Human Small Maf Proteins Form Heterodimers with CNC Family Transcription Factors and Recognize the NF-E2 Motif

Human small Maf proteins form heterodimers with CNC family transcription factors and recognize the NF-E2 motif

Tsutomu Toki1, Jugou Itoh2, Jun'ichi Kitazawa1, Koji Arai1, Koki Hatakeyama3, Jun-itsu Akasaka4, Kazuhiko Igarashi5, Nobuo Nomura6, Masaru Yokoyama1, Masayuki Yamamoto5 and Etsuro Ito1

1Department of Pediatrics, 2Medicine, School of Medicine; 3Department of Biology, Faculty of Sciences, Hirosaki University, Hirosaki 036; 4Department of Biochemistry, Tohoku University School of Medicine, Sendai 980-77; 5Center for TARA and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305; 6Kazusa DNA Institute, Kisarazu 292, Japan

The transcription factor NF-E2, a heterodimeric protein Talbot et al., 1990; Talbot and Grosveld, 1991; complex composed of p45 and small Maf family Kotkow and Orkin, 1995). Recent analyses demon- proteins, is considered crucial for the regulation of strated that NF-E2 is composed of two subunits erythroid gene expression and platelet formation. To (Andrews et al., 1993a,b; Igarashi et al., 1994). The facilitate the characterization of NF-E2 functions in large p45 subunit belongs to a family of basic leucine- human cells, we isolated cDNAs encoding two members zipper (bZip) proteins that is closely related to the of the small Maf family, MafK and MafG. The human Drosophila Cap`n'colar (the CNC family) factor mafK and mafG genes encode proteins of 156 and 162 (Mohler et al., 1991). It cannot bind to the NF-E2 amino acid residues, respectively, whose deduced amino sequence as a homodimer, but does do after forming acid sequences show approximately 95% identity to their heterodimers with chicken small Maf family proteins, respective chicken counterparts. Expression of mafK MafF, MafG or MafK (Igarashi et al., 1994). The mRNA is high in heart, skeletal muscle and placenta, heterodimers activate transcription, whereas in the whereas mafG mRNA is abundant in skeletal muscle and absence of p45 the small Maf family proteins act as is moderately expressed in heart and brain. Both are repressors of transcription by forming homodimers expressed in all hematopoietic cell lines, including those (Igarashi et al., 1994, 1995a). MafK has been shown to of erythroid and megakaryocytic lineages. In electro- be an authentic subunit of murine NF-E2 (Andrews et phoretic gel mobility shift assays binding to NF-E2 sites al., 1993a; Igarashi et al., 1995a). In man, two other was found to depend on formation of homodimers or CNC family proteins, Nrf1/LCR-F1/TCF11 (Caterina heterodimers with p45 and p45-related CNC family et al., 1994; Chan et al., 1993b; Luna et al., 1994) and proteins. The results suggest that the small Maf family Nrf2 (Moi et al., 1994) have been identi®ed, although proteins function in human cells through interaction with their DNA binding activities have not been character- various basic-leucine zipper-type transcription factors. ized in detail. Retrovirus insertional mutagenesis plays an impor- Keywords: NF-E2; small Maf; MafK; MafG; Nrf1; tant role in the induction of erythroleukemia caused by Nrf2 various strains of the Friend murine leukemia virus. Recently, a genomic sequence adjacent to the common retrovirus integration site, designed Fli-2, was found to correspond to the coding region of p45 (Lu et al., Introduction 1994). In one erythroleukemic cell line, the expression of p45 was inhibited due to proviral integration in Expression of the ®ve globin genes in the human b- Fli-2, suggesting that p45 may act as a tumor globin gene cluster is tightly regulated in a tissue- suppressor gene whose destruction or dysfunction speci®c and developmental stage-speci®c manner confers a growth advantage leading to the leukemic (Orkin, 1990). The b-globin locus control region phenotype (Lu et al., 1994). On the other hand, (LCR), which plays an essential role in this regulation overexpression of MafK in MEL cells can induce (Grosveld et al., 1987), is divided into four distinct erythroid dierentiation without additional chemical 200 ± 300 bp core regions (HS-1, HS-2, HS-3 and HS-4) inducers (Igarashi et al., 1995b). These data suggest all showing DNase I hypersensitivity (Grosveld et al., that p45 and its dimeric partner