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Conditional Expression of the Ubiquitous Transcription Factor

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Conditional Expression of the Ubiquitous Transcription Factor Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7445-7449, August 1995 Biochemistry Conditional expression of the ubiquitous transcription factor MafK induces erythroleukemia cell differentiation (erythroid/NF-E2/maf) KAZUHIKO IGARASHI*, KEN ITOH*, NORIo HAYASHI*, MAKOTO NISHIZAWAt, AND MASAYUKI YAMAMOTO* *Department of Biochemistry, Tohoku University School of Medicine, 2-1 Seiryomachi, Aoba-ku, Sendai 980-77; and tDepartment of Molecular Oncology, Kyoto University School of Medicine, Yoshida-Konoecho, Sakyo-ku, Kyoto 606, Japan Communicated by Irving M Klotz, Northwestern University, Evanston, IL, March 20, 1995 ABSTRACT Transcription factor NF-E2 activity is expression accompanying the in vitro differentiation of eryth- thought to be crucial for the transcriptional regulation of roleukemia cells. One of the key mediators appears to be many erythroid-specific genes. The three small Maf family NF-E2, since during such induced differentiation, both DNA proteins (MafF, MafG, and MafK) that are closely related to binding activity to, and enhancer activities of, NF-E2 sites the c-Maf protooncoprotein constitute half of the NF-E2 increase (15-20). However, the level of p45 RNA does not activity by forming heterodimers with the large tissue- change significantly during DMSO-induced MEL cell differ- restricted subunit of NF-E2 called p45. We have established entiation (21). These observations suggested that hetero- and characterized murine erythroleukemia cells that condi- dimeric partners of p45 (i.e., the small Maf family proteins) tionally overexpress MafK from a metallothionein promoter. might be limiting in MEL cells prior to DMSO treatment. The conditional expression of MafK caused accumulation of To analyze the potential regulatory role of the small Maf hemoglobin, an indication of terminal differentiation along family proteins during erythroid differentiation, we prepared the erythroid pathway. Concomitantly, DNA binding activities MEL-cell lines that conditionally overexpress MafK and then containing MafK were induced within the MafK-overexpress- analyzed their properties. The results of these analyses strongly ing cells. These results demonstrate that MafK can promote suggest that MafK is one of the key regulatory molecules the erythroid differentiation program in erythroleukemia governing the differentiation of the erythroleukemia cells and cells and suggest that the small Maf family proteins are key that MafK exerts its effect by forming both homodimers and regulatory molecules for erythroid differentiation. heterodimers with unknown proteins (in addition to its estab- lished counterpart, p45) within erythroid cells. Six members of the maf protooncogene family have been identified (1-4). The translation products of the genes possess MATERIALS AND METHODS a conserved basic region-leucine zipper (b-zip) domain that mediates dimer formation and DNA binding (5). While Construction ofPlasmids. The prokaryotic plasmid used for chicken v-Maf (6), MafB (4), and human NRL (2) contain mouse MafK protein expression encodes a fusion protein of putative transcription activation domains, chicken MafF, maltose binding protein and the entire mouse MafK (22) MafK, and MafG (3, 7) lack canonical trans-activation do- except the first methionine. pHMTmMafK, where mouse mains. MafF, MafG, and MafK are essentially composed of mafK cDNA was fused to the human metallothionein IIA gene b-zip domains and are collectively referred to as the small Maf promoter, was constructed by inserting a 0.76-kb Sma I-Pst I family proteins. mRNAs for these small Maf family proteins fragment of mouse mafK cDNA into the BamHI site of are expressed in a wide range of tissues in the chicken (3, 8). pSVneoHMTIIdelTer (23) (a gift of W. Shoji and M. Obinata, Recent analyses of the erythroid transcription factor NF-E2 Tohoku University, Sendai, Japan). revealed that it is a heterodimer formed between the hema- Conditional Forced Expression of malK in MEL Cells. The topoietic cell-restricted b-zip protein termed the p45 subunit MEL cells utilized in this experiment were clone B8 and grown of NF-E2 and one of the small Maf family proteins (8-11). in ES medium supplemented with 10% (vol/vol) fetal bovine NF-E2 recognizes an 11-bp consensus sequence: TGCT- serum. MafK-overexpressing cell lines were established as GA(G/C)TCA(T/C) (10). Whereas p45 alone cannot bind to described (24). For Zn treatment of the stably transformed the NF-E2 site, each of the small Maf family proteins alone can cells, cells were seeded at 0.5-1.0 x 105 cells per ml and, 1 day act as transcriptional repressors, dependent on the presence of later, ZnSO4 was added to 120 AM. Dianisidine staining of the NF-E2 sites, in transient transfection assays (8). Heterodimers cells was performed as described (25). consisting of p45 and one of the small Maf family proteins RNA Blot Hybridization. Total RNAs were prepared from (NF-E2) bind to NF-E2 sites (8-10) and activate transcription