kinase activation and ATF2 phosphorylation, potentiating the transactivation activity of ATF2. RA Treatment and p38β Expression Increases ATF2 Transactivation by AS" @default.
• W1988028386 created "2016-06-24" @default.
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• W1988028386 date "2004-04-01" @default.
• W1988028386 modified "2023-09-29" @default.
• W1988028386 title "Activation and Interaction of ATF2 with the Coactivator ASC-2 Are Responsive for Granulocytic Differentiation by Retinoic Acid" @default.
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