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J. Biol. Chem., Vol. 279, Issue 17, 16996-17003, April 23, 2004

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Activation and Interaction of ATF2 with the Coactivator ASC-2 Are Responsive for Granulocytic Differentiation by Retinoic Acid^*

SunHwa Hong, Hyun Mi Choi, Min Jung Park, Young Hee Kim, Yoon Ha Choi, Hyung Hoi Kim, Young Hyun Choi¶, and JaeHun Cheong||

From the Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan 609-735, the Department of Clinical Pathology, College of Medicine, Pusan National University, Busan 602-739, and the ^¶Department of Biochemistry, College of Oriental Medicine, Dong-Eui University, Busan 614-054, Korea

Received for publication, October 27, 2003 , and in revised form, January 5, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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/EBP), and ASC-2, which function in a cooperative manner. The interaction between ATF2 and C/EBP 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 p38 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/EBP and coativator ASC-2 and synergizes to induce target gene transcription during granulocytic differentiation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (1). The vitamin A metabolite, all-trans retinoic acid, induces the granulocytic differentiation of the promyelocytic cell line U937, similar to HL60. C/EBP,¹ C/EBP, PU.1, etc. have been known to contribute in granulopoiesis (2). In particular C/EBP 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 (3).

Among the ATF/CREB family, ATF2 (initially called CREBP1, Refs. 4 and 5) 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 (6). ATF2 is capable of forming homodimers or heterodimers with c-Jun for binding to cAMP-response element (CRE) (5'-TGACGTCA-3') (6). In particular, ATF2 is known to play an important role in inducing cell differentiation including cardiomyocyte (7), adipogenesis in early stage (8), and central nervous system development (9).

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 (10). ATF2 exhibits intramolecular inhibitory interaction between N-terminal transactivation domain and C-terminal DNA-binding domain under normal growth conditions (11). Stability, transcriptional activity, and histone acetyltransferase activity of the ATF2 transcription factor are regulated by phosphorylation and dephosphorylation (12–14). 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. 15 and 16), p38 mitogen-activated protein kinase (17), and extracellular single-regulated kinases (ERK, Ref. 18) and also by Thr-73 by Ca²⁺-/calmodulin-dependent protein kinase IV (CaMKIV) (19).

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/EBP and increases its transactivation activity in granulocyte differentiation (21). Lee et al. (22) 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 (23–25) and was also identical to AIB-3, which was amplified in breast and other human cancers (26). 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-, thyroid receptor, NF-B, AP-1, and serum response factor (27).

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 p38 kinase pathway leading to phosphorylation and activation of ATF2, thereby enhancing PEPCK gene transcription and glucose production (20). 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/EBP 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 x 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 1x 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 1x 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/EBP (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/-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 -galactosidase activities were determined as described (28). 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 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 , 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 -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 2x 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 (1x Tris/EDTA buffer). Immunocomplexes were extracted in 1x 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 10x 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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/EBP is expressed during early granulocytic differentiation induced by RA exposure. In addition to this, our previous two different results showed that C/EBP physically and functionally interacts with ATF2 in vitro and in vivo in liver cells (3). 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 (20). 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/EBP protein was increased at early times after RA treatment, although C/EBP is highly expressed and transcriptionally active in untreated U937 cells. Consistent with the finding of ASC-2 in blood cells (25), the protein expression of ASC-2 here may provide a clue for a functional role in granulocytic differentiation.

View larger version (41K): [in this window] [in a new window]   FIG. 1. Protein expression of ATF2 in granulocyte differentiation of U937 cells. U937 cells were grown to confluency as described under "Experimental Procedures." The cells were refed with complete growth medium containing 1 µM RA for the times indicated. Approximately 50 µg of cell lysate protein from each sample was separated on 10% SDS-PAGE and transferred to Immobilon-P membranes. Duplicate membranes were subjected to Western blot analysis by using antibodies specific for ATF2, C/EBP, and ASC-2 as indicated. Western blot detection of TATA-binding protein estimated protein-loading control for each lane. AtRA, all-trans retinoic acid; TBP, TATA-binding protein.

View larger version (14K): [in this window] [in a new window]   FIG. 2. The coactivator ASC-2 interacts with ATF2 increasingly depending on granulocytic differentiation. A, HeLa cells were transfected with -galactosidase expression vector and an increasing amount of ATF2 expression vector, either in the absence () or in the presence () of ASC-2, along with 3x CRE-luciferase reporter. B, the mammalian expression plasmids encoding a series of ASC-2, ATF2, and Jun were transfected with G-CSFR reporter plasmid into U937 cells, as indicated. 48 h after transfection in the absence or presence of 1 µM RA, cells were harvested for luciferase activities.

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.

