BBRC
Biochemical and Biophysical Research Communications 312 (2003) 831–842
www.elsevier.com/locate/ybbrc

Mouse MafA, homologue of zebraﬁsh somite Maf 1, contributes
to the speciﬁc transcriptional activity through the insulin promoter
Miwako Kajihara,a,d Hirohito Sone,b Michiyo Amemiya,b Yasutake Katoh,cMasashi Isogai,a Hitoshi Shimano,b Nobuhiro Yamada,b and Satoru Takahashia,*aInstitute of Basic Medical Sciences, University of Tsukuba, 1-1-1, Tennodai, Tsukuba 305-8575, Japan
Department of Internal Medicine, University of Tsukuba, 1-1-1, Tennodai, Tsukuba 305-8575, Japanc
Center for Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, 1-1-1, Tennodai, Tsukuba 305-8575, Japand
Bio-oriented Technology Research Advancement, Institution (BRAIN), 1-40-2, Nisshin-cho, Saitama-city, Saitama 331-8537, Japanb

Received 20 October 2003

Abstract
Large Maf transcription factors, which are members of the basic leucine zipper (b-Zip) superfamily, have been reported to be
involved in embryonic development and cell diﬀerentiation. Previously, we isolated a novel zebraﬁsh large Maf cDNA, somite Maf1
(SMaf1), which possesses transactivational activity within its N-terminus domain. To elucidate SMaf1 function in mammals, we
tried to isolate the mouse homologue of zebraﬁsh SMaf1. We isolated the mouse homologue of zebraﬁsh SMaf1, which is the same
molecule as the recently reported MafA. MafA mRNA was detected in formed somites, head neural tube, and liver cells in theembryos. In the adult mouse, MafA transcript was ampliﬁed in the brain, lung, spleen, and kidney by RT-PCR. MafA mRNA wasalso detectable in b-cell line. Next, we analyzed the transcriptional activity of MafA using rat insulin promoters I and II (RIPI andII), since a part of RIP sequence was similar to the Maf recognition element (MARE) and MafA was expressed in pancreatic b-cells.
MafA was able to activate transcription from RIPII, but not RIPI, in a dose dependent manner and the activity was dependent on
RIPE3b/C1 sequences. In addition, the amount of MafA protein was regulated by glucose concentration. These results indicate that
MafA is the homologue of zebraﬁsh SMaf1 and acts as a transcriptional activator of the insulin gene promoter through the RIPE3b
element.
Ó 2003 Published by Elsevier Inc.
Keywords: Maf; b-Zip; Transcription factor; Insulin

The Maf transcription factors are basic-leucine zipper(b-Zip) family proteins and are homologues of thev-Maf oncoprotein, which was isolated as a transforming component of the avian musculoaponeurotic ﬁbrosarcoma virus, AS42 [1,2]. Maf family proteins are
divided into two subgroups, the large Maf and the small
Maf proteins. The large Maf proteins, MafA/L-Maf/
SMaf1 [3–5], MafB [6], c-Maf [7], and Nrl [8], contain anacidic domain in their N-termini that acts as a transcriptional activation site. By contrast, the small Maf
proteins, MafK, MafF [9], and MafG [10], lack such anacidic domain.

*
Corresponding author. Fax: +81-298-53-6965.
E-mail address: satoruta@md.tsukuba.ac.jp (S. Takahashi).

0006-291X/$ - see front matter Ó 2003 Published by Elsevier Inc.
doi:10.1016/j.bbrc.2003.10.196

Several experiments have demonstrated that the large
Mafs are key factors involved in cellular diﬀerentiation.
MafA/L-Maf/SMaf1 takes part in lens diﬀerentiation,induces crystallin expression in chicken embryos [4], andis expressed in the somite during zebraﬁsh development[5]. The MafB member has been identiﬁed as the aﬀectedgene in mice carrying the kreisler (kr) mutation [11], andthe gene product is required for segmentation of thehindbrain by controlling Hoxb-3 expression [12]. In
addition, MafB down-regulates the expression of thetransferrin receptor and inhibits erythroid diﬀerentiation in myelomonocytic cells [13]. Furthermore, ENU
induced kreisler mutant (kreislerENU ) displayed inadequate podocyte development in the kidney and caused
nephrosis [14]. It has been reported that c-Maf activates
the transcription of L7 gene expression in developing

