Molecular Cloning and Functional Characterization of a New Cap'n' Collar Family Transcription Factor Nrf3
1999; Elsevier BV; Volume: 274; Issue: 10 Linguagem: Inglês
10.1074/jbc.274.10.6443
ISSN1083-351X
AutoresAkira Kobayashi, Etsuro Ito, Tsutomu Toki, Keiji Kogame, Shinichiro Takahashi, Kazuhiko Igarashi, Norio Hayashi, Masayuki Yamamoto,
Tópico(s)RNA Research and Splicing
ResumoThe NF-E2-binding sites or Mafrecognition elements (MARE) are essentialcis-acting elements in the regulatory regions of erythroid-specific genes recognized by the erythroid transcription factor NF-E2, composed of p45 and MafK. Recently, two p45-related factors Nrf1 and Nrf2 were isolated, and they are now collectively grouped as the Cap'n' collar (CNC) family. CNC factors bind to MARE through heterodimer formation with small Maf proteins. We report here the identification and characterization of a novel CNC factor, Nrf3, encoding a predicted 73-kDa protein with a basic region-leucine zipper domain highly homologous to those of other CNC proteins. In vitro and in vivo analyses showed that Nrf3 can heterodimerize with MafK and that this complex binds to the MARE in the chicken β-globin enhancer and can activate transcription. Nrf3 mRNA is highly expressed in human placenta and B cell and monocyte lineage. Chromosomal localization of human Nrf3 is 7p14–15, which lies near the hoxAgene locus. As the genetic loci of p45, nrf1, andnrf2have been mapped close to those ofhoxC, hoxB, and hoxD, respectively, the present study strongly argues for the idea that a single ancestral gene for the CNC family members may have been localized near the ancestral Hox cluster and have diverged to give rise to four closely related CNC factors through chromosome duplication. The NF-E2-binding sites or Mafrecognition elements (MARE) are essentialcis-acting elements in the regulatory regions of erythroid-specific genes recognized by the erythroid transcription factor NF-E2, composed of p45 and MafK. Recently, two p45-related factors Nrf1 and Nrf2 were isolated, and they are now collectively grouped as the Cap'n' collar (CNC) family. CNC factors bind to MARE through heterodimer formation with small Maf proteins. We report here the identification and characterization of a novel CNC factor, Nrf3, encoding a predicted 73-kDa protein with a basic region-leucine zipper domain highly homologous to those of other CNC proteins. In vitro and in vivo analyses showed that Nrf3 can heterodimerize with MafK and that this complex binds to the MARE in the chicken β-globin enhancer and can activate transcription. Nrf3 mRNA is highly expressed in human placenta and B cell and monocyte lineage. Chromosomal localization of human Nrf3 is 7p14–15, which lies near the hoxAgene locus. As the genetic loci of p45, nrf1, andnrf2have been mapped close to those ofhoxC, hoxB, and hoxD, respectively, the present study strongly argues for the idea that a single ancestral gene for the CNC family members may have been localized near the ancestral Hox cluster and have diverged to give rise to four closely related CNC factors through chromosome duplication. Regulation of biological processes is accomplished through interactions among various tissue- or developmental stage-specific transcription factors. For instance, the transcription factor NF-E2 is critical for erythroid-specific expression of the porphobilinogen deaminase gene (1Mignotte V. Navarro S. Eleouet J.F. Zon L.I. Romeo P.H. J. Biol. Chem. 1990; 265: 22090-22092Abstract Full Text PDF PubMed Google Scholar). NF-E2 is composed of two subunits; the p45 component contains the Cap' n' collar (CNC) 1The abbreviations used are: bZip, basic region-leucine zipper; CNC, Cap'n' collar; EMSA, electrophoretic gel mobility shift analysis; EST, expressed sequence tag; FISH, fluorescence in situ hybridization; MARE, Maf recognition element; MBP, maltose-binding protein; Nrf3, NF-E2-related factor 3; LCR, locus control region; UTR, untranslated region; bp, base pair(s); kbp, kilobase pair(s); PCR, polymerase chain reaction; GAD, GAL4 activation domain; GBD, GAL4 DNA-binding domain.