Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7445-7449, August 1995 Biochemistry

Conditional expression of the ubiquitous MafK induces erythroleukemia cell differentiation (erythroid/NF-E2/) 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 . The three family NF-E2, since during such induced differentiation, both DNA (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- (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 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 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. from the exogenous mafK genes exceeded that transcribed To further clarify the effect of MafK forced expression on from the endogenous mafKgene by >20-fold (K1-3) or 70-fold erythroid differentiation, total RNA was isolated from the test (K9-4). Although the expression level of the mafK transgenes and control cells at various times after ZnSO4 induction, and declined somewhat thereafter, abundant exogenous mafK ex- levels of the mRNAs encoding a- and j9-globin were deter- pression continued for at least 3 days after the addition of mined by blot hybridization analysis. Both mRNAs are tran- ZnSO4. scribed abundantly upon DMSO-induced differentiation of The relative levels of MafK protein within the control cells MEL cells (27). Zn treatment caused an increase in the level and the overexpressing cells were compared by immunoblot of a- and f3-globin mRNAs in the test cell lines (Fig. 3), analysis with an anti-MafK antiserum (Fig. 1B). In the absence although the increase was not as great as that observed in MEL of ZnSO4 in the medium, clone K9-4 accumulated twice as cells treated with DMSO, the conventional differentiation much MafK as control cells (lanes 1 and 3). This difference inducer (27). The differences in the steady-state levels of a- probably reflects the low-level expression of the mafK trans- and 13-globin mRNAs in the two test cell clones may reflect the gene in uninduced cells (see above). In clone K9-4 cells, MafK difference in the expression levels ofthe exogenous majKgene. increased after treating the cells with ZnSO4 for 1 day (lanes The expression of mRNA for erythroid S-aminolevulinate A B SVneo K9-4 clone SVneo K1-3 K9-4 clone m m - + - + m II ZnSO4 0.5 0.5 0.5 1 3 3 0.5 0.5 1 3 3 day

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1 2 3 4 5 6 7 8 9 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~10 11 1 2 3 4 FIG. 1. Conditional overexpression of MafK in MEL cells. (A) Inducible overexpression of the mafK transgene. The test clones (B8/K1-3 and B8/K9-4) and the control clone (B8/SVneo) derived from MEL B8 cells were grown in the absence or presence of 120 JIM ZnSO4 for the indicated periods. Total RNAs were extracted and analyzed for mRNAs from endogenous and exogenous maJK genes by blot hybridization. Positions of endogenous and exogenous mafK RNA are indicated. (B) Accumulation of MafK protein in MEL cells. Nuclear extracts from control (lanes 1 and 2) and K9-4 clone (lanes 3 and 4) cells cultured in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of ZnSO4 were separated by SDS/PAGE, transferred onto a membrane, and reacted with the anti-MafK antiserum. The position of MafK is indicated by an arrow. Downloaded by guest on September 27, 2021 Biochemistry: Igarashi et aL Proc. Natl. Acad. Sci. USA 92 (1995) 7447 Al

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FIG. 2. Induction of MEL cell differentiation by MafK overexpression. The B8/K1-3 (A and B), B8/K9-4 (C and D), and B8/SVneo (E and F) cells were cultured for 3 days in the absence (A, C, and E) or presence (B, D, and F) of 120 ,uM ZnSO4 and stained with dianisidine to detect hemoglobin. Counterstaining was with methyl green. Frequencies of dianisidine-positive cells (mean of five experiments) were 3.8, 24, 7.8, 51, 1.4, and 3.2% for A-F, respectively. synthase, which is essential for erythroid cell differentiation complexes. Appearance of these complexes was dependent on (27, 30, 31), also increased severalfold in the test cell lines after the overexpression of MafK since they were not detected in treatment with ZnSO4 (data not shown). nuclear extracts from the control cells treated with or without MafK Participates in DNA Binding Activities. To demon- ZnSO4 or from the K9-4 cells without ZnSO4. Inclusion