Oncogene (1997) 14, 745 ± 750 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 SHORT REPORT Rat maf related genes: speci®c expression in chondrocytes, lens and spinal cord Masaharu Sakai1, Junko Imaki2, Kazuhiko Yoshida1, Akihiko Ogata1, Yuko Matsushima-Hibiya1, Yoshinori Kuboki3, Makoto Nishizawa4 and Shinzo Nishi1 1Department of Biochemistry, School of Medicine and 3Department of Biochemistry, School of Dentistry, Hokkaido University N15 W7, Kita-ku, Sapporo 060; 2Department of Anatomy, Nippon Medical College, Sendagi, Bunkyo-ku, Tokyo 113; 4Department of Molecular Oncology, Kyoto University, Faculty of Medicine, Yoshida-Konoecho, Sakyo-ku, Kyoto 606, Japan maf is a family of oncogenes originally identi®ed from tion domain is truncated. MafK forms a heterodimer avian oncogenic retrovirus, AS42, encoding a nuclear with NF-E2 p45, a transcription factor that regulates bZip transcription factor. We have isolated two maf the globin gene (Andrews et al., 1993; Igarashi et al., related cDNA clones, maf-1 and maf-2, from a rat liver 1994). Recently it was found that MafB interacts with cDNA library. Comparison of the sequence homologies Ets-1, a transcription factor containing a helix ± turn ± of the proteins encoded by maf-1 and maf-2 with those of helix DNA binding domain, inhibits Ets-1 activity, c-maf and chicken mafB indicated that maf-1 and maf-2 thus interrupting erythroid dierentiation (Sieweke et are the rat homologues of mafB and c-maf, respectively. al., 1996). Maf-family transcription factors heterodi- Both genes are expressed at low levels in a wide variety merize not only with bZip proteins, but also with of rat tissues, including spleen, kidney, muscle and liver. helix ± turn ± helix proteins. Thus, a wide variety of Immunohistochemical studies and in situ hybridization Maf-heterodimers are formed, which in turn could analyses show that maf-1 and maf-2 are strongly regulate a wide variety of target genes depending on expressed in the late stages of chondrocyte development which heterodimer is formed. The mouse mafB gene in the femur epiphysis and the rib and limb cartilage of was identi®ed as the gene responsible for the 15 day old (E15) embryo in rat. Cartilage cells, induced segmentation of hind brain during early development by subcutaneous implantation of bone morphogenic by analyses of kreislar (Kr) mutant mice (Cordes and protein, also expressed maf-1 and maf-2. In situ Barsh, 1994). These ®ndings indicate that Maf hybridization analyses of E15 embryos show that both oncogene products play an important role in morpho- genes are expressed in the eye lens and the spinal cord as genic processes and cellular dierentiation. well as the cartilage. However, the expression patterns of Determination of maf expression should facilitate maf-1 and maf-2 in lens and spinal cord are dierent. identi®cation of target genes and would thus further clarify maf and target gene functions. In this report, we Keywords: maf; transcription factor; chondrocyte; lens; isolated two maf related cDNA clones, maf-1 and maf- spinal cord 2, from a rat liver cDNA library and studied the expression of these genes. Approximately 16106 recombinant phages from a The maf oncogene (v-maf) was initially identi®ed from rat liver cDNA library were screened with a v-maf an avian oncogenic retrovirus, AS42, which induces probe, and two clones, maf-1 and maf-2, were obtained musculoaponeurotic ®brosarcoma in vivo and trans- (nucleotide sequences of both cDNAs were submitted forms chicken embryo ®broblast cells in vitro to GenBank/EMBL/DDBJ DNA database, accession (Nishizawa et al., 1989). The v-maf oncogene product numbers are U56241 and U56242 for maf-1 and maf-2, is a transcription factor containing a typical bZip DNA respectively). binding domain. The binding consensus sequence of The predicted amino acid sequences of Maf-1 and the v-maf product (Maf recognition element, MARE, - Maf-2 show great similarity with that of v-Maf. The TGCTGACTCAGCA-) overlaps with the TPA respon- overall amino acid sequence homologies of Maf-1 and sive element (TRE), and cyclic AMP responsive Maf-2 with those of chicken MafB and c-Maf element (CRE) (Kataoka et al., 1994b). The maf (Kataoka et al., 1994a) are 84% and 81%, respec- oncogene products can form homo- and hetero-dimers tively; thus rat Maf-1 and Maf-2 appear to be the rat with transcription factors having bZip structures, such homologues of MafB and c-Maf. The bZip structures as Jun and Fos (Kataoka et al., 1994a,b, 1996; of all Mafs are conserved at more than 80% homology. Kerppola and Curran, 1994). Furthermore, several This suggests that these Maf-related proteins recognize maf-related genes have been identi®ed (i.e. mafB, similar sequence elements. The DNA binding analyses mafK, mafG, mafF and Nrl) (Fujiwara et al., 1993; using Ecoliexpressed proteins show that both Maf-1 Kataoka et al., 1994a, 1995; Swaroop et al., 1992). and Maf-2 bind with almost