-type basic region-leucine zipper (bZip) domain, which is highly conserved with the bZip domain of the Drosophila transcription factor CNC (2Mohler J. Vani K. Leung S. Epstein A. Mech. Dev. 1991; 34: 3-10Crossref PubMed Scopus (108) Google Scholar). Recently, several mammalian transcription factors carrying the CNC/bZip domain were identified, such as Nrf1/LCR-F1/TCF11 (3Chan J.X. Han X.-L. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11366-11370Crossref PubMed Scopus (111) Google Scholar, 4Caterina J.J. Donze D. Sun C.-W. Ciavatta D.J. Townes T.M. Nucleic Acids Res. 1994; 22: 2383-2391Crossref PubMed Scopus (124) Google Scholar, 5Luna L. Johnsen O. Skartlien A.H. Pedeutour F. Turc-Carel C. Prydz H. Kolsto A. Genomics. 1994; 22: 553-562Crossref PubMed Scopus (85) Google Scholar), Nrf2/ECH (6Moi P. Chan K. Asunis I. Cao A. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9926-9930Crossref PubMed Scopus (1252) Google Scholar, 7Itoh K. Igarashi K. Hayashi N. Nishizawa M. Yamamoto M. Mol. Cell. Biol. 1995; 15: 4184-4193Crossref PubMed Scopus (365) Google Scholar), Bach1, and Bach2 (8Oyake T. Itoh K. Motohashi H. Hayashi N. Hoshino H. Nishizawa M. Yamamoto M. Igarashi K. Mol. Cell. Biol. 1996; 16: 6083-6095Crossref PubMed Scopus (525) Google Scholar). The other subunit of the NF-E2 complex is composed of the small Maf proteins, members of the Maf proto-oncoprotein family (9Andrews N.C. Kotkow K.J. Ney H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11488-11492Crossref PubMed Scopus (238) Google Scholar, 10Igarashi K. Kataoka K. Itoh K. Hayashi N. Nishizawa M. Yamamoto M. Nature. 1994; 367: 568-572Crossref PubMed Scopus (401) Google Scholar). To date, three small Mafs, MafF, MafG, and MafK, have been identified (11Toki T. Itoh J. Kitazawa J. Arai K. Hatakeyama K. Akasaka J. Igarashi K. Nomura N. Yokoyama M. Yamamoto M. Itoh E. Oncogene. 1997; 14: 1901-1910Crossref PubMed Scopus (95) Google Scholar, 12Igarashi K. Itoh K. Motohashi H. Hayashi N. Matuzaki Y. Nakauchi H. Nishizawa M. Yamamoto M. J. Biol. Chem. 1995; 270: 7615-7624Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 13Blank V. Kim M.J. Adrews N.C. Blood. 1997; 89: 3925-3935Crossref PubMed Google Scholar, 14Fujiwara K.T. Kataoka K. Nishizawa M. Oncogene. 1993; 8: 2371-2380PubMed Google Scholar). A homodimeric complex of small Maf proteins recognizes a DNA sequence motif called Maf recognitionelement (MARE, TGCTGA(G/C)TCAGCA or TGCTGACGTCAGCA), which contains either a 12-O-tetradecanoylphorbol-13-acetate-responsive element (TGA(G/C)TCA) or cAMP-responsive element (TGACGTCA) (15Kataoka K. Fujiwara T. Noda M. Nishizawa Mol. Cell. Biol. 1994; 14: 7581-7591Crossref PubMed Scopus (198) Google Scholar, 16Kataoka K. Igarashi K. Itoh K. Fujiwara T. Noda M. Yamamoto M. Nishizawa M. Mol. Cell. Biol. 1995; 15: 2180-2190Crossref PubMed Scopus (200) Google Scholar). Homodimeric complexes of small Mafs repress transcription through binding to MARE, since they lack canonical trans-activation domains. On the other hand, CNC proteins require a small Maf protein for their DNA binding activities. A CNC-small Maf heterodimer complex binds to both the NF-E2 consensus sequence (TGCTGA(G/C)TCA (T/C)) and MARE (in particular to the 12-O-tetradecanoylphorbol-13-acetate-responsive element-type