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Purkinje cells [15] and controls the tissue-speciﬁc expression of interleukin-4 (IL-4) in CD4þ T helper 2 cells[16]. In addition, severe defects occur in the lens ﬁber
cells of c-maf gene knock-out mice [17] and mutation of
the c-maf gene is the cause of abnormal human oculardevelopment [18]. Nrl, the last member of the large
Mafs, is well known as the gene responsible for human
Retinitis Pigmentosa [19]. Nrl contributes to the maturation and establishment of neuronal cells [20] andregulates the expression of the rhodopsin gene in theretina [21]. This accumulated information indicates thatthe large Maf proteins play indispensable regulatory
roles in cellular diﬀerentiation, morphogenesis, andseveral distinct developmental processes.
It has been reported that large Mafs bind to the target
DNA sequence by their basic domain and form either
homodimers or heterodimers with other Mafs, Jun, or
Fos [6,22,23] through their leucine-zipper domains. Maf
dimers bind to TRE (TPA-responsive element)-type Maf
recognition elements, T-MARE (TGCTGACTCAG
CA), or CRE (cAMP-responsive element)-type MARE,
C-MARE (TGCTGACGTCAGCA) [22,24]. The
chicken L-Maf was shown to recognize the core site
(TGCTGAC, )108 to )102) within the aCE2 sequence(CTCCGCATTTCTGCTGACCAC, )119 to )99),
which was deﬁned as the lens-speciﬁc enhancer elementin the chicken aA-crystallin promoter [25]. The hoxb-3
r5 enhancer sequences (TGTCATCCCCCTAAGTCAG
CAGTTAC and CCAAATTTGCAGACACCTACATTCTTGGC) are recognized by MafB [12]. The mouse
IL-4 promoter (CTCATTTTCCCTTCCTTTCAGC
AACTTTAACTC) [16] is associated with c-Maf and therhodopsin promoter (TGCTGATTCAGCA) is the target site of Nrl [26]. These known Maf target sequences
are conserved at least half sequences of MARE, indicating that MARE is the adequate consensus sequenceof Maf target elements in vivo.
We previously isolated novel large Mafs named
SMaf1 and SMaf2 transcription factors [5], in addition
to previously isolated MafB/Val and c-Maf factors fromzebraﬁsh [27,28]. At the amino acid level, SMaf1 isclassiﬁed as MafA/L-Maf-type large Maf. SMaf1
showed transcriptional activation activity and themRNAs were detected in the paraxial mesoderm cells
after somite boundary formation. Our investigation ofzebraﬁsh large Mafs indicated that SMafs might play
important roles in somitogenesis. Very recently, it has
been reported that human and mouse MafA are expressed in the pancreatic b cells of the islet of Langerhans and are the potential regulators of insulin gene
expression [29,30]. To elucidate the detailed function of
MafA/L-Maf/SMaf1 in mammals, we isolated mouse
MafA, the homologue of zebraﬁsh SMaf1, and analyzed
its expression proﬁle. Furthermore, we analyzed thetranscriptional activity of MafA from several insulin
promoter constructs as compared with other large Mafs.

Our data indicate that MafA is the potential transcriptional regulator of the mammalian insulin gene.