molecules play vital 1987; Tuan, 1985). Three types of DNA binding sites, roles in both normal dierentiation of hematopoietic i.e., GATA, Sp1 and NF-E2-like motifs, are found cells and leukemic transformation. However, in man, within the core sequences (Philipsen et al., 1990; Talbot while CNC family members have been cloned and et al., 1990; Talbot and Grosveld, 1991). characterized (Ney et al., 1993; Chan et al., 1993a; NF-E2 activity was originally identi®ed as a type of Luna et al., 1994), none of the small Maf family AP-1-like DNA-binding, with recognition of an 11 bp proteins have so far been isolated. motif (Mignotte et al., 1989). This NF-E2-binding As a ®rst step toward understanding the function of sequence has been shown to be crucial for expression the human NF-E2 factor, we carried out molecular of various erythroid genes (Mignotte et al., 1989; cloning and characterization of human cDNAs encoding small Maf family proteins, isolating for the Correspondence: E Ito ®rst time clones for MafK and MafG and demonstrat- Received 13 August 1996; revised 8 January 1997; accepted 9 January ing by Northern blot analysis their expression in all 1997 tissues including hematopoietic cells. We also showed Human small Mafs and CNC family TTokiet al 1902 that both p45-MafK and p45-MafG heterodimers We next examined the expression levels of human activate transcription from the NF-E2 site. Further- mafK and mafG mRNA in various hematopoietic cell more, we established that MafK and MafG form lines. Both mRNAs were found to be expressed in all heterodimers with Nrf1 and Nrf2, and that these cases examined but the levels for mafK were generally heterodimers can also bind to NF-E2 sites. These the higher of the two (Figure 4b). Taking the results results suggest that the small Maf family factors play together, the expression pro®les of mafK and mafG important roles in transcriptional regulation in mRNAs overlapped substantially, suggesting redun- hematopoietic cells by forming a bZip regulatory dant roles for MafK and MafG in various cell lineages. network together with the CNC family proteins. Both MafK and MafG bind to NF-E2 sites by forming homodimers or heterodimers with p45 Results To examine the DNA binding properties of human MafK and MafG, these bZip proteins were expressed Isolation and structures of the human mafK and mafG in E. coli. We placed 66His anity tags at the N- cDNA clones termini of the recombinant proteins to allow one-step We screened a human T cell cDNA library under puri®cation. As shown in Figure 5a and b we could not relaxed hybridization conditions using a mouse mafK detect any DNA binding activity of these small maf cDNA as a probe, and isolated 25 positive clones. homodimers using a classic PBGD probe containing Upon sequence analysis, two clones were found to encode human mafK and four clones were found to encode human mafG, based on similarity to their chicken counterparts (see below). We determined the entire nucleotide sequence of the longest mafK cDNA insert (1.1 kb in l9 clone) and mafG cDNA insert (1.7 kb in l4 clone) (Figures 1 and 2). These human cDNAs were found to encode proteins consisting of 156 and 162 amino acid residues, respectively, with molecular masses of approximately 18 kDa. As is the case for chicken small Maf proteins (Fujiwara et al., 1993; Kataoka et al., 1995) and mouse MafK (Andrews et al., 1993b; Igarashi et al., 1995a), human MafK and MafG contain a basic domain and a leucine zipper domain, but lack any canonical transactivation domains. The human MafK sequence was established to show 96% and 95% identity with its chicken and mouse counterparts, respectively, while the Figure 1 The nucleotide and deduced amino acid sequences of human MafG sequence showed 94% identity with that the human mafK cDNA. The deduced amino acid sequence is of chicken MafG (Figure 3). This comparison further indicated below the line in the standard one-letter amino acid revealed one major dierence between MafK and code. The termination codon is indicated by an asterisk MafG. A 14 amino acid insertion was found to exist in both chicken and human MafGs (between 130 and 144 amino acids) but not in MafK in either species. The MafKs of all species, on the other hand, demonstrated the same eight amino acid extension at the C-terminals.