MARE). These regulatory motifs are frequently observed in the regulatory regions of erythroid-specific genes, such as the locus control region (LCR) of β-globin genes, as well as in several non-erythroid gene promoters (17Kataoka K. Noda M. Nishizawa M. Mol. Cell. Biol. 1994; 14: 700-712Crossref PubMed Google Scholar). To assess the biological roles of the CNC family proteins,p45, nrf1 and nrf2 genes were disrupted individually (18Shivdasani R.A. Rosenblatt M.F. Zucker-Franklin D. Jakson C.W. Hunt P. Saris C.J.M. Orkin S.H. Cell. 1995; 81: 695-704Abstract Full Text PDF PubMed Scopus (632) Google Scholar, 19Farmer S.C. Sun C.-W. Winner G.E. Hogan B.L.M. Townes T.M. Genes Dev. 1997; 11: 786-798Crossref PubMed Scopus (99) Google Scholar, 20Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3226) Google Scholar). Whereas the loss of p45 function impaired megakaryocyte maturation and platelet formation, erythropoiesis in p45-null mice was normal. Nrf2 is essential for the induction of phase II-detoxifying gene expression such as glutathione S-transferase and NADPH-quinone oxidoreductase (NQO1) by phenolic antioxidants, but its loss (20Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3226) Google Scholar), even in combination with p45 (21Kuroha T. Takahashi S. Komeno T. Itoh K. Nagasawa T. Yamamoto M. J. Biochem. (Tokyo). 1998; 123: 376-379Crossref PubMed Scopus (37) Google Scholar, 22Martin F. Deursen J.M.V. Shivdasani R.A. Jackson C.W. Troutman A.G. Ney P.A. Blood. 1998; 91: 3459-3466Crossref PubMed Google Scholar), did not cause anemia. The results of thenrf1 gene disruption are somewhat controversial; one report showed early embryonic lethality (19Farmer S.C. Sun C.-W. Winner G.E. Hogan B.L.M. Townes T.M. Genes Dev. 1997; 11: 786-798Crossref PubMed Scopus (99) Google Scholar), whereas another study showed loss of definitive hematopoiesis (23Chan J.Y. Kwong M. Lu R. Chang J. Wang B. Ten T.S.B. Kan Y.W. EMBO J. 1998; 17: 1779-1787Crossref PubMed Scopus (222) Google Scholar). These results indicate that compensation among CNC family proteins may exist in erythroid-specific gene regulation and that lineage-specific regulation of gene expression through MAREs appears to be accomplished by multiple CNC family proteins. Fluorescence in situ hybridization (FISH) analyses revealed that the chromosomal localization of the p45,nrf1, and nrf2 genes are mapped close to the hoxC (12q13.1–13.3), hoxB (17q21), andhoxD (2q31) gene clusters, respectively (24Chan J.Y. Cheung M.-C. Moi P. Chan K. Kan Y.W. Hum. Genet. 1995; 95: 265-269Crossref PubMed Scopus (58) Google Scholar), suggesting that the p45-related factors may be derived from a single ancestral gene through chromosome duplication, as is the case for thehox gene clusters. Since no CNC factor has been identified near the hoxA locus (7p14–15), this also implies the existence of an additional CNC factor. We report here the identification and characterization of this new CNC family member, Nrf3 (NF-E2 related factor3). Expressedsequence tag (EST) cDNA fragment for Nrf3 (THC181377) was isolated by PCR using genomic DNA of HeLa cells as a template. 30 cycles of PCR were performed at 94 °C for 30 s; 60 °C for 30 s; 72 °C for 1 min. Primers used were 5′-GATATTTTTAGTAGATTAAGAGATGACC-3′ and 5′-GCACTTCATGAAAAAGTTGTGGC-3′. A human placenta λgt11 cDNA library (a generous gift from Dr. Shigeru Taketani, Kansai Medical University) was plated on 150-mm Petri dishes at a density of 5 × 104 plaque-forming units per plate, and 1 × 106 plaques were screened with labeled EST fragment (25Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). To isolate mouse Nrf3, a mouse brain cDNA library (Stratagene) was screened using a 0.5-kbp HindIII fragment of human Nrf3 cDNA as a probe. Hybridization was carried out at 65 or 50 °C in 50 mm Tris-HCl (pH 7.5), 1 mNaCl, 10 mm EDTA, 1× Denhardt's solution, 0.1% SDS, and 0.1 mg/ml salmon sperm DNA. Membranes were washed twice for 30 min at 65 °C in 0.1 × SSC and 0.1% SDS solution (human cDNA library screening) or 50 °C in 2 × SSC and 0.1% SDS solution (mouse cDNA library screening). Positive plaques were isolated and purified. 