Materials and methods
Isolation of the mouse homologue of zebraﬁsh SMaf1. The coding
sequence of the mouse homologue of zebraﬁsh SMaf1 was ampliﬁed
from genomic DNA extracted from the tail of C57BL/6J mouse, using
sense (50 -TTCTCCGACGACCACCTG) and antisense (50 -CTTCTCG
TA(C/T)TTCTCCTTG) oligonucleotide primers. These primers correspond to peptide sequences FSDDQL and KEKYEK, respectively,
which are conserved between human EST (BE676631), chicken L-Maf
and zebraﬁsh SMaf1. Polymerase chain reaction was performed with 1
cycle at 94 °C for 2 min, followed by 35 cycles at 94 °C for 30 s, 55 °C
for 30 s and 72 °C for 30 s, then 1 cycle at 72 °C for 3 min in 20 ll ofreaction mixture. The PCR product was cloned into pCRII-TOPO
vector (Invitrogen) and sequenced. The genomic and cDNA clones
were isolated from a 129/SvJ genomic library (Stratagene) and 10.5 dpc
mouse embryo cDNA library (Stratagene), respectively, using the PCR
fragment as probe. Approximately 8.64  105 genomic and 2.4  106
cDNA phages were screened, and positive clones were subcloned into
pBluescript II SK+ vector (Stratagene), and then the nucleotide
sequences were determined.
In situ hybridization. In situ hybridization was performed as described previously [31]. E12.5 mice embryos were ﬁxed overnight in 4%
buﬀered paraformaldehyde (PFA) at 4 °C. Following cryoprotection
with 30% sucrose in PBS, 10 lm sections were cut using a cryostat.
They were treated with protease K and ﬁxed with 4% PFA. After
postﬁxation, the sections were hybridized with digoxigenin (DIG)labeled RNA probes (50 ng/ml) in hybridization solution (50% formamide, 5 SSC, pH 4.5, 1% SDS, 50 lg/ml heparin, and 50 lg/ml yeastRNA) at 65 °C for 16 h. The samples were washed with 50% formamide, 5 SSC, and 1% SDS at 65 °C for 30 min, and three times with
50% formamide, 2 SSC at 65 °C for 30 min, and subsequently incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody(Roche) at 4 °C overnight. After washing with Tris-buﬀered saline with
0.1% Tween 20 (TBST), hybridization signals were visualized using
nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphatase as
chromogen. For in situ hybridization of sectioned mouse embryos, theprobes corresponding to the b-Zip coding region (229–359 amino acid)of the mouse mafA gene were synthesized using T3 and T7 RNApolymerases with DIG-11-uridin-50 -triphosphate labeling kit (Roche).
RT-PCR analysis of MafA gene expression. The total RNAs ofindividual tissues, cultured cell-line, and transfected cells were extracted from adult C57BL/6J mouse (27 w), INS-1 cells, and NIH3T3
cells, respectively, using TRIzol Reagent (Invitrogen). After DNase
treatment, 1 lg of the total RNA was used for cDNA synthesis using
SuperScript First-Strand Synthesis System (Invitrogen). Polymerase
chain reaction was performed with 1 cycle at 94 °C for 2 min, followed
by 50 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, and then1 cycle of 72 °C for 3 min in 20 ll of reaction mixture. The MafA
cDNA fragments (405 bp) were ampliﬁed using sense (50 -CACC
ACGTGCGCTTGG) and antisense (50 -CAGAAAGAAGTCGGG
TG) oligonucleotide primers.
Cell culture and transient transfection assay. MafA, MafB, andc-Maf cDNAs were subcloned into pEFX3-FLAG eukaryotic expression plasmid [4]. The constructs DMafA, DMafB, and Dc-Maf
encode amino acids 229–359 of mouse MafA, amino acids 207–323 ofmouse MafB, and amino acids 252–370 of mouse c-Maf, respectively.
The reporter plasmids, RIPII-251 (II-251), RIPII-126 (II-251), RIPII100 (II-251), RIPI-746 (I-746), and RIPI-135 (I-135), were ampliﬁed by
PCR from rat genomic DNA and cloned into pGL2-Basic vector(Promega). Mutated RIPII-251 (mII-251) was synthesized from II-251
using a QuickChange Site-Directed Mutagenesis Kit (Stratagene).
NIH3T3 cells were maintained in Dulbecco’s modiﬁed Eagle’s

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medium, supplemented with 10% fetal bovine serum. The cells weretransfected by the lipofection method using FuGENE6 Transfection
Regent (Roche) and then harvested at 48 h post-transfection. The luciferase assay was performed according to the supplier’s protocol using
the Dual-Luciferase Reporter Assay System (Promega). Transfection
eﬃciencies were routinely normalized by the co-expressed Renilla
reniformis luciferase activity, which was expressed by pRL-TK (Promega) expression plasmid.
Immunostaining and Immunoblotting. Immunostaining of cultured
cells was carried out as previously described [4]. The cells were ﬁxed
and incubated with anti-FLAG M2 antibody (Kodak), and then withgoat anti-mouse IgG FITC conjugated antibody (Zymed). As much as
10 lg/ml Hoechst (bisBENZIMIDE, Sigma) was added to ﬁnal wash
solution. Immunoblotting was performed as described previously [32].
Lysates of transfected NIH3T3 cells were generated with cell lysis
buﬀer. For electrophoretic gel mobility shift assay (EMSA), Mafs orDMafs were synthesized with TNT coupled reticulocyte lysate or TNT
coupled wheat germ extract in vitro transcription/translation systems(Promega), respectively, using pEFX3-FLAG-Mafs and pEFX3FLAG-DMaf constructs. As much as 100 lg of extract from NIH3T3
cells or 1 ll of in vitro translated protein lysate was applied to SDS–
polyacrylamide gel. To detect the immunoreactive proteins, we usedhorseradish peroxidase-conjugated anti-FLAG M2 (Sigma) and ECL
blotting reagents (Amersham).
Electrophoretic gel mobility shift assay. Maf and truncated DMaf
proteins were synthesized with in vitro transcription/translation
systems (Promega). RIPE3b and RIPE3b-I (RIPE3b of RIPI) oligonucleotides, (50 -GATCCGTTTGGAAACTGCAGCTTCAGCCCCA)
and (50 -GATCCGTTTGGAAATTACAGCTTCAGCCCCA), corresponding to the RIP II ()129 to )105) and the RIP I ()141 to )117),
respectively, were labeled with [32 P-a]dCTP using Klenow DNApolymerase. An aliquot of 7 ml of in vitro translated proteins was
mixed with 2 ng of a radiolabeled oligonucleotide in a reaction mixture
containing 10 mM Hepes–KOH (pH 7.9), 100 mM NaCl, 2 mM
EDTA, 80 mM DTT, and 50 lg/ml of poly(dI–dC), and analyzed by a
4% polyacrylamide gel in 0.25 TAE (10 mM Tris–HCl (pH 8.0),
10 mM acetate, and 0.25 mM EDTA). The gels were dried up and833