of the strate an emergence of DNA binding activities in MafK- anti-MafK antiserum, but not the control preimmune serum, overexpressing cells, we carried out EGMSA with nuclear in the binding reactions inhibited formation of all three extracts prepared from control cells, untreated K9-4 cells, or complexes (Fig. 4B, lanes 2 and 3). The much lower mobility K9-4 cells treated with ZnSO4. The probe DNAs were either band in Fig. 4B, lane 2, may represent a supershift of MafK ofthe two variants ofthe T-MARE (5). Variant sequence 9 can complexes by the anti-MafK antibody. The anti-p45 NF-E2 bind the MafK homodimer but cannot bind either NF-E2 or antiserum showed no effect on formation of these complexes Jun-Fos heterodimers. Variant sequence 25 contained the (Fig. 4B, lane 4) even though the same antiserum abolished the NF-E2 site of the PBGD gene and can bind MafK homodimers DNA binding activity of a p45-MafK heterodimer reconsti- and MafK-p45, MafK-Fos, and Jun-Fos heterodimers (5, 7, tuted in quail fibroblasts (data not shown). These results 22). As shown in Fig. 4A4, three nucleoprotein complexes with therefore indicated that all three EGMSA complexes (recov- distinct mobilities were detected in nuclear extracts prepared ered from mafK-induced MEL cells) contained MafK but not from the K9-4 cells treated with ZnSO4, irrespective of the p45 NF-E2. probe used. Appearance of the fastest migrating complex To examine which DNA binding activity represented pos- among the three preceded appearance of the slower migrating sible homodimeric association of MafK with DNA, the mo- Downloaded by guest on September 27, 2021 7448 Biochemistry: Igarashi et al. Proc. Natl. Acad. Sci. USA 92 (1995) SVneo K1-3 K9-4 clone The results described here showed that overexpression ofmafK in MEL cells induced hemoglobin synthesis, which is an ZnSO4 indication of terminal differentiation of erythroid cells. This is somewhat surprising in view of the fact that MafK can act as a globin a repressor of NF-E2 site-dependent transcription in the absence of its partner protein, p45 NF-E2, in transient trans- fection assays (8). Several points should be addressed in consideration of the results described herein. Several lines of evidence indicate that, besides p45 NF-E2, p globin p45-related transcription factors such as Nrf-1 (33) and LCR-F1 (34) can regulate transcription through binding to NF-E2 sites. Since p45, Nrf-1, and LCR-F1 possess conserved b-zip domains, it seems possible that MafK may form het- erodimers with Nrf-1 and LCR-F1, as it does with p45. FIG. 3. Induction of erythroid-specific genes by MafK overexpres- Consistent with this hypothesis, we have identified another sion. Total RNAs were prepared from the test and control cells grown distinct member of this p45-related protein family that is in the absence or presence of 120 ,uM ZnSO4 for 3 days. Expressions expressed specifically in erythroid cells, forms heterodimers of a- and ,B-globin mRNAs were analyzed by blot hybridizations. with the small Maf family proteins, and activates transcription bilities of the MEL EGMSA complexes were compared to through binding to the NF-E2 site (42). Aside from these those produced by in vitro translation of MafK. When MafK various p45-related proteins, there is a possibility that tran- protein was translated in wheat germ extracts and tested in scription factors of the AP-1 family actively participate in gene EGMSA, a complex was detected (Fig. 4C, lanes 2 and 3). This regulation through the NF-E2 site, because the NF-E2 site complex represents a homodimer of MafK (7). Comparative encompasses a phorbol 12-0-tetradecanoate 13-acetate- analysis shows that the lowest mobility band of the three responsive element [TGA(G/C)TCA]. Indeed, MafK was ZnSO4-induced MEL K9-4 complexes (Fig. 4C, lane 4) mi- shown to form a heterodimer with Fos and bind to the NF-E2 grated with a mobility similar to that of the MafK homodimer site (7). Thus, the NF-E2 site can be a target for a variety of complex generated in wheat germ extracts (Fig. 4C, lane 3), different transcriptional regulatory proteins, and MafK can suggesting that the slowest complex detected in conditionally participate in this regulation as a partner in multiple het- mafK-transformed ZnSO4-induced MEL cells represents a