Tissue distribution of human mafK and mafG mRNAs To shed light on the functional roles of MafK and MafG in human cells, we examined their mRNA expression by Northern blot analysis (Figure 4). The levels of mafK and mafG mRNAs were found to be low as compared to the p45 case but nonetheless appreciable in all the tissues examined (Figure 4a). The mafK mRNA was relatively abundantly expressed in heart, skeletal muscle and placenta, whereas mafG mRNA was expressed at a high level in skeletal muscle and moderately in heart and brain. We also found large size transcripts of mafK and mafG in all tissues, which became apparent upon longer exposure of the Northern blot ®lters. In particular, the large size transcript of mafK was quite strong in placenta. These transcripts may re¯ect an alternative RNA Figure 2 The nucleotide and deduced amino acid sequence of the human mafG cDNA. The deduced amino acid sequence is processing or represent a derivative of a closely indicated below the line in the standard one-letter amino acid related gene. code. The termination codon is indicated by an asterisk Human small Mafs and CNC family TTokiet al 1903

Figure 3 Comparison of the amino acid sequences of chicken, mouse and human small Maf proteins. Conserved amino acids among the six small Maf family proteins are boxed. The leucine repeats are indicated by asterisks above the line

the NF-E2 site of the human PBGD gene promoter p45-expression plasmid with pRBGP2 markedly (Mignotte et al., 1989). In contrast, strong and very stimulated transcriptional activity of the reporter speci®c formation of DNA-protein complexes was plasmid (Figure 6b). Since the expression of p45 detected in the presence of p45 (lane 4). The cDNA in COS cells is sucient to generate NF-E2 speci®city was con®rmed by the inhibition of observed binding activity by heterodimerizing with endogenous complex formation when excess unlabeled NF-E2 small Maf proteins (Andrews et al., 1993a,b) and since oligonucleotide (lane 5), but not excess cold AP-1 p45 alone does not bind to the NF-E2 motif eciently oligonucleotide (lane 6) was added. The complex (see Figure 5), this transcriptional activation was most contained p45, since anti-p45 monoclonal antibodies likely mediated by p45-small Maf heterodimers. More- super-shifted the complex (lanes 7 and 8). These results over, co-expression of p45 and MafK or MafG resulted indicated that the human MafK and MafG form in a much stronger reporter gene expression than that heterodimers with p45, which can then eciently bind observed in cells transfected with p45 alone. These to NF-E2 sites. results indicated that p45-MafK and p45-MafG Although homodimers of human small Mafs were heterodimers act as transcriptional activators through found not to bind to the PBGD probe, they did bind binding to NF-E2 sites. to other NF-E2 sites as earlier found to be the case for mouse MafK (Igarashi et al., 1995a). For instance, the MafK and MafG bind to NF-E2 sites by forming human MafG and MafK homodimers demonstrated heterodimers with Nrf1 and Nrf2 binding to the NF-E2 site of the chicken b-globin gene enhancer (Figure 5c). p45 NF-E2 is a member of the CNC family of factors that share a 63 amino acid `CNC domain' overlapping the basic region (Mohler et al., 1991). Recently, new Regulation of transcription by small Maf proteins and CNC family members, Nrf1 (Caterina et al., 1994; p45 in vivo Chan et al., 1993b; Luna et al., 1994) and Nrf2 (Moi et We next addressed the functions of human MafG and al., 1994) were identi®ed in human erythroleukemic cell MafK as transcriptional regulators using a transient lines. Nrf1 has been shown to be a potent transcrip- cotransfection assay. Human MafK- and MafG- tional activator in erythroid cells, as compared with expression plasmids were transfected into COS-1 cells p45 (Caterina et al., 1994). Therefore, these proteins with a reporter plasmid pRBGP2 that contains three may potentially regulate erythroid-speci®c gene expres- copies of the chicken b-globin enhancer NF-E2 site in sion through NF-E2 sites. front of the ®re¯y luciferase gene (Igarashi et al., 1994). To test the possibility that MafK and MafG can Signi®cant reporter activity was observed when bind to NF-E2 sites by forming heterodimers with pRBGP2 was transfected into COS-1 cells, as was the Nrf1, or Nrf2, bZip domains of these factors were case for transfection of the same reporter plasmid into expressed in E. coli and heterodimer formation with NIH3T3 and QT6 cells (Igarashi et al., 1994, 1995a). small Maf proteins was examined by electrophoretic gel This endogenous cellular activity was predictable, since mobility shift assay (EGMSA). As shown in Figure 7, the binding sequence of NF-E2 encompasses an AP-1 no binding activity was detected when the radiolabeled site. When excess amounts of MafK- or MafG- NF-E2 probe was incubated with the bZip domains of expression plasmids were co-transfected with the Nrf1 and Nrf2 alone. We have already shown that pRBGP2 reporter, the reporter gene activity was small Maf proteins alone do not bind to the PBGD/ repressed signi®cantly (Figure 6a), suggesting that the NF-E2 probe (see above and Figure 7a, lanes 1 and 2). small Mafs can act as sequence-speci®c negative However, addition of small Maf proteins to Nrf1 or regulators of transcription. Co-transfection of the Nrf2 produced pronounced binding activity to the NF- Human small Mafs and CNC family TTokiet al 1904 a E2 site, although binding activity of Nrf1-MafK heterodimer was much weaker than the other heterodimers. The mobility of the complexes formed in the presence of Nrf1, or Nrf2, with MafK was greater than with MafG, in line with slightly larger size of the latter. kb Heart Brain Placenta Lung Liver Skeletal muscle Pancreas Kidney The NF-E2 site binding speci®city of the heterodimers of CNC proteins with small Maf proteins was examined by adding excess unlabeled oligonucleotide 9.5 — competitors (Figure 7a, lanes 6 to 11 and b, lanes 4 to 7.5 — 9). Formation of the complexes was inhibited by adding excess unlabeled NF-E2 oligonucleotide 4.4 — (Figure 7a, lanes 7 and 8 and b, lanes 5 and 6), but not by excess cold AP-1 oligonucleotide (Figure 7a, lanes 10 and 11 and b, lanes 8 and 9). These results 2.4 — demonstrated that Nrf1 and Nrf2 can interact with small Maf proteins, and suggest that CNC family members act as the partner molecules for the small 1.35 — Maf proteins in various tissues.