5′-Rapid amplification of the cDNA end analyses were carried out with the MarathonTM cDNA amplification kit (CLONTECH). DNA sequences were determined on both strands by the dideoxynucleotide method using an ABI377 automated sequencer (Perkin-Elmer). NALL-1, NALM-6, BALM-2, NAMALWA, and RPMI8226 cells were gifts of Dr. Kenji Ohtani (Fujisaki Cell Center of Hayashibara Biochemical Laboratories, Inc.), and KOPT-K1, THP6, and NALM-17 cells were gifts of Dr. Yasuhide Hayashi (University of Tokyo). MGS cells were a gift of Dr. Shinkichi Yokoyama (Yamagata University). KG-1, HL60, U937, and THP1 were obtained from Japanese Cancer Resources Bank. Poly (A)+ RNA samples from various cultured cell lines were prepared by the guanidine-acidified phenol chloroform method (26Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar) and purified with oligotex (dT)30 column chromatography (Roche). RNA samples were electrophoretically separated on a 1.0% agarose gel containing 1.1m formaldehyde and transferred onto ZetaProbe membranes (Bio-Rad). RNA blots containing multiple human tissue RNAs were purchased from CLONTECH (2 μg of poly (A)+ RNA per sample). Radiolabeled probe was prepared from the 500-bp HindIII fragment of the human clone, SKhNrf3/1-1. BosNrf1, BosNrf2, and BosNrf3 were generated by subcloning the 2.6-kbp HindIII/XbaI fragment from pcDNAI/Neo-Nrf1 (11Toki T. Itoh J. Kitazawa J. Arai K. Hatakeyama K. Akasaka J. Igarashi K. Nomura N. Yokoyama M. Yamamoto M. Itoh E. Oncogene. 1997; 14: 1901-1910Crossref PubMed Scopus (95) Google Scholar), the 2.6-kbp BamHI/XbaI fragment from pcDNAI/Neo-Nrf2 (11Toki T. Itoh J. Kitazawa J. Arai K. Hatakeyama K. Akasaka J. Igarashi K. Nomura N. Yokoyama M. Yamamoto M. Itoh E. Oncogene. 1997; 14: 1901-1910Crossref PubMed Scopus (95) Google Scholar), and the 2.5-kbp fragment from SKmNrf3–1, respectively, into the blunt-ended XbaI site of pEFBos (27Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5332Crossref Scopus (1499) Google Scholar). The quail fibroblast cell line QT6 (28Moscovici C. Moscovici M.G. Jimenez H. Lai M.M.C. Hayman M.J. Vogt P.K. Cell. 1977; 11: 95-103Abstract Full Text PDF PubMed Scopus (280) Google Scholar) was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and plated onto 24-well dishes (5 × 104 cells per well with 0.3 ml medium) 24 h before transfection by the calcium phosphate precipitation method (29Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5292) Google Scholar). 10 ng of firefly luciferase (Luc) reporter plasmid, 50 ng of pENL (an internal control), and various combinations of effector plasmids were used (see figure legends). Luc reporter plasmids pRBGP2 and pRBGP4 were previously described (10Igarashi K. Kataoka K. Itoh K. Hayashi N. Nishizawa M. Yamamoto M. Nature. 1994; 367: 568-572Crossref PubMed Scopus (401) Google Scholar). QT6 cells were washed twice with phosphate-buffered saline 12 h after transfection, fed with fresh media, and incubated for an additional 24 h. Luc activity was measured following the supplier's protocol (Promega) with a Biolumat luminometer (Berthold). Assays were performed in triplicate in independent transfection experiments, and the results were normalized with respect to pENL β-galactosidase activity. Expression plasmids of GADNrf1, GADNrf2, and GADNrf3 were generated by inserting the 2.6-kbpDraI/XbaI fragment from pcDNAI/Neo-Nrf1 (11Toki T. Itoh J. Kitazawa J. Arai K. Hatakeyama K. Akasaka J. Igarashi K. Nomura N. Yokoyama M. Yamamoto M. Itoh E. Oncogene. 