equal anity to the Interestingly, mafK, mafG and mafF encode small MARE consensus sequence. However, when subtle proteins in which the N-terminal putative transactiva- variations of this sequence were used, Maf-1, Maf-2 and v-Maf showed signi®cant dierences in binding anity (manuscript in preparation). Maf-1 is almost identical (98% homology) with the Correspondence: M Sakai Received 23 May 1996; revised 7 October 1996; accepted 8 October Kr gene product, a mouse mafB homologue (Cordes 1996 and Barsh, 1994). Expression of Rat maf-related oncogenes MSakaiet al 746 The transactivation functions of the proteins hybridization studies using 15 day old (E15) rat encoded by maf-1 and maf-2 were con®rmed by a co- embryos clearly show that maf-2 is strongly expressed transfection assay using a fusion construct consisting of in the cartilage of ribs (Figure 3c) and limbs (Figure a MARE and luciferase gene. Figure 1 shows both 3d). Almost identical results were obtained using the maf-1 and maf-2 activate the gene containing MARE maf-1 probe. These ®ndings suggest that Mafs are with activities similar to those of v-maf. The activities closely associated with a chondrocyte dierentiation were much stronger than c-jun, although MARE process not only in the adult epiphysis but also in contained complete TRE (TPA responsive element, - embryonic cartilage tissue. To con®rm the expression TGACTCAG-) sequence. of Mafs in chondrocytes, we used a BMP (bone We investigated the tissue distribution of maf-1 and morphogenic protein) implantation system to induce maf-2 mRNAs by RNase protection analysis (Figure cartilage formation. BMP stimulates immature me- 2). maf-1 is expressed at low levels in a variety of senchymal cells to create a process similar to organs including spleen, muscle and liver. The tissue endochondral ossi®cation when they are subcuta- distribution of maf-2 mRNA is similar to that of maf- 1, but not identical. Immunohistochemical staining by speci®c antisera against Maf-1 and Maf-2 did not detect any signi®cant positive signals in liver, though mRNA is expressed there (Figure 2). However, anti-Maf antibodies strongly and speci®cally stained chondrocyte nuclei M located in the hypertrophic chondrocytes of the femur — epiphysis, and the immature proliferating chondrocytes — Brain — Heart — Lung — Spleen — Liver — Kidney — Stomach — Intestine — Uterus — Muscle — Bone marrow were stained very weakly (Figure 3a and b). Hypertrophic chondrocytes are maturated non-divid- ing cells in chondrocyte dierentiation. The antiserum ¨ against Maf-1 yields almost the same results as maf1 obtained with anti-Maf-2 antiserum. RNase protection analyses detected mRNA of both mafs from femur epiphysis but signals were weak (data not shown). This is probably due to the fact that the expressed cells are low in number in the dissected samples of femur epiphysis as shown in Figure 3a and b. In situ ¨ maf2 ¨ GAPDH Figure 2 Expression of maf-1 and maf-2 mRNA in rat tissues, examined by RNase protection analysis. The RNAs from various tissues were prepared using an RNA extraction kit (Nippon Gene Figure 1 Transactivation of MARE-luciferase gene by Mafs. Co. Toyama, Japan). Nucleotides 829 ± 1037 and 1248 ± 1417 of MARE (-TGCTGACTCAGCA-) was ligated into the luciferase the maf-1 and maf-2 clones, respectively, were inserted into expression vector, pGV-P (Nippon Gene, Toyama, Japan), and pBluescript II vector (Stratagene, California, USA), and anti- co-transfected with indicated expression plasmids into HepG2 sense RNA probes were prepared (approximately 16109 c.p.m./ cells, a human hepatoma cell line. Transfection and determination mg). Ten micrograms of the total RNAs from indicated tissues of luciferase activity were done as described (Sakai et al., 1995). were used for RNase protection analysis as described previously The v-maf and c-jun expression plasmids were previously (Sakai et al., 1992). The same preparations of RNAs were described (Sakai et al., 1995). Expression plasmids of maf-1 and quantitated using an anti-sense probe for glyceraldehyde 3- maf-2 were constructed by inserting the cDNA fragments phosphate dehydrogenase (GAPDH). HpaII digested pUC18 corresponding to amino acids 18-C-terminus and 19-C-terminus DNA, terminally labeled by 32P, was used as a size marker for maf-1 and maf-2, respectively, into a human b-actin expression (M). Autoradiograms were exposed for 2 days and overnight for vector (Gunning et al., 1987). Vec indicates expression vector mafs and GAPDH, respectively. This experiment was done three without insert times with nearly identical results Expression of Rat maf-related oncogenes M Sakai et al 747 Figure 3 Maf expression in chondrocytes. (a and b): The speci®c antibodies of Maf-1 and Maf-2 were prepared as follows: Fragments of Maf-1 and Maf-2 proteins were produced in E coli as fusion proteins with maltose binding protein and glutathione S- transferase, respectively.