Probe : mafK Regulation of transcription by CNC family members in vivo Because all three CNC family members bind to NF- E2 sites as heterodimers with small Maf proteins, we examined their regulatory activities with a reporter plasmid containing NF-E2 sites. Co-transfection of the Nrf1 expression plasmid with the pRBGP2 reporter kb Heart Brain Placenta Lung Liver Skeletal muscle Pancreas Kidney increased the luciferase activity, to a level which is almost equivalent to that observed with the p45 expression plasmid (Figure 8a). In contrast, co- 9.5 — transfection of the Nrf2 expression plasmid increased 7.5 — the reporter gene expression more than tenfold (Figure 8a). These results indicate that all these CNC family 4.4 — proteins are transcriptional activators through binding to the NF-E2 site and that Nrf2 may have most potent transcriptional activity among the small Maf 2.4 — partners. Since neither Nrf1 nor Nrf2 can themselves bind eciently to NF-E2 sites, the transcription activation 1.35 — activity is most likely mediated by heterodimers with other molecules within transfected cells. To test the possibility that small Maf proteins are the partner Probe : mafG molecules of Nrf1 and Nrf2, we next co-transfected the small Maf expression plasmids and Nrf1 and Nrf2 b expression plasmids with the pRBGP2 reporter (Figure 8b and c). Co-expression of Nrf1 and MafG expression plasmid resulted in stronger reporter gene activities than in the cells transfected with Nrf1 alone (Figure HL-60 KG-1 CMK86 CMK11-5 Meg 01 HEL K562 YN-1 THP-1 Raji SCMC-L1 CEM 8b, left). However, in the presence of the excess amounts of MafG expression plasmid, the reporter activity was reduced (Figure 8b, left). Similar results mafK were obtained with MafK (Figure 8b, right). No signi®cant dierence was observed between MafK and MafG in terms of transcriptional activity with Nrf1, although a slight dierence in DNA binding activity between Nrf1-MafG and Nrf1-MafK heterodimers was noted (Figure 7a). mafG These results were very similar to those with p45 and small Mafs as described above, indicating that Nrf1-

Figure 4 mafK and mafG mRNA expression. (a) Northern blot analysis with poly (A)+ RNAs derived from human adult tissues. lines. CMK86, CMK11-5, Meg01, HEL, K562 and YN-1 are (b) Northern blot analysis with poly(A)+ RNAs derived from erythroid/megakaryocytic cell lines. THP-1 is a monocytic cell human leukemic cell lines. HL-60 and KG-1 are myeloid cell line. Raji, SCMC-L1 and CEM are lymphoid cell lines Human small Mafs and CNC family TTokiet al 1905 small Maf heterodimers act as transcriptional activa- Discussion tors through binding to the NF-E2 site. In contrast, we could not observe any stimulation of transcription In the present report, we describe for the ®rst time the upon co-transfection of the Nrf2 and MafG expression isolation and characterization of human cDNAs plasmids (Figure 8c, left). Reporter activity was already encoding the small Maf family proteins MafK and decreased in the presence of very small amount of the MafG. Both Maf proteins were found to interact with MafG expression plasmid (1 ng per transfection) and the bZip protein p45 as well as two other p45-related this inhibitory eect proved dose dependent. Similar CNC family proteins, Nrf1 and Nrf2. This analysis is results were obtained using the MafK expression the ®rst step toward understanding the functional roles plasmid (Figure 7c, right). These results suggest that of NF-E2 in normal dierentiation processes as well as Nrf2 may form heterodimers with novel endogenous in pathologic disorders of erythropoiesis and thrombo- factors within COS-1 cells, which possesses more poiesis in man. potent transcriptional activity than the Nrf2-small One of the striking features of the small Maf Maf heterodimer. proteins is the conservation of their primary structures

a c P45 + + + + + + MafK MafG Maf K + + + + + + cold NF-E2 + HS-2 + + + + cold AP-1 + PBGD + + + + MoAb #1 + MoAb #10 +

1 2 3 4 5 6 7 8

b P45 + + + + + + Maf G + + + + + + cold NF-E2 + cold AP-1 + MoAb #1 + MoAb #10 +