erodimeric proteins and as a homodimer. MafK homodimer. The other two faster mobility complexes In the MafK-overexpressing cells, the DNA binding activity are, therefore, likely to represent heterodimers formed be- of NF-E2 (p45-small Maf heterodimer) did not increase tween MafK and other MEL-cell protein(s). significantly. Instead, three distinct DNA binding activities of MafK were induced. One of the three DNA binding activities appears to be a MafK homodimer, while the nature of the DISCUSSION other two MafK-containing DNA binding activities is not clear Levels of various erythroid-specific proteins increase during at the present time. Although these complexes migrated faster the induced terminal differentiation of MEL in vitro (27, 32). than the MafK homodimer complex, mobilities in EGMSA do

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9 25 25 25 Probe FIG. 4. DNA binding activities generated by MafK overexpression. (A) Induction of DNA binding activities by overexpression of MafK. The control SVneo clone and the test clone K9-4 were grown in the absence or presence of 120 j±M ZnSO4 for the indicated times (in days), and then nuclear extracts were prepared. EGMSA was carried out with either probe 9 (lanes 1-12) or 25 (lanes 13-16). (B) Effect of anti-MafK antisera on EGMSA activities. Nuclear extract from the K9-4 cells grown for 3 days in the presence of ZnSO4 was analyzed with an EGMSA using probe 25. Binding reactions were carried out in the absence (lane 1) or presence (lanes 2-4) of indicated sera in the binding reactions. (C) Comparison of mobilities of in vitro-translated MafK- and MEL-cell-derived MafK-DNA complexes. Translation reactions in wheat germ extract were carried out in the absence (lane 1) or presence (lanes 2 and 3) of increasing amounts of mafK cDNA and then analyzed with an EGMSA using probe 25. MafK-DNA complexes in nuclear extract prepared from K9-4 cells (grown for 3 days in the presence of ZnSO4) and electrophoresed in parallel are shown in lane 4. Downloaded by guest on September 27, 2021 Biochemistry: Igaras'hi et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7449 not necessarily reflect the sizes of proteins involved. These 6. Kataoka, K., Nishizawa, M. & Kawai, S. (1993)J. Virol. 67, 2133-2141. complexes may thus be heterodimers of MafK and some other 7. Kataoka, K., Igarashi, K., Itoh, K., Fujiwara, K. T., Noda, M., Yamamoto, M. & Nishizawa, M. larger proteins such as Nrf-1 or LCR-F1. None of the com- (1995) Mol. Cell. Bio. 15,2180-2190. 8. Igarashi, K., Kataoka, K., Itoh, K., Hayashi, N., Nishizawa, M. & plexes, however, is a heterodimer with Fos, since the three Yamamoto, M. (1994) Nature (London) 367, 568-572. complexes were not reactive with anti-Fos antibody (data not 9. Andrews, N. C., Kotkow, K. J., Ney, P. A., Erdjument-Bromage, H., shown). Tempst, P. & Orkin, S. H. (1993) Proc. Natl. Acad. Sci. USA 90, An important question regarding the effect of MafK over- 11488-11492. expression in MEL cells is how MafK induced hemoglobin 10. Andrews, N. C., Ergjument-Bromage, H., Davidson, M. B., Tempst, P. & Orkin, S. H. (1993) Nature (London) 362, 722-728. accumulation. The overexpression of MafK did not inhibit the 11. Ney, P. A., Andrews, N. C., Jane, S. M., Safer, B., Purucker, M. E., DMSO-induced differentiation of the MEL cells (data not Weremowicz, S., Morton, C. C., Goef, S. A., Orkin, S. H. & Nienhuis, shown), suggesting that excess MafK did not repress NF-E2 A. W. (1993) Mol. Cell. Biol. 13, 5604-5612. site-dependent transcription in these cells. Thus, one possibil- 12. Marks, P. A. & Rifkind, R. A. (1978) Annu. Rev. Biochem. 47, ity is that the overall effect of the overproduced MafK within 419-448. the MEL cells was activation of NF-E2 site-dependent tran- 13. Martin, P. & Papayannopoulou, T. (1982) Science 216, 1233-1235. 14. Charnay, P. & Maniatis, T. (1983) Science 220, 1281-1283. scription. The other possibility is that MafK repressed expres- 15. Mignotte, V., Wall, L., deBohr, E., Grosveld, F. & Romeo, P.-H. sion of some genes that inhibit differentiation of MEL cells (1989) Nucleic Acids Res. 17, 37-54. and, hence, induced differentiation indirectly. These two pos- 16. Mignotte, V., Eleouet, J. F., Raich, N. & Romeo, P.