1997; 14: 1901-1910Crossref PubMed Scopus (95) Google Scholar), the 2.6-kbp BglII/XbaI fragment from pcDNAI/Neo-Nrf2 (11Toki T. Itoh J. Kitazawa J. Arai K. Hatakeyama K. Akasaka J. Igarashi K. Nomura N. Yokoyama M. Yamamoto M. Itoh E. Oncogene. 1997; 14: 1901-1910Crossref PubMed Scopus (95) Google Scholar), and the 1.8-kbp EcoRI fragment from SKhNrf3/1–1, respectively, into the appropriate sites of GAD424. GBD-MafK was generated by subcloning the 1.0-kbpBamHI/HindIII fragment from pQE30-MafK (11Toki T. Itoh J. Kitazawa J. Arai K. Hatakeyama K. Akasaka J. Igarashi K. Nomura N. Yokoyama M. Yamamoto M. Itoh E. Oncogene. 1997; 14: 1901-1910Crossref PubMed Scopus (95) Google Scholar) into the blunt-ended ClaI site of GBD-pCla which was created by inserting a ClaI linker (8-mer, 5′-catcgatg-3′) into the EcoRI site of GBT9. Saccharomyces cerevisiae SFY526 were transformed with several combinations of plasmids by the lithium acetate method. Measurement of β-galactosidase activity of transformants was performed as described (30Kobayashi A. Numayama-Tsuruta K. Sogawa K. Fujii-Kuriyama Y. J. Biochem. (Tokyo). 1997; 122: 703-710Crossref PubMed Scopus (153) Google Scholar). MBPNrf1, MBPNrf2, MBPNrf3, and MBPMafK were constructed by inserting 500-bp PCR-generatedBamHI/XbaI fragments from pcDNAI/Neo-Nrf1, pcDNAI/Neo-Nrf2, and SKhNrf3/1–1, and the 1.0-kbpBamHI/HindIII fragment from pQE30-MafK (11Toki T. Itoh J. Kitazawa J. Arai K. Hatakeyama K. Akasaka J. Igarashi K. Nomura N. Yokoyama M. Yamamoto M. Itoh E. Oncogene. 1997; 14: 1901-1910Crossref PubMed Scopus (95) Google Scholar) into appropriate sites of pMAL-C2 (New England Biolabs). Primers used were 5′-CGGGATCCAAAGGCAGCAAGGAGAAGCA-3′/SP6 primer (Nrf1), 5′-CGGGATCCCCATTCACAAAAGACAA-3′/SP6 primer (Nrf2), and 5′-CGGGATCCCAGAAGATAAGGAGTAGATA-3′/T3 primer (Nrf3). All constructs were confirmed by sequencing. BL21(DE3) pLysS strain of E. coli was transformed with each expression vector and incubated overnight. Each culture was diluted 10-fold with fresh LB medium containing 100 μg/ml ampicillin and incubated for additional 2 h at 37 °C. The cells were harvested 3 h after the addition of isopropyl-1-thio-β-d-galactopyranoside at a final concentration 0.2 mm and mildly sonicated in extraction buffer (20 mm Tris-HCl (pH 7.5), 500 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 1 mmphenylmethylsulfonyl fluoride, and 1 mm dithiothreitol). Lysates were prepared by centrifugation at 5,000 × gfor 20 min at 4 °C and loaded on columns of amylose resin (New England Biolabs). The columns were washed with extraction buffer and subsequently with buffer B (20 mm Hepes-NaOH (pH 7.9), 20 mm NaCl, 4 mm MgCl2, 1 mm EDTA, 20% glycerol, 1 mmphenylmethylsulfonyl fluoride, and 1 mm dithiothreitol). Recombinant proteins were eluted with buffer B containing 10 mm maltose and analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Oligonucleotides encoding the chicken β-globin enhancer MARE sequence (5′-TCGCCCGAAAGGAGCTGACTCATGCTAGCCC-3′) were labeled with γ[32P]ATP by T4 polynucleotide kinase. The reaction mixture contained 20 mm Hepes-NaOH (pH 7.9), 1 mm EDTA, 60 mm NaCl, 1 mmdithiothreitol, 4 mm MgCl2, 25 μg of poly(dI-dC) and poly(dA-dT)/ml, 100 μg of bovine serum albumin/ml, and 50 ng of purified proteins. The reactions were electrophoresed at 4 °C, 150 V on a 4.5% polyacrylamide gel in 0.5 × TBE (45 mm Tris-HCl (pH 8.2), 45 mm borate, and 1.25 mm