View larger version (20K): [in this window] [in a new window]   FIG. 3. Identification of the protein-protein interaction of ATF2 and ASC-2 and each interacting region. A, ASC-2 coprecipitates with ATF2 from the RA-treated U937 cells nuclear extracts. ATF2 and ASC-2 were immunoprecipitated (IP) from RA-treated U937 cell nuclear extract by incubation with anti-ATF2 IgG. Antibody complexes were captured on protein A-Sepharose. The beads were washed three times with binding buffer and eluted into SDS sample buffer. WB, Western blot. B, a schematic diagram of ATF2 showing different functional domains and regions encoding ATF2. ASC-2 were labeled with [35S]methionine by in vitro translation and incubated with glutathione beads containing GST alone, GST-ATF2 (1–492), GST-ATF2 (1–323), and GST-ATF2 (323–492) as indicated. The bound proteins were resolved by SDS-PAGE and autoradiography. Beads were washed, and specifically bound material was eluted with reduced glutathione and resolved by SDS-PAGE. Approximately 10% of the labeled proteins used in binding reactions were loaded as input. HAT, histone acetyltransferase, bZIP, basic leucine zipper region. C, a schematic representation of ASC-2 polypeptides encoded by expression constructs is shown at the bottom. Domains unique to Q-rich, the receptor interacting domain (RID), and S/T rich are shown by different bars. The mammalian expression plasmids encoding GAL4-ASC-2 series and VP16-ATF2 were transfected into U937 cells, as indicated. 48 h after transfection, cells were harvested for luciferase activities. All transfection results were normalized to -galactosidase activity and represent the average of three independent experiments, with fold induction over the level observed with the reporter alone.

The Coactivator ASC-2 Enforces the Interaction between ATF2 and C/EBP a—From the previous results, ATF2 and C/EBP 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/EBP in U937 cells. For this, in the absence or presence of ASC-2 transfection, the mammalian expression plasmids for ATF2 and C/EBP were ectopically expressed in U937 cells. As shown in Fig. 4A, ASC-2 expression significantly increased the protein interaction of ATF2 and C/EBP. 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/EBP (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/EBP. As shown by a mammalian two-hybrid assay, ASC-2 increased the interaction of ATF2 and C/EBP (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/EBP, 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/EBP.

View larger version (15K): [in this window] [in a new window]   FIG. 4. The ASC-2 enforces the interaction between ATF2 and C/EBP. A, U937 cells were transfected with vectors expressing Gal4-ATF2 and VP16-C/EBP along with 100 ng of a reporter gene Gal4-tk-luc as indicated. Lanes 6 and 7 were added with the 393–930 domain and 849–1057 domain and of ASC-2, respectively. All the transfection results were normalized to -galactosidase activity, and the presented results represented the average of three independent experiments, with fold induction over the level observed with the reporter alone. B, chromatin immunoprecipitation analysis of factor occupancy on G-CSFR promoters. Following formaldehyde cross-linking, soluble chromatin was prepared. After immunoprecipitation with antibodies against the indicated proteins (Myo-D, C/EBP, ATF2, and ASC-2), precipitated DNAs were used in PCR analysis. The input lane shows the starting chromatin extracts.

RA Potentiates the Physical Association of ATF2 and C/EBP in Granulocytic Differentiation—The results shown in Fig. 4 indicate that the temporally increased coactivator increases the association of ATF2 and C/EBP. 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/EBP 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/EBP transactivation prompted us to examine the direct effect on the protein-protein interaction of ATF2 and C/EBP by RA. To identify the possibility, we applied the mammalian two-hybrid assay with the cognate ATF2 and C/EBP constructs (Fig. 5). The physical interaction of ATF2 and C/EBP was increased considerably by the treatment of 1 µM RA, but not Me₂SO, as a vehicle.

View larger version (17K): [in this window] [in a new window]   FIG. 5. RA treatment increases the protein interaction of ATF2 and C/EBP. The mammalian expression plasmids encoding GAL-ATF2 and VP16-C/EBP were transfected into U937 cells, as indicated. Then, 48 h after transfection in the absence or presence of 1 µmol/l RA, cells were harvested for luciferase activities. All the transfection results were normalized to -galactosidase activity, and the presented results represented the average of three independent experiments, with fold induction over the level observed with the report alone. AtRA, all-trans retinoic acid; DMSO, Me2SO.

View larger version (50K): [in this window] [in a new window]   FIG. 6. Phosphorylation of ATF2 and p38 kinase in the early phase of granulocytic differentiation. U937 cells were incubated with 1 µM RA for the indicated times. Total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of ATF2 and p38 kinase. The protein amount of ATF2 and p38 kinase was probed with ATF2- and p38 kinase-specific antibodies as a loading control, respectively.