-H. (1989) Proc. sibilities are not mutually exclusive. Natl. Acad. Sci. USA 86, 6548-6552. One criticism of forced expression experiments is that 17. Ney, P. A., Sorrentino, B. P., McDonagh, K. T. & Nienhuis, A. W. overexpression of a particular protein within cells may perturb (1990) Genes Dev. 4, 993-1006. 18. Ney, P. A., Sorrentino, P., Lowrey, C. H. & Nienhuis, A. W. (1990) cellular homeostasis in a nonphysiological way, resulting in Nucleic Acids Res. 18, 6011-6017. altered phenotypes. We believe, based on the following con- 19. Talbot, D., Philipsen, S., Fraser, P. & Grosveld, F. (1990) EMBO J. 9, siderations, that this is not the case for the cells described here 2169-2178. and that the observed phenotypes of these cells reflect bona 20. Walters, M. & Martin, D. I. (1992) Proc. Natl. Acad. Sci. USA 89, fide activities of MafK as a transcriptional regulator. (i) Even 10444-10448. though the cells overexpress mafK mRNA by 30- to 70-fold 21. Lu, S. J., Rowan, S., Bani, M. R. & Ben-David, Y. (1994) Proc. Natl. Acad. Sci. USA 91, 8398-8402. over endogenous mRNA at peak accumulation, the MafK 22. Igarashi, K., Itoh, K., Motohashi, H., Hayashi, N., Matuzaki, Y., protein level increased by only -10-fold. (ii) It is well known Nakauchi, H., Nishizawa, M. & Yamamoto, M. (1995) J. Biol. Chem. that overexpression of transcription factors per se does not 270, 7615-7624. necessarily induce differentiation of MEL cells. On one hand, 23. Shoji, W., Yamamoto, T. & Obinata, M. (1994) J. Biol. Chem. 269, the known examples of induction of differentiation of MEL 5078-5084. 24. Ohmori, M., Tanabe, J., Takada, S., Lee, W. M. F. & Obinata, M. cells by forced expression of transcription factors are those of (1993) Oncogene 8, 379-386. SCL and (35, 36). On the other hand, several transcription 25. Minegishi, N., Minegishi, M., Tsuchiya, S., Fujie, H., Nagai, T., factors, such as c-, c-Myb, members of the Jun family, and Hayashi, N., Yamamoto, M. & Konno, T. (1994) J. Biol. Chem. 269, Id, were shown to block, rather than induce, differentiation of 27700-27704. MEL cells when they were overexpressed (23, 37-41). 26. Chomczynski, I. & Sacchi, N. (1987) Anal. Biochem. 162, 156-159. In conclusion, the results here suggest a regulatory role of 27. Fujita, H., Yamamoto, M., Yamagami, T., Hayashi, N. & Sassa, S. (1991) J. Biol. Chem. 266, 17494-17502. MafK during differentiation of erythroleukemia cells. How- 28. Harlow, E. & Lane, D. (1988)Antibodies:A Laboratory Manual (Cold ever, they do not necessarily indicate that the level of MafK Spring Harbor Lab. Press, Plainview, NY). within erythroid precursor cells normally determines the ex- 29. Andrews, N. C. & Faller, D. V. (1991) Nucleic Acids Res. 19, 2499. tent or time of terminal erythroid differentiation. In this 30. Yamamoto, M., Yew, N. S., Federspeiel, M., Dodgson, J. B., Hayashi, regard, it should be noted that expression of maJK does not N. & Engel, J. D. (1985) Proc. Natl. Acad. Sci. USA 82, 3702-3706. increase significantly during DMSO-induced differentiation of 31. Lake-Bullock, H. & Dailey, H. A. (1993) Mol. Cell. Biol. 13, 7122- 7132. MEL cells (unpublished observation). It is possible that other 32. Fujita, H., Yamamoto, M., Yamagami, T., Hayashi, N., Bishop, T. R., small maf family genes may be induced upon treatment of DeVernuil, H., Yoshinaga, T., Shibahara, S., Morimoto, R. & Sassa, MEL cells with DMSO. Analysis of the molecular mechanisms S. (1991) Biochim. Biophys. Acta 1093, 311-316. of MafK-induced erythroleukemia differentiation should lead 33. Chan, J. Y., Han, X.-L. & Kan, Y. W. (1993) Proc. Natl. Acad. Sci. to continued enlightenment into the mechanisms of normal USA 90, 11371-11375. erythroid differentiation. 34. Caterina, J. J., Donze, D., Sun, C. W., Ciavatta, D. J. & Towns, T. M. (1994) Nucleic Acids Res. 12, 2383-2391. 35. Aplan, P. D., Nakahara, K., Orkin, S. H. & Kirsch, I. R. (1992) EMBO We thank M. Obinata, W. Shoji, and J. D. Engel for help. This work J. 11, 4073-4081. is supported in part by Grants-in-Aid from the Ministry of Education, 36. Johnson, P. A, Chung, S. & Benchimol, S. (1993) Mol. Cell. Biol. 13, Science and Culture. 1456-1463. 37. Coppola, J. A. & Cole, M. D. (1986) Nature (London) 320, 760-763. 1. 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(1995) Mol. Cell. Biol. 15, in press. Downloaded by guest on September 27, 2021