exposed to X-ray ﬁlms for 12 h. In the competition assay, the EMSA
reaction mixture was ﬁrst incubated with a radiolabeled probe at 25 °C
for 15 min and then unlabeled RIPE3b, RIPE3b-I or mutated RIPE3b
oligonucleotide (50 -GATCCGTTTGGAAACTCAGGCTTCAGCCCCA) was added to the reaction mixture.

Results
Mouse MafA is a homologue of zebraﬁsh SMaf1
To isolate the mouse gene fragments homologous tozebraﬁsh Smaf1 encoding the b-Zip domain, we performed PCR analysis using mouse genomic DNA andoligonucleotides corresponding to the conserved regionof known SMaf1/L-Maf homologues (Fig. 1A). The
resultant PCR products were subcloned and sequenced(Fig. 1B). Half of the clones encoded part of the basic
and leucine-zipper domains of mouse SMaf1 homologue. The Smaf1 genomic DNA and cDNA were obtained by screening a mouse genomic library preparedfrom 129Sv mice and a mouse cDNA library preparedfrom 10.5 dpc CD1 mouse embryo, respectively, using
the partial mouse SMaf1 clone as probe. In the result,
we isolated a mouse SMaf1 genomic clone that is similar
to the known large Maf sequences. And the cDNA clone
implicated that this mouse SMaf1 gene is expressed in
the mouse embryo.
Sequence analysis indicated this SMaf1 homologue
clone as mouse MafA, which has been reported very
recently [30]. The clone encodes a protein containing
acidic, basic, and leucine zipper domains conserved

Fig. 1. The mouse homologue of zebraﬁsh SMaf1 exists in the mouse genome. (A) Conserved amino acid sequences of known MafA/L-Maf/SMaf
members. The human EST is the data of BE676631. The primer-set was constructed for the b-Zip sequence of L-Maf/SMaf members. Arrows depict
the location of sense and antisense primers. (B) DNA fragments derived from mouse genomic mafA gene. FSD and KEK lanes show the PCR
experiments with a single primer. The arrow indicates the expected PCR product.

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Fig. 2. Mouse MafA is a zebraﬁsh SMaf1 homologue. (A) Schematic representation of MafA and other mouse large Mafs. Hatched boxes, acidic
domains; gray-boxes, basic domains; and solid boxes, leucine zipper (Lz) domains. Percentages of amino acid sequences identical to MafA are
indicated in each domain box. (B) Sequence alignment of mouse MafA and zebraﬁsh SMaf1. Horizontal bars and asterisks represent identical aminoacid residues to mouse MafA and gaps, respectively. The acidic, basic, and Lz domains are indicated. (C) Phylogenetic tree of vertebrate large Mafs.
The tree was generated by the UPGMA method using the whole amino acid sequences of the known large Mafs.

amongst the large Maf subfamily, and its basic domain
showed high homology (>75%) to that of other mouselarge Maf members (Fig. 2A). Mouse MafA cDNA
shares 85%, 92%, and 86% amino acid sequence identities with the individual acidic, basic and leucine zipper
moieties, respectively, of zebraﬁsh SMaf1 (Fig. 2B). A
phylogenetic tree based on the entire amino acid sequences of each Maf protein (Fig. 2C) indicated that theisolated clone belongs to the chicken L-Maf/zebraﬁsh
SMaf1 class. According to these observations, we concluded that mouse MafA is a homologue of zebraﬁsh
SMaf1. Based on the sequence similarity, the amino acidsequence of mouse MafA is closer to that of zebraﬁsh
SMaf1 than that of chicken L-Maf (Fig. 2C).
Expression of mouse MafA
In order to analyze the spatial expression proﬁle ofmouse MafA during the fetal period (12.5 dpc), we
performed whole mount in situ hybridization using
DIG-labeled probe (Fig. 3). MafA mRNA was detectedin each formed somite and its expression pattern appeared to be in the myotomal cells (Fig. 3C arrows). The
expression pattern of mouse MafA in the somite issimilar to that of zebraﬁsh SMaf1. MafA mRNAs werealso observed in the head neural tube (Fig. 3E arrows)
and liver cells (Fig. 3G arrows). A low level of MafA
transcripts was detected in some mesenchyme-like cells.
Next, to reveal the expression of mouse MafA in
the adult mouse, we performed RT-PCR using cDNA