RA Treatment and p38 Expression Increases ATF2 Transactivation by ASC-2 and the Protein Interaction of ATF2 and ASC-2 in Vivo—To further characterize the functional meaning of ATF2 phosphorylation by p38 kinase, we examined the effects of p38 on the ASC-2-stimulated transactivation of the ATF2-dependent reporter gene. U937cells were transiently transfected with plasmids encoding ATF2, p38, p38m, and/or the coactivator ASC-2, along with CRE reporter plasmid. Ectopic expression of p38 alone did not significantly increase the ATF2-dependent transactivation (Fig. 7A). Interestingly, additional expression of ATF2 with p38 synergistically enhanced the ATF2-driven transactivation 6-fold relative to transfection of p38 alone (Fig. 7A). When compared with this increased -fold effect of co-transfection of ATF2 and ASC-2, p38 contributed a highly enhanced activation of ATF2-driven transcription. RA treatment significantly increased the ATF2-ASC-2-driven transactivation, but the p38m kinase expression inhibited the enhanced transactivation activity as shown in Fig. 7A. In addition to this, the p38 kinase-specific inhibitor, SB203580, also blocked the RA-mediated transactivation by ATF2 and ASC-2 synergistic manner. These results suggest that the granulocytic differentiation by RA activates the p38 kinase pathway followed by increasing ATF2-ASC-2-mediated transactivation.

View larger version (16K): [in this window] [in a new window]   FIG. 7. RA treatment and p38 kinase expression increases ATF2 transactivation by ASC-2 and the protein interaction of ATF2 and ASC-2 in vivo. A, synergistic activation of p38 kinase in ATF2-ASC-2-driven transcription. U937 cells were co-transfected with CRE-Luc and -galactosidase expression vector, along with ATF2, ASC-2, p38, and p38m expression vectors in the presence or absence of heat shock treatment and p38 kinase inhibitor, SB203580. All transfections were determined by the -galactosidase activity assay system to normalize results for transfection efficiency. Luciferase activity was determined in cell lysates 48 h later, and the values (± S.E.) from at least two independent experiments performed in triplicate are shown in the form of a bar graph. AtRA, all-trans retinoic acid. B, the mammalian expression plasmids encoding GAL-ATF2 and VP16-ASC-2 were transfected into U937 cells, as indicated. 48 h after transfection in the absence or presence of RA treatment, cells were harvested for luciferase activities. All the transfection results were normalized to -galactosidase activity, and the presented results represented the average of three independent experiments, with fold induction over the level observed with the reporter alone. The transient protein expression of p38 kinase was confirmed by using Western blotting with p38 kinase-specific antibody. T, ATF2, A, ASC-2.

Dominant Negative Mutant ATF2, ASC-2, and p38 Expression Inhibits the Induction of Graulocytic Differentiation—To study the physiological role of ATF2, we generated U937 cell lines that express the wild-type ATF2 and the dominant negative mutant ATF2 (ATF2-Nd), which is deleted with the ASC-2-binding domain and transactivation domain. All genes were expressed under the control of a constitutive promoter. After selection with neomycin, cells were induced to differentiate. Immunoblot analysis revealed that two ATF2 constructs were expressed in transfected U937 cells but absent in untransfected cells (Fig. 8A). Cells expressing the wild-type ATF2 were able to produce Mac-1 protein (Fig. 8A). ATF2-Nd expression inhibited the induction of late markers of granulocytic differentiation, Mac-1. The differentiation status correlated well with expression levels of ATF2. We also confirmed that the interaction of ATF2 and ASC-2 was not detected in the ATF2-Nd-expressing U937 cells (Fig. 8A). When clones expressing wild-type ATF2 highly were treated with RA, they likewise underwent granulopoietic development, as assessed by detection of respiratory burst activity with NBT, with 95% of the cells staining positively for NBT (Fig. 7C), suggesting that the ATF2 overexpression is sufficient to mediate granulopoiesis induced by RA. In marked contrast, when the ATF2-Nd expression was unable to induce granulopoiesis (Fig. 7C), only 22% of the cells were positive upon NBT staining in the RA-treated cell fraction. Taken together, these results indicate that ATF2 is a positive regulator of granulocytic differentiation.