cultured cells by using guanidine-acidified phenol/chloroform (8). In addition to the interactions with p45, the small Maf (26), and expression of mafK, a-globin, and f3-globin mRNAs family proteins can form heterodimers with one another and were examined as described (27). with Fos (7). Antisera and Immunoblot Analysis. The maltose binding Mouse Friend virus-induced murine erythroleukemia protein-MafK fusion protein purified from overexpressing (MEL) cells represent committed erythroid precursor cells, Escherichia coli cells was utilized to immunize a Japanese white but the expression of a mature erythroid phenotype is normally rabbit. The antiserum was collected after two successive blocked in MEL cells. MEL cells undergo differentiation upon immunizations (28). The anti-p45 antiserum was as described addition of dimethyl sulfoxide (DMSO) or other chemicals (22). Nuclear extracts were size-fractionated by SDS/PAGE (12). A similar induced differentiation in vitro also occurs in (15% gels); proteins were transferred to poly(vinylidene di- human erythroleukemia cells (13, 14). However, it is unclear fluoride) membranes (Waters) and processed for reactions what nuclear events mediate the changes in patterns of gene with the primary and secondary antibodies as described (28). The publication costs of this article were defrayed in part by page charge Abbreviations: b-zip, basic region-leucine zipper; MEL, murine eryth- payment. This article must therefore be hereby marked "advertisement" in roleukemia; EGMSA, electrophoretic gel mobility shift assay; DMSO, accordance with 18 U.S.C. §1734 solely to indicate this fact. dimethyl sulfoxide. 7445 Downloaded by guest on September 27, 2021 7446 Biochemistry: Igarashi et al. Proc. Natl. Acad. Sci. USA 92 (1995) Detection of peroxidase activity was carried out with the ECL 2 and 4). Consistent with the results of RNA analysis, the system (Amersham). amount of MafK protein in ZnSO4-treated K9-4 cells declined Electrophoretic Gel Mobility Shift Analysis (EGMSA). thereafter but was still more abundant than in control cells or Nuclear extracts were prepared from cells as described (29). in untreated K9-4 cells (data not shown). Addition of ZnSO4 The oligonucleotide DNA probes were probe 9 (5'-TCG- at the concentrations used did not significantly affect the AGCTCGGAATTGCCGACTCGGCATTACTC-3') and growth or viability of the cells during the term of experiments probe 25 (5'-TCGAGCTCGGAATTFGCTGACTCATCAT- (data not shown). TACTC-3') (where underlined sequences match the Maf Forced MafK Expression Induces Terminal Differentiation recognition element determined by PCR selection analysis), as of Erythroleukemia Cells. To assess the effects of the over- described (5). EGMSA with cell extracts was carried out as expression of mafK on the terminal differentiation of MEL described (5). Where indicated, the rabbit preimmune, the cells, the control cell line and the two test cell lines were anti-MafK, or the anti-p45 sera were added to the binding cultured in the presence or absence of ZnSO4, and the cells reaction mixtures at a 1:10 dilution and the reactions were were then stained with dianisidine 3-5 days later (Fig. 2); incubated for 10 min on ice before addition of probe DNAs. dianisidine specifically stains assembled hemoglobin tetram- ers. By the third day after the addition of ZnSO4, significant fractions of the two mafK-expressing cell lines, K1-3 and K9-4, RESULTS were dianisidine-positive (Fig. 2 B and D). The appearance of Conditional Overexpression of MafK in MEL Cells. MEL hemoglobin-positive cells is a consequence of overexpression cells were transfected with a plasmid in which the mouse mafK of the mafK transgene, since (i) the number of dianisidine- cDNA was placed under the control of a metallothionein positive cells did not change significantly after the addition of promoter; two stably transformed clones were examined fur- ZnSO4 to the control cells (Fig. 2 E and F) and (ii) the ther. As a control, one clone that was transformed with the percentage of dianisidine-positive cells increased markedly same vector lacking the mafK coding sequence was examined after the addition of ZnSO4 only in the two test cell lines that in parallel. Expression of the exogenous mafK gene was expressed the transfected malK gene. Even in the absence of monitored by RNA blot hybridization (Fig. 1A). In the absence added ZnSO4, the fraction of hemoglobin-positive cells in the of ZnSO4 in the culture medium, one of the clones (K1-3) two test cell lines (Fig. 2 A and C) was higher than in the expressed similar amounts of mafK mRNA from the transgene control cell line (Fig. 2E) or in parent MEL cells (data not and the endogenous gene, whereas the other clone (K9-4) shown). These spontaneously differentiating cells may, there- expressed 4-fold more mRNA from the transgene than from fore, be due to the low-level expression of the mafK transgene the cellular gene (lanes 2 and 7). However, 10 h after the prior to ZnSO4 addition (Fig. 1). Thus, overexpression of addition of 120 AM ZnSO4 to the medium, mRNA transcribed MafK induces hemoglobin synthesis in MEL cells.
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