EDTA). Competition assays using various cold probes were performed as described previously (8Oyake T. Itoh K. Motohashi H. Hayashi N. Hoshino H. Nishizawa M. Yamamoto M. Igarashi K. Mol. Cell. Biol. 1996; 16: 6083-6095Crossref PubMed Scopus (525) Google Scholar). In off-rate kinetic experiments, EMSA reaction mixture was first incubated with radiolabeled probe at 37 °C for 5 min. After the addition of cold double-stranded oligonucleotides (560-fold molar excess), an aliquot of the mixture was taken and analyzed by polyacrylamide gel electrophoresis at several time points (described in figure legend). Amounts of binding complexes were quantified by an imaging analyzer (Fuji BAS-2000). Metaphase spreads for FISH were prepared from phytohemagglutinin-stimulated normal human lymphocytes using a thymidine synchronization/deoxybromouridine release technique. Nrf3 cDNA was labeled by nick translation with biotin-16-dUTP (Boehringer Mannheim). In situ hybridization was performed with the protocol of Inazawa et al. (32Inazawa J. Fukunaga R. Seto Y. Nakagawa H. Misawa S. Abe T. Nagata S. Genomics. 1991; 10: 1075-1078Crossref PubMed Scopus (53) Google Scholar) with minor modifications. Briefly, for hybridization, 200 ng of probe was added to 10 ml of a hybridization mixture containing 50% formamide, 2× SSC, 10% dextran sulfate, and 20 μg/ml bovine serum albumin at 37 °C for 90 min. Slides were denatured in 70% formamide, 2× SSC at 72 °C for 3 min, dehydrated, and hybridized overnight in a moist chamber at 37 °C with the probe. The slides were washed in 50% formamide, 2× SSC and in 1× SSC at 37 °C for 10 min each, and then in 4× SSC at room temperature for 10 min. After washing, the slides were incubated with avidin-fluorescein isothiocyanate at 37 °C for 50 min and then counterstained with DAPI in anti-fade solution. Analysis was carried out using an Olympus BX50-FLA fluorescence microscope and photographs were taken with Kodak Ektachrome ASA 400 film. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBankTM nucleotide sequence data bases with the following accession numbers, AB010812 (human) and AB013852 (mouse). Loci for the p45, nrf1, andnrf2 genes have been mapped near the hoxC,hoxB, and hoxD gene clusters, respectively (24Chan J.Y. Cheung M.-C. Moi P. Chan K. Kan Y.W. Hum. Genet. 1995; 95: 265-269Crossref PubMed Scopus (58) Google Scholar), but no CNC factor mapping close to the hoxA locus had yet been identified, suggesting that a novel CNC factor may exist. We searched for CNC factors in the TIGR human EST data base and found a 668-bp EST clone (GenBankTM accession number THC181377) encoding an amino acid sequence homologous to that of the C-terminal end of Nrf1. As this EST clone may encode a novel CNC factor, we set out to isolate the corresponding or related DNA fragments. We first attempted to isolate a genomic DNA fragment corresponding to this EST clone, since nrf1 and nrf2 genes are known to contain a large C-terminal exon (5Luna L. Johnsen O. Skartlien A.H. Pedeutour F. Turc-Carel C. Prydz H. Kolsto A. Genomics. 1994; 22: 553-562Crossref PubMed Scopus (85) Google Scholar, 6Moi P. Chan K. Asunis I. Cao A. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9926-9930Crossref PubMed Scopus (1252) Google Scholar). The C-terminal exon sequence was amplified by PCR using genomic DNA of HeLa cells as a template, and a fragment with sequence matching exactly to the EST clone was successfully obtained (data not shown). To obtain a full length cDNA clone, a human placenta cDNA library was screened using the PCR clone, and four positive overlapping phage clones were isolated (data not shown; see below). To isolate the mouse homologue, we screened a mouse brain cDNA library using one of the human partial cDNA clones and obtained eight positive phage clones. As shown in Fig.1 A, the longest mouse cDNA clone encodes a 1983-bp open reading frame flanked by 237 and 339 bp of 5′- and 3′-untranslated regions (UTRs), respectively. Although no preceding in-frame stop codon was noted in the 5′-UTR of this cDNA, we identified a termination codon 15 nucleotides upstream from 5′-terminal of this cDNA in the mouse genomic clone, 2A. Kobayashi, unpublished observations. supporting our assigned translation initiation codon. The nucleotide sequence GAGATGA surrounding the ATG codon (underlined) matches the translation initiation site consensus sequence reported by Kozak (33Kozak M. J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2810) Google Scholar). The open reading frame encodes a predicted protein of 660 amino acid residues with a calculated molecular mass of 72,706 Da (Fig.1 A). Two copies of RNA destabilizing signal (ATTTA) (underlined; Ref. 34Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3124) Google Scholar) reside in the 3′-UTR.Figure 1Cloning and structure of Nrf3. A, nucleotide sequence and deduced amino acid sequence of mouse Nrf3 cDNA. The CNC and basic domains and leucine residues in zipper domains are indicated by the dotted line,box, and circles, respectively. A polyadenylation consensus sequence is indicated by the thick underline. Two mRNA destabilizing sequences in 3′-UTR and one CNC factor-conserved amino acid sequence (DSGLSL) are underlined. B, comparison of amino acid sequences of mouse and human Nrf3. Sequence information from human Nrf3 genomic sequence data of bacteria artificial chromosome clone (AC004520) is underlined.View Large Image Figure ViewerDownload (PPT) Sequence analysis of the longest human clone (number 1–1) and comparison of the predicted protein with that of the mouse revealed that the human clone is likely to be partial (Fig. 1 B), since the N-terminal coding region was shorter than that of the mouse protein. Despite extensive 5′-rapid amplification of the cDNA end analyses using human placental mRNA, we were unable to isolate any additional upstream sequence (data not shown). However, the genomic sequence data of a bacteria artificial chromosome clone containing this locus was recently entered in the GenBankTM data base from the Genome Sequencing Center of Washington University (accession numberAC004520) and that sequence information has been incorporated into the human cDNA sequence (Fig. 1 B). Comparison of the N-terminal structure of the human protein with that of the mouse revealed that their deduced coding sequences are less conserved (68% homology) than those of other CNC family members (89, 97, and 80% overall identities, respectively, for p45, Nrf1, and Nrf2) between mouse and human. Inspection of the domain structure of the new mouse clone revealed the presence of a bZip domain which is homologous to that of other CNC transcription factors: 53, 56, and 50% identity, respectively, for p45, Nrf1, and Nrf2 (Fig.2 A), indicating that this protein is a member of the CNC family. We therefore named this factor Nrf3 (NF-E2 relatedfactor-3). In the bZip region, the basic region of Nrf3 was especially highly conserved with that of other CNC factors (see Fig. 2 B), suggesting that Nrf3 may share their DNA binding specificity. A short region “DSGLSL” that is absolutely conserved among the CNC family members (24Chan J.Y. Cheung M.-C. Moi P. Chan K. Kan Y.W. Hum. Genet. 