View larger version (28K): [in this window] [in a new window]   FIG. 8. Dominant negative mutant ATF2, ASC-2, and p38 expression inhibits the induction of graulocytic differentiation. A, the wild-type and the C-terminal truncated mutant of ATF2 was constitutively expressed in U937 cells. After harvesting two stable transfectants, the cell extracts were applied Western blot analysis by using antibodies against Mac-1 and TATA-binding protein (TBP, used as loading control). ASC-2 coprecipitates with ATF2 from the RA-treated normal and ATF2-Nd-expressing U937 cell nuclear extracts (NE). ATF2 and ASC-2 were immunoprecipitated (IP) from RA-treated cell nuclear extracts by incubation with anti-ATF2 IgG. Western blot (WB) assay was determined by using anti-ASC-2 IgG. HA, hemagglutinin; DMSO, Me2SO. B, the dominant negative mutants of ASC-2 and p38 kinase were constitutively expressed in U937 cells. After treatment of RA, the cell extracts were applied Western blot as the same as in A. Co-immunoprecipitation assay was done as the same as in A. C, wild-type (WT) and mutant cells were treated with RA and determined the differentiation yield by scoring reduction of NBT, and quantified data are presented.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell differentiation appears to be mediated by an orchestrated series of genetically controlled events. To understand specific lineage commitment and maturation of multipotent hematopoietic progenitors, it is necessary to identify transcription factors required during differentiation. Distinct C/EBPs are specially expressed in myeloid and eosinophil maturation by various differentiating inducers. Promyeloid leukemia cells were differentiated to granulocytic lineage by treatment of retinoic acid. This retinoic acid is also known to promote ubiquitination and proteolysis of cyclin D1 during induction of tumor cells (30).

It is considered one of the difficult answers about the poor understanding of parallel mechanisms between differentiation and proliferation. Moreover, the cyclin D1 gene is a direct target of ATF2 in chondrocytes because ATF2 binds as a complex with the CREB family to the CRE in the cyclin D1 promoter (31). In addition to regulation of cell cycle gene expression, ATF2 has been known as a regulator in adipogenesis (8) and components of differentiation regulatory factor complex, which regulates retinoic acid- and E1A-mediated transcription of the c-jun gene in differentiation of F9 cells (32) and stimulates transcription of genes related to oncogenic transformation, apoptosis, and adaptive responses of the cells against a large number of stimuli, including cytokine, viruses, and cellular stresses (29, 33, 34).

Heterodimerization of ATF2 appears to be crucial for its functions. Previously, we observed that ATF2 and C/EBP could form a heterodimeric DNA-binding complex for transcriptional regulation in vitro (3). Taken together, it is strongly suggested that the implication of ATF2 in RA mediated the differentiation of promyeloid leukemia cells. As a result, the protein expression and phosphorylation pattern of ATF2 by RA treatment were increased. ASC-2 exhibited as a coactivator in cell-specific lineage differentiation in ATF2-mediated transactivation such as C/EBP-mediated transactivation. Through a mammalian two-hybrid assay, the direct interaction of ATF2 and C/EBP was identified by retinoid signal and enforced by ASC-2. Since the dominant negative ASC-2 proteins, Co2C and Co-2, were associated with ATF2 and C/EBP, respectively, the expression of those constructs inhibited ASC-2-mediated coactivation. This implies that ASC-2 is a strong mediator for the interaction between ATF2 and C/EBP and can promote granulocytic differentiation. Future investigations will determine the functional ordering between C/EBPs and ATF2 during granulopoiesis.

Our observations demonstrate that the phosphorylation of ATF2 by p38 may facilitate interactions between C/EBP and coactivators through inducing a conformational change that unmasks the domains for interaction. Establishment of a stable interaction of ATF2 with C/EBP or coactivators may be a crucial prerequisite for efficient transcription by ATF2. These results suggest that the phosphorylation of ATF2 by p38 may be necessary for the recruitment of specific transcription factors and coactivators to the ATF2-DNA transcription site.

For active transcription process upon cell differentiation, chromatin modification by several coactivators including CBP/p300, p300- and CBP-associated factor, and SRC-1 may be necessary. Although ASC-2 has a homology with the activation domain of CBP/p300, it lacks an inherent histone acetyltransferase activity (30, 31, 40). As CBP/p300 was found to interact with ASC-2, the recruitment of ASC-2 into the ATF2 transcription complex may drive active chromatin structure for transcription by histone acetyltransferase proteins, such as CBP/p300. Finally, it is interesting to note that the G-CSFR promoter may be a typical enhancesome, comprised of a series of cis-elements, including binding sites for ATF2 family proteins and several different classes of transcription factors (5, 6). Our findings that ASC-2 interacts with ATF2 protein may shed some light into how the granulocyte-specific target gene enhanceosome is regulated upon differentiation inducing stimuli, since ASC-2 functionally associates with other classes of transcription factors including AP-1, serum response factor, NF-B, and nuclear receptors. ASC-2 can stabilize the assembly of the granulocyte-specific target gene enhancesome through association with ATF2 and other transcription factors. In addition, ASC-2 may enhance the function of target gene enhancesome by juxtaposing components of the transcriptional machinery in a more favorable orientation and may also play a role in recruiting transcriptional coactivators.

	FOOTNOTES

 
^* This research was supported by a grant of the Korea Health Ministry of Health and Welfare, Republic of Korea (Grant. 02-PJ1-PG3-20908-0009) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 82-51-510-2277; Fax: 82-51-513-9258; E-mail: molecule85{at}pusan.ac.kr.

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