synthesized from adult C57BL/6J brain, thymus, heart,
lung, liver, spleen, pancreas, trunk muscle, and kidney(Fig. 4A). We used pancreas cDNA at three times thevolume of the others, in order to obtain equivalentamounts of control HPRT PCR product. In this experiment, the fragments derived from MafA mRNA
were detected in the extracts of brain, lung, spleen,pancreas, and kidney. These PCR products were subcloned and the sequences were veriﬁed (data not shown).
Mouse MafA transcripts were undetectable in the trunk
muscle; a similar result was found for zebraﬁsh SMaf1
mRNA, which diminishes as maturation proceeds in thesomite cells.
Recently, it has been reported that MafA is a transcriptional activator of the human insulin gene [30]. To
assess the expression of MafA in pancreatic b cells,
where insulin peptide hormone is produced, we tested
RT-PCR using cDNA synthesized from the INS-1 cells
derived from mouse pancreatic b cells (Fig. 4B). MafA
transcripts were certainly detected in the cDNA of INS1 cells and also slightly recognized in the cDNA of
NIH3T3 mouse ﬁbroblast cells (data not shown).
Large Mafs activate the rat insulin promoter II
It is well known that mouse and rat have two insulin
genes and that each gene has a speciﬁc expression proﬁle[33–36]. To assess the contribution of large Mafs to thetransactivation activity of the insulin gene through therat insulin promoters I and II (RIPI and II), we ﬁrst

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Fig. 3. Expression of Mouse MafA is detected in the mouse fetus. (A) The mouse MafA mRNA expression of longitudinal section with antisense
probes. (B) This represents a sense probe control. (C,E,G) and (D,F,H) show higher magniﬁcations of (A,B), respectively, and the frames exhibit eachrange. Expression of MafA in the somite cells (C,D), the anterior neural tube cells (E,F), and the liver cells (G,H). Each arrow indicates one of thestained cells. A 12.5 dpc mouse fetus was used, sectioned laterally. Anterior to the top (A,B).

Fig. 5. Large Mafs activate reporter gene expression. (A) Schematic representation of the FLAG-Maf fusion protein and reporter construct (II-251).
(B–D) Transactivation activity of large Maf proteins. Rat insulin promoter II (RIPII) reporter plasmid was transfected into NIH3T3 cells with eachvolume of Maf expression plasmids. (B–D) represent the result of MafA, MafB, and c-Maf, respectively. Luciferase activity in the absence of theeﬀector plasmids was arbitrarily set at 1 and the mean values of triplicate samples are shown. (E) Detection of transfected Maf proteins in NIH3T3
cells by immunostaining. Transfected cells were incubated for 48 h and immunostained with an anti-FLAG Antibody (green). The nuclei were stainedwith Hoechst (blue).

analyzed the activities of MafA, MafB, and c-Maf in a
co-transfection/transactivation assay using RIPII in
NIH3T3 ﬁbroblast cells. The eﬀector Mafs were pre-pared as constructs with FLAG at the N-termini, while
the reporter construct RIPII-251 (II-251) was cis-linked
to a luciferase gene and contained the rat insulin

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Fig. 4. MafA mRNA is expressed in various organs of adult mice.
MafA mRNAs were detected in the cells of brain, lung, spleen, pancreas, kidney, and INS-1 cells (A,B; upper panels). HPRT was used as
an internal control (A,B; lower panels). Reverse transcriptase(RTase)—samples were examined to expose DNA contamination ofeach RNA source.