1995; 95: 265-269Crossref PubMed Scopus (58) Google Scholar) is also present in Nrf3 (see Fig. 1 A, underlined). The leucine-zipper sequence of Nrf3, however, was much more variable than those of other CNC factors. The three CNC factors currently analyzed (i.e. p45, Nrf1, and Nrf2) have all been shown to bind MAREs as heterodimers with one of the small Maf proteins, but not as homodimers. Since one of these small Mafs, MafK (9Andrews N.C. Kotkow K.J. Ney H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11488-11492Crossref PubMed Scopus (238) Google Scholar, 11Toki T. Itoh J. Kitazawa J. Arai K. Hatakeyama K. Akasaka J. Igarashi K. Nomura N. Yokoyama M. Yamamoto M. Itoh E. Oncogene. 1997; 14: 1901-1910Crossref PubMed Scopus (95) Google Scholar, 12Igarashi K. Itoh K. Motohashi H. Hayashi N. Matuzaki Y. Nakauchi H. Nishizawa M. Yamamoto M. J. Biol. Chem. 1995; 270: 7615-7624Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), is highly coexpressed with Nrf3 (see below) in the placenta, these two factors are likely to be partners, at least in this tissue. We therefore examined whether a complex of Nrf3 and MafK could bind to the MARE of the chicken β-globin enhancer by EMSA. Bacterially expressed MBP-fused bZip domains of the CNC proteins were tested for their ability to bind MARE. In the absence of MafK, no DNA binding was observed (Fig. 3 A,lanes 2–4). Although a homodimer of MafK could bind the probe (lane 5), the affinity appeared to be much weaker than the CNC-MafK heterodimers (lanes 6–8). The binding of Nrf3-MafK was efficiently competed out with addition of cold double-stranded oligonucleotides encoding the MAREs of the chicken β-globin enhancer and the hypersensitive site 2 (HS-2) of human β-globin LCR (Fig. 3 B, lanes 3 and 5), but not by mutated MAREs (lane 4). Similarly, the MARE sequence of the erythroid 5-aminolevulinate synthase gene promoter, to which NF-E2 cannot efficiently bind, 3K. Igarashi and M. Yamamoto, unpublished observations. did not compete with the binding of the labeled probe (lane 6). These results demonstrate the specificity of the recognition for MAREs by the Nrf3-MafK heterodimer complex. To assess binding affinities of the three CNC factors to MARE, a series of off-rate kinetic experiments was carried out (Fig. 3 C). These CNC factors were mixed with MafK and radiolabeled MARE probe, and the mixtures were subjected to the addition of cold double-stranded oligonucleotides. The mixtures were then separated at several time points by polyacrylamide gel electrophoresis. Fig. 3 D shows half-times of Nrf1-, Nrf2- and Nrf3-MafK-DNA complexes. The half-time of dissociation of Nrf1- and Nrf3-MafK complex from MARE was approximately 5 min, while that of Nrf2-MafK complex was within 3 min. These results suggest that Nrf1- and Nrf3-MafK complexes could bind the MARE probe more tightly than Nrf2-MafK complex in this experimental condition. We then examined dimerization affinities between Nrf3 and MafK using the yeast two-hybrid system. For this purpose the S. cerevisiae strain SFY526 was utilized which carries a lacZ reporter gene with binding sites for GAL4 (upstream activating sequence). Hybrid proteins of human Nrf1, Nrf2 (both full length), and Nrf3 (lacking the N-terminal) fused to the GAL4 activation domain (GAD) were used for “prey,” whereas a fusion of human MafK to the GAL4 DNA-binding domain (GBD) was used as “bait” (Fig.4). Dimerization activities between these chimeric proteins were determined by measuring the LacZ activity. As shown in Table I, control experiments using GBT9 plasmid (GBD alone) and GAD-Nrf1, GAD-Nrf2, or GAD-Nrf3 did not exhibit LacZ reporter activity. Similarly, GBD-MafK and GAD424 plasmid (GAD alone) showed no LacZ activity. In contrast, considerable enhancement of LacZ activity was observed when th
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