Figure 5 DNA binding of human MafK and MafG. (a) and (b) autoradiographic images of EGMSA with the authentic PBGD probe. Binding reactions were carried out with the proteins expressed in bacteria as follows: reaction buer alone (lanes 1), 50 ng p45 alone (lane 2), and 50 ng MafK (a) or MafG (b) in the presence (lanes 4 to 8) or absence (lane 3) of 50 ng p45. A 100- fold excess of the cold PBGD probe or the AP-1 probe was included in lanes 5 and 6, respectively. Anti-p45 monoclonal antibodies no. 1 and no. 10 were included in the binding reactions for lanes 7 and 8, respectively. (c) autoradiographic image of EGMSA with the HS-2 (chicken b-globin gene) probe and the authentic PBGD probe. Reaction buer alone (lanes 1, 2, 5 and 6), 50 ng MafK (lanes 3 and 4), and 50 ng MafG (lanes 7 and 8) with the HS-2 probe (lanes 1, 3, 5 and 7) and the authentic PBGD 1 2 3 4 5 6 7 8 probe (lane 2, 4, 6 and 8) Human small Mafs and CNC family TTokiet al 1906 among various species. We found, out of the 156 of NF-E2. The overlapping expression pro®les of the amino acid residues of human MafK, 148 and 149 small Mafs in human (Figure 4) and in chicken residues to be identical to those of mouse and chicken (Fujiwara et al., 1993) strongly suggest that they MafKs, respectively. Out of 162 amino acid residues of together constitute a regulatory network with consider- human MafG, 152 were shown to be shared by chicken able redundancy. For example, because mafK and MafG. In addition, the three small Maf proteins mafG mRNAs are both expressed in human erythroid demonstrate signi®cant similarity to each other. Such and megakaryocytic lineage cells, it is plausible that a high degree conservation of the primary structure both MafK and MafG form NF-E2 complexes that suggests that all the small Maf family proteins play contribute to completion of the late stage of important roles. As expected from the structural megakaryocyte dierentiation. similarity, both human MafK and MafG can interact In man, two other p45-related molecules, Nrf1/LCR- with p45 to form heterodimers which preferentially F1/TCF11 and Nrf2, have been identi®ed. The results recognize the consensus sequence of NF-E2 and of the present EGMSA demonstrated for the ®rst time probably participate in the regulation of NF-E2 that Nrf1 and Nrf2 cannot bind to NF-E2 sites activities. We could not detect any signi®cant eciently on their own, but bind as heterodimers dierences between MafK-p45 and MafG-p45 hetero- with small Maf proteins, thus participating in the dimers in terms of DNA binding activity or transcrip- regulation of NF-E2 activities (Figures 7 and 8). In this tional activity (Figures 5 and 6). Based on the present regard, it is interesting to note that there are some ®ndings and previous observations regarding the dierences among the CNC family members in terms chicken small Maf proteins (Igarashi et al., 1994; of transcriptional activity. First, we observed very Kataoka et al., 1995), we conclude that the small Maf strong activation of the reporter gene expression by family proteins share similar biochemical function(s). Nrf2 alone in COS cells, while only modest trans- The results of a gene knockout experiment recently activation was observed with p45 and Nrf1 (Figure 8a). indicated that p45 is essential for platelet formation Recently, a chicken CNC type bZip protein ECH, that (Shivdasani et al., 1995), whereas MafK (the p18 is closely related to the mammalian Nrf2, was subunit) appears to be dispensable for normal demonstrated to be a very potent transcriptional hematopoiesis as well as other developmental pro- activator aecting the NF-E2 site (Itoh et al., 1995). grams (Kotkow and Orkin, 1996). Considering the Our current observation is consistent with this ®nding. present results, not only MafK but also other small Secondly, transactivation by Nrf2 was suppressed by Maf proteins may contribute to the small p18 subunit the addition of exogenous small Mafs in a dose