promoter II ()251 to )1), including the RIPE3b element
which is a putative Maf binding site (Fig. 5A). The
transcriptional activity of MafA increased with an increasing transfection volume of eﬀector (Fig. 5B). The
transcriptional activities of MafB and c-Maf were six toseven times stronger than that of MafA (Fig. 5C). Taken
together, these results indicate that the large Mafs possess transcriptional activity for the RIPII. Since c-Maf
and MafB knock-out +/) mice did not show dysglycemia, whereas MafA knock-out +/) mouse showed
exceptional sugar metabolism (our unpublished observation), further analysis was restricted to MafA and
MafB was used as a positive control. To conﬁrm theexpression of transfected genes, we performed immunostaining of culture cells transfected with MafA or
MafB using anti-FLAG antibody (Fig. 5E). The Maf
proteins induced to cells were detected in the nucleus.
RIPE3b is the target site of MafA and MafB
To designate the site of MafA recognition, two reporter constructs were prepared. II-126 and II-100
contain 126 bp ()126 to )1) and 100 bp ()100 to )1) of
RIPII, respectively (Fig. 6A). We also constructed
DMafs, where DMafA (amino acid 229–359) and DMafB(amino acid 207–323) lack acidic domains, in order toestimate the functional importance of the acidic domain
for transactivation (Fig. 6B). The DMafs were taggedwith N-terminal FLAGs. Whereas MafA and MafB
exhibited similar luciferase activities with II-251 and II126 reporters, II-100 was not activated by either Maf

Fig. 6. The RIPE3b element is the target sequence of Mafs. (A) The 50
end deletion reporters and (B) truncated Maf mutants are shown
schematically. (C,D) Results of transient transfection assay using
MafA and MafB eﬀector, respectively. The large Maf proteins activated the expression of reporter constructs, II-251 and II-126, but notof II-100. (E) The mutant sequences of RIPII-251 reporter are shown
compared with MARE motif. (F,G) The mII-251 reporter was not
suﬃciently activated with both MafA and MafB. (C–F) Both DMafAand DMafB, which lacked the acidic domains, did not cause obvious
activation.

factor (Figs. 6C and D). These results indicate that the
responsible elements for Maf activation are locatedbetween )126 and )101 of RIPII. Since this region
includes the RIPE3b element, which contains the Maf
recognition element (MARE) like sequence [22](Fig. 6E), we focused on the core site ()119 to )113) of
the RIPE3b element as the Maf binding sequence. To
elucidate the importance of the sequence, we constructed a mutated RIPE3b (mII-251) reporter based on
the II-251 construct, whose mutation abolishes bindingof Mafs [5] (Fig. 6E). When MafA or MafB was transfected with mII-251 reporter plasmid, the luciferase activity decreased by 90–95% (Figs. 6F and G). These data
indicated that the core sequence ()119 to )113) of theRIPE3b element is the target site for Mafs, and the two

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837

nucleotides GC are crucial for transactivation of RIPII
by Mafs. In addition, neither DMafA nor DMafB
activated the transcription of the reporter in any ofthe experiments (Figs. 6C, D, F, and G). These results
suggested that activation of MafA and MafB throughthe insulin promoter depends on acidic domainfunction.
MafA and MafB speciﬁcally bind to the RIPE3b element
To conﬁrm that Mafs directly bind to the RIPE3b
element, we carried out an electrophoresis gel mobility shift assay (EMSA) using RIPE3b oligonucleotide
as probe. Maf and DMaf proteins were synthesizedusing in vitro systems and the proteins were conﬁrmed by immunoblotting with FLAG epitope antibody (Fig. 7A). The results showed that all MafA
(lane 3), MafB (lane 7), DMafA (lane 13), and
DMafB (lane 17) proteins could bind to the RIPE3b
oligonucleotide (Figs. 7B and C). To examine the
DNA binding speciﬁcity, unlabeled RIPE3b oligonucleotide or mRIPE3b oligonucleotide was added tothe binding reaction as competitor. Addition of unlabeled RIPE3b oligonucleotide, but not mRIPE3b
oligonucleotide, reduced the shifted DNA–protein
complexes containing Maf or DMaf factors in eachcase. We concluded that MafA and MafB bind to
RIPII and recognize the core site of RIPE3b speciﬁcally. Our results also demonstrated that the reduced
activity observed using acidic domain deletionmutants is not due to a loss in DNA-binding (Figs.
6C, D, F, and G).
MafA and MafB do not activate the RIPI
There is a high similarity between the sequences of
RIPI and RIPII. To assess the transcriptional activityof Maf factors with the RIPI, we carried out luciferase
assays using an RIPI reporter (Fig. 8A). The luciferase
activity of the RIPI (I-746) was not of an adequatelevel compared with that of RIPII (II-251) with either
MafA or MafB (Figs. 8B and C). Further examination
was carried out using I-135 reporter (Fig. 8A) to exclude the possibility that the I-746 construct contains
negative regulatory elements. The transactivation activity of MafA or MafB was not detected for I-135(Figs. 1B and C). Taken together, we concluded thatboth MafA and MafB could activate the RIPII, but
not RIPI. The nucleotide diﬀerence in the core sequence of RIPE3b between RIPII and RIPI is a possible explanation for this. In the RIPE3b of RIPI
(RIPE3b-I), the crucial nucleotide G required for recognition of Maf proteins (see Fig. 6) was substituted
with A (Fig. 8D). To conﬁrm this hypothesis, we did
EMSA using RIPE3b-I and RIPE3b oligonucleotides
as competitors. RIPE3b-I showed signiﬁcantly lessFig. 7. MafA and MafB bind to RIPE3b. (A) Immunoblotting forthe conﬁrmation of Maf and DMaf proteins synthesized in vitro.
(B,C) DNA binding activity of MafA and MafB (lanes 3–10) or
DMafA and DMafB (lanes 13–20) was analyzed using RIPE3b oligonucleotides as probe. Lanes indicated as (—), RL or WG contained no proteins (lanes 1 and 11), the crude proteins in rabbit
reticulocyte lysate (lane 2), and those in wheat germ extract (lane12), respectively. We used 100- (lanes 4, 8, 14, and 18) or 200- (lanes
5, 9, 15, and 19)-fold excess of RIPE3b oligonucleotides, or 200-foldexcess of mRIPE3b oligonucleotides (lanes 6, 10, 16, and 20) as theunlabeled competitors. Arrows denote the positions of the DNA–
Mafs complexes.

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M. Kajihara et al. / Biochemical and Biophysical Research Communications 312 (2003) 831–842

Fig. 8. Rat insulin promoter I (RIPI) reporter is not activated by Mafs. (A) Schematic representation of RIPI reporter constructs. (B,C) Mafs do notpossess adequate transcriptional activity for RIPI. (D) Conservation of the nucleotide sequence of insulin promoters and MARE. Focus on the)130th nucleotide, “A,” of RIPI. (E) DNA binding activity of MafA and MafB (lanes 3–12) was analyzed using RIPE3b oligonucleotides as probe.
Lanes indicated as (—) and RL contained no proteins (lanes 1) and the crude proteins in rabbit reticulocyte lysate (lane 2), respectively. We used 100(lanes 4 and 9) or 200- (lanes 5 and 10)-fold excess of RIPE3b-I oligonucleotides (RIPE3b of RIPI), or 100- (lanes 6 and 11) or 200- (lanes 7 and 12)fold excess of RIPE3b oligonucleotides as the unlabeled competitors. Arrows denote the positions of the DNA–Mafs complexes.

competing activity as compared with RIPE3b (Fig. 8E).
These data indicate that transcription from RIPI is notunder large Maf regulation.
The amount of MafA protein increases by high glucose
level
It has been shown that the transcriptional activity of
MafA was lower than those of MafB and c-Maf (Figs.

5B–D). To conﬁrm the expression of Maf proteins intransfected NIH3T3 cells, we performed immunoblotting and immunostaining of transfected NIH3T3 cellswith anti-FLAG antibody. MafA protein was least detected as compared with other Mafs and DMafs (Figs.
9A and C). To assess transcriptional level from transfected MafA plasmids, we carried out RT-PCR analysis(Fig. 9B). MafA transcripts were suﬃciently expressed
as well as DMafA. Since insulin expression was induced

M. Kajihara et al. / Biochemical and Biophysical Research Communications 312 (2003) 831–842

839

Fig. 9. High glucose state is required for the accumulation of MafA protein. (A) Immunoblot detection of Mafs and DMafs. Mafs and DMaf expression plasmids were transfected into NIH3T3 cells cultured in normal glucose (5.6 mM) medium. Whole-cell extracts of NIH3T3 were subjected toimmunoblot detection by anti-FLAG antibody. (B) Expression levels of MafA and DMafA in the transfected cells cultured in normal glucose
medium. MafA and DMaf mRNAs were detected by RT-PCR analysis. (C) Immunostaining of transfected cells. Maf expression plasmids weretransfected into NIH3T3 cells. At 24 h after transfection, normal glucose medium was replaced with high glucose medium (25 mM) for cells in highglucose condition. Then immunostaining at 48 h after transfection was performed. (D) Immunoblot detection of Maf proteins using transfected
NIH3T3 cells. The band density was measured by densitometer (Bio-Rad) and normalized with laminB. The relative value is depicted at the bottom.

by high glucose concentration in vivo, we checked theamount of MafA protein in high glucose condition(25 mM). Immunostaining and Western blot analyses
clearly showed that MafA protein was more abundantin high glucose condition than in normal glucose condition (5.6 mM), while that of MafB or DMafA was notchanged by glucose concentration (Figs. 9C and D).
These results indicated that high glucose state induced
the accumulation of MafA protein. Taken together, weconcluded that the amount of MafA protein was regulated by glucose concentration, and the acidic domain of
MafA protein might be responsible for this regulation.