a b

Figure 6 Regulation of transcription by MafK or MafG with or without p45 in COS cells. (a) transcriptional repression by MafG and MafK. Aliquots of 10 mg of either pcDNAIneo-MafG or pcDNAIneo-MafK were transfected into COS cells with 1 mgof pRBGP2. The results of two independent experiments are shown, each carried out in triplicate. Luciferase activity of pRBGP2 in the absence of any eector plasmid was set as 100% and relative activity was obtained by addition of the eector plasmids. Standard errors are indicated. (b) reporter gene activities in the presence of various combinations of the MafK, MafG and p45 expression plasmids (1 mg) with 1 mg of pRBGP2 Human small Mafs and CNC family TTokiet al 1907 dependent manner, while those observed with p45 and the CNC family of factors and the small Maf family of Nrf1 were stimulated. These results suggest that Nrf2- proteins mutually restricts their activity as functional small Maf heterodimers may act as trans-repressors activators in certain cell lineages. In chicken erythroid through the NF-E2 site, whereas Nrf1 and p45-small cells, for example, ECH predominates as the CNC Maf heterodimers act as transcriptional activators. family protein which functions by forming hetero- Alternatively, Nrf2 may form heterodimers with novel dimers with the small Maf proteins (Itoh et al., 1995). molecules within COS cells with more potent activities Redundancy in the regulatory network provided by the than Nrf2-small Maf heterodimers and therefore small Maf proteins and the CNC family presumably competing as transcriptional activators aecting the also operate in other tissues. NF-E2 site. In summary, our present investigation showed that Whereas Nrf1 and Nrf2 are expressed ubiquitously the human small Maf proteins, MafK and MafG, can (Chan et al., 1993b; Moi et al., 1994), our results bind to authentic NF-E2 motif eciently by forming suggest that their functions depend on the presence of heterodimers with three human CNC family proteins partner molecules. Thus, it is likely that expression of (p45, Nrf1 and Nrf2). These results suggest that small Maf family proteins have very important partner roles in gene regulation in erythroid and megakaryocytic lineages as well as in non-hematopoietic tissues. a cold cold NF-E2 AP-1 Nrf1 + + + + + + + + + Materials and methods MafG + + + + MafK + + + + Cell culture

Cells were cultured at 378Cin5%CO2. The human erythroid/megakaryocytic cell line UT-7 (Komatsu et al., 1991) was maintained in Iscove's Modi®ed Dulbecco's Medium (IMDM) with 10% FBS and 1 ng/ml of granulocyte macrophage-colony stimulating factor (GM- CSF, kindly provided by SANDOZ). The other cell lines were grown in RPMI 1640 medium with 10% fetal bovine serum (FBS).

Isolation and sequencing of human small maf cDNAs Ahumanlgt11 T cell cDNA library was plated on 150 mm 1 2 3 4 5 6 7 8 9 10 11 Petri dishes at the density of 56104 plaque-forming units (PFU) per plate. Screening was performed using a mouse b mafK cDNA fragment as a probe under non-stringent cold cold conditions. Duplicate phage lift ®lters (Colony/Plaque NF-E2 AP-1 Screeen, Du Pont) were made from each plate and processed for hybridization to the probe as described Nrf2 + + + + + + + + + previously (Ito et al., 1993). This was carried out in the MafG + + + presence of 66SSC (16SSC is 0.15 M NaCl plus 0.015 M MafK + + + sodium citrate) and 0.25% of milk at 508Covernight. Filters were washed in 16SSC/0.1% SDS at 508Cfor 30 min. Positive clones were puri®ed by three additional plaque hybridization screenings. Inserts of positive phage clones were then subcloned into the EcoRI site of the pBluescript KS(7) plasmid (Stratagene, La Jolla, CA). DNA sequences were determined on both strands by the dideoxynucleotide method using a Sequenase sequence system (USB, Cleveland) and deletion subclones of the plasmid as templates.

Production of bZip proteins in E. coli 1 2 3 4 5 6 7 8 9 Prokaryotic expression plasmids for human MafK were Figure 7 Interaction with Nrf1 or Nrf2 and DNA binding of constructed by generating mafK cDNA by the poly- human MafK and MafG. Autoradiographic images of EGMSA merase chain reaction (PCR). A set of primers with the authentic PBGD probe. Binding reactions were carried (5'-CAGGATCC TT AAA GGT CAA GAA GGA GGC - 3' out with the proteins expressed in and puri®ed from E. coli as and 5' -CA AAGCTTCTAGGATGCAGCCGAGAAGG- follows. (a) 100 ng MafG alone (lane 1), 100 ng MafK alone (lane 3') was designed to amplify cDNA with BamHI and 2), 300 ng of Nrf1 alone (lane 3), and 300 ng of Nrf1 in the HindIII sites (underline) at the two ends. A small deletion presence of 100 ng MafG (lanes 4, 7 and 10) or 100 ng MafK (10 amino acid residues) was introduced into the N- (lanes 5, 8 and 11). 100-fold excess of cold PBGD probe and AP- 1 probe were included in lanes 6 to 8 and lanes 9 to 11, terminus to give stable expression of the protein in E. respectively. (b) 300 ng of Nrf2 alone (lane 1), and 300 ng of Nrf2 coli. The cDNA was then subcloned into the BamHI and in the presence of 100 ng MafG (lanes 2, 5 and 8) or 100 ng HindIII sites of the pQE-30 vector (QIAGEN, USA). MafK (lanes 3, 6 and 9). 100-fold excess of cold PBGD probe and Prokaryotic expression plasmids for human MafG were AP-1 probe were included in lanes 4 to 6 and lanes 7 to 9, constructed similarly. cDNA encoding MafG was generated respectively by PCR using a set of primers (5'-AAGGATCCTT- Human small Mafs and CNC family TTokiet al 1908 GAAGGTGAAGCGGGAGCC-3' and 5'-ACAAGCTTCC- bZip regions of Nrf1 and Nrf2 were then generated by PCR TACGATCGGGCATCCGTC-3'). A small deletion (11 using the single strand cDNA and following primers. Nrf1; 5'- amino acid residues) was again introduced into the N- CAGATGAGCCGGGATGAGCAC-3' and 5'-CTCACT- terminus. The ampli®ed cDNA was then subcloned into TTCTCCGGTCCTTTG-3'. Nrf2; 5'-GGCTCATCTCACAA- pQE-30. For p45 expression, the PstI fragment of p45 cDNA GAGATGA-3' and 5'-GGCTTCTTACTTTTGGGAACA-3'. (Toki et al., 1996) was excised and subcloned into pQE. The amplicons were subcloned into TA cloning vector Prokaryotic expression plasmids for Nrf1 and Nrf2 were according to the manufacturer's recommendations (Invitro- constructed as follows. First, single strand cDNA was gen, San Diego) and the sequences were determined to exclude synthesized using RNA isolated from K562 cells and random any possible mutation. Then the BamHI and HindIII primers as described earlier (Ito et al., 1993). cDNAs encoding fragments were subcloned into pQE-31 vectors.

b Human small Mafs and CNC family TTokiet al 1909 Recombinant proteins tagged with 66His at the N-termini reactions were incubated for 10 min on ice before addition were expressed in E. coli strain M15 (pREP4) and puri®ed with of probe DNAs. The following oligonucleotides were used the Ni-NTA resin (QIAGEN, USA) under denaturing for EGMSA. NF-E2(PBGD), 5'-TGGGGAACCTGTGCT- conditions. Puri®ed proteins were dialyzed against refolding GAGTCACTGGAG-3',AP-1:5'-TGGGGAACCTGTT- buer containing 25 mM HEPES-KOH (pH 7.5), 100 mM CTGAGTCACTGGAG-3'; NF-E2 (chicken b-globin) 5'-

KCl, 10 mM ZnSO4, 0.1% NP40, 1 mM DTT and 5% Glycerol. CCCGAAAGGAGCTGACTCATGCTAGCCC-3'.

Monoclonal antibody preparation Plasmid construction A female Balb/c mouse was immunized with the puri®ed An eukaryotic expression plasmid for MafK was con- recombinant p45 protein containing 66His tag. Spleen structed by excising the HindIII and XbaI fragment from cells from the immunized mouse were then fused with the the parental clone in Bluescript KS(7) and subcloning it murine myeloma cell line P3X63Ag8U.1 (P3U1) using into the HindIII and XbaI sites of the pcDNAI/Neo vector polyethylene glycol. The cells were distributed into 96 well (Invitrogen, San Diego, CA). Similarly, expression vectors plates at about 16105 cells/well in serum free media S- for MafG and p45 were constructed by excising the Clone CM-B (Sanko Junyaku Co Ltd, Tokyo) with HAT- HindIII and XbaI fragments from the parental clones media supplement (Boehringer Mannheim). Hybridoma and recloning them into the HindIII and XbaI sites of the colonies secreting anti-p45 NF-E2 antibodies were cloned pcDNAI/Neo vector. The luciferase reporter gene pRBGP2 by limiting dilution. The speci®city of the monoclonal contains three copies of the chicken b-globin enhancer NF- antibodies was determined by enzyme-linked immunosor- E2 site, upstream of the minimal promoter of the rabbit b- bent assay (ELISA). globin gene (Igarashi et al., 1994). The eukaryotic expression plasmids for Nrf1 and Nrf2 were constructed as follows. Nrf1 and Nrf2 cDNA clones Electrophoretic gel mobility shift analysis (EGMSA) were isolated from a human lgt11 T cell cDNA library. Double-stranded oligonucleotide DNAs were radiolabeled Screenings of the library were performed using PCR with g-[32P]ATP and used as probes for EGMSA. The ampli®ed partial cDNA fragments (see above) as probes binding mixture (25 ml) contained 26104 c.p.m. of the under stringent conditions. Positive clones were puri®ed by radiolabeled oligonucleotide, 20 mM HEPES-KOH three additional plaque hybridization screenings. The insert (pH 7.9), 0.2 mM EDTA, 60 mM KCl, 1 mM DTT, 6 mM of the Nrf1 phage clone (no. 237) was subcloned into the

MgCl2,0.5mMPMSF, 40 mg of poly(dI-dC) per ml, 1 mg/ EcoRI site of the pBluescript KS(7) plasmid and veri®ed by ml of BSA, 10% of glycerol (v/v), and 50 ± 100 ng of the sequencing. An eukaryotic expression plasmid for Nrf1 was bZip proteins synthesized in bacteria. Where indicated, a then constructed by subcloning the insert into the HindIII MoAb against human NF-E2 (no. 1 and no. 10) was added and XbaI sites of the pcDNAI/Neo vector (Invitrogen, San to the binding reactions at a 1 : 12.5 dilution, and the Diego, CA). The Nrf2 expression plasmid was constructed as

Figure 8 Regulation of transcription by CNC family proteins with or without small Mafs in COS cells. (a) transcriptional activation of CNC family proteins. Aliquots of 2 mg of the p45, Nrf1 or Nrf2 expression plasmids were transfected into COS cells with 1 mg of pRBGP2. The results of two-independent experiments are shown, each carreid out in triplicate. Luciferase activity of pRBGP2 in the absence of any eector plasmid was set as 100% and the relative activities obtained with addition of various combinations of eector plasmids are shown. Standard errors are indicated. (b) reporter gene activities in the presence of various amounts of the MafG (left) or MafK (right) expression plasmids wit 2 mg of the Nrf1 expression plasmid and 1 mg of pRBGP2. Luciferase activity of pRBGP2 in the presence of the Nrf1 expression plasmid alone was set as 100%. Results are presented as described above. (c) reporter gene activities in the presence of various amounts of the MafG (left) or MafK (right) expression plasmid with 2 mg of the Nrf2 expression plasmid and 1 mg of pRBGP2. The results are presented as described above Human small Mafs and CNC family TTokiet al 1910 follows. The insert of the Nrf2 phage clone (no. 85) was RNA blot analysis ampli®ed by PCR using the phage DNA and the following Total cellular RNAs were isolated from leukemic cell lines set of primers. 5'-CAGCGAATTCGGTGGCGACGAC- by the guanidine-acidi®ed phenol chloroform method TCCTGGAGC-3' and 5'-CAGCGAATTCTTGACACCA- (Chomczynski and Sacchi, 1987) and puri®ed by the GACCAACTGGTA-3'. Then the cDNA was subcloned Oligotex dT30 system (Nippon Roche, Kamakura). Ali- into the PCR2.1 plasmid using a TA Cloning Kit quots of the RNA samples (2 mg) were electrophoresed on (Invitrogen) and veri®ed by sequencing. An eukaryotic 1% agarose/formaldehyde gels and transferred to nylon expression plasmid for Nrf2 was then constructed by ®lters as described previously (Ito et al., 1993). Northern subcloning the insert into the BamHI and XbaI sites of the blots containing multiple human tissue RNA samples were pcDNAI/Neo vector. purchased from CLONTECH; (each tissue sample contains 2 mgofpoly(A)+ RNA). The blots were probed with Transfection radiolabeled cDNA coding for human MafK and MafG according to the standard procedure. Filters were washed COS-1 cells were maintained in Dulbecco's modi®ed at 658Cin0.26SSC and 0.1% SDS solution. Eagle's medium (DMEM) supplemented with 10% FBS and plated at 26105 cells/60 mm culture dish 24 h before transfection. They were transfected using the DEAE- dextranmethodwith1mg of reporter plasmid along with various combinations of eector plasmids, e.g. pcDNAI/ Neo-MafK, pcDNAI/Neo-MafG, pcDNAI/Neo-p45, pcDNAI/Neo-Nrf1 or pcDNAI/Neo-Nrf2. 0.5 mgof pCH110 which expresses b-galactosidase was also included Acknowledgements as an internal control. The transfected cells were then We would like to thank Drs Makoto Nishizawa, Tsukasa washed with Dulbecco's phosphate-buered saline (PBS) Iwata, Ken Itoh, Hozumi Motohashi, Norio Hayashi for and incubated with 1.5 ml of Tris-serum free media (Tris- their help. Part of this work was carried out using the SFM) solution containing DNA and 0.4 mg/ml of DEAE facilities of the Department of Molecular Genetics at dextran for 4 h. After washing with Tris-SFM, they were Hirosaki University. This work was supported by Grants- incubated in 100 mM chroloquin in Tris-SFM for 3 h, in-Aid for Scienti®c Research and a Grant-in-Aid for washed with Tris-SFM twice, and cultured in DMEM Encouragement of Young Scientists from the Ministry of containing 4% FBS. The cells were harvested 48 h after Education, Science, Sports and Culture, by the Japanese transfection, and cell extracts were assayed with Picagene Society for Promotion of Sciences, and by the Uehara Luciferase Assay System (Toyo Ink, Tokyo). Memorial Foundation.

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