Discussion
In this paper, we isolated mouse MafA as thehomologue of zebraﬁsh SMaf1. In fact, mouse MafA

belongs to the L-Maf/SMaf1 class (Fig. 2C). We showedthat mouse MafA transcripts were located in several tissues, using whole mount in situ hybridization (Fig. 3) and
RT-PCR analysis (Fig. 4). The MafA mRNA was expressed in mouse somite cells, where the detected level ofzebraﬁsh SMaf1 expression was striking [5]. Additionally,
the transcripts of zebraﬁsh SMaf1 were also detected inhindbrain cells during zebraﬁsh embryogenesis [5],whereas, in our experiments, the mRNAs of mouse MafA
were observed in the developing mouse fetus (12.5 dpc)
(Fig. 3). Chicken L-Maf is expressed in the developing
lens cells of the chicken embryo, whereas quail MafA,
which is almost the same in structure to L-Maf except for
one amino acid diﬀerence, is expressed in retinal cells [3].
We also detected MafA mRNA in the lens and retina of
the mouse embryo (our unpublished observation). These
results manifest the various functions of the MafA/LMaf/SMaf1 class of Maf factors in the vertebrate.

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M. Kajihara et al. / Biochemical and Biophysical Research Communications 312 (2003) 831–842

In this study, we indicated that MafA and MafB
activate the RIPII and bind to the core site of RIPE3b.
Mouse and rat possess two insulin promoters each (insulin promoters I and II), whereas human possesses only
one promoter; these insulin promoters have uniquetranscriptional regulations [33–36]. Two signiﬁcant differences appeared when the RIPE3b sequences of theseinsulin promoters were aligned with the MARE sequence (Fig. 8D). One diﬀerence was in the human insulin promoter sequence and the other was in the RIPI.
Since nucleotide )122 “C” in the human sequence is
identical to the one in MARE, although the nucleotide is
diﬀerent from that in other insulin promoters, human
RIPE3b seems to be easily activated by Maf transcription factors. On the other hand, nucleotide )130 “A” in
RIPI obviously diﬀers from nucleotide “G” in the otherinsulin promoters and in the MARE sequence. As wehave demonstrated in this paper and as others have reported previously [22,24], “G” is crucial for Maf activity. This diﬀerence in the RIPI must be the reason why
the Mafs did not activate the RIPI reporter in our experiments. Taken together, we conclude that MafA is
not a suﬃcient transactivator for the RIPI. Furthermore, these data indicate that both the mouse andhuman insulin promoters contain Maf target sites andcan be activated by Maf transcription factors. We haverevealed that MafA activates the rat insulin promoter II,
but not promoter I. It has been reported that the responses of rat insulin genes to stimulation are diﬀerent[33,34]. Our results imply that, under conditions ofstimulation, the diﬀerence between rat insulin promoters
I and II depends on Maf regulation. It has been reported
that mouse insulin promoters I and II are also diﬀerentially regulated [35,36]. Since the RIPE3b elements ofboth genes are identical, other elements except for RIPE3b may be responsible for the regulational diﬀerence
between mouse insulin promoters I and II.
The RIPE3b/C1 element has been identiﬁed as one of
the most important elements for pancreatic b-cell speciﬁc insulin expression [37–39]. Other important elements are known as E1 and A3 [40–45]. The pancreatic
islet restricted transcription factors BETA2/NeuroD
and PDX1, which bind to the E1 and A3 elements, havebeen isolated. Mice deﬁcient in these transcription factors revealed these factors to be indispensable for islet
cell development and insulin gene expression [46–49]. In
addition, mutations in the BETA2 and PDX1 genes
were found in some patients with maturity-onset diabetes of the young (MODY) [50,51]. The RIPE3b has
also been shown to be involved in b-cell-speciﬁc insulin
gene transcription as well as in its glucose-regulated
expression. Previous studies have identiﬁed a b-cell-restricted RIPE3b-binding factor, called the RIPE3b1
activator, that appears in response to glucose in pancreatic b-cell nuclear extracts [38]. Very recently, twopapers have been published stating that the RIPE3b1

activator is MafA [29,30]. Our data conﬁrmed theirobservations and added new information about RIPE3b
recognition by large Maf transcription factors.
It was shown that the transactivation ability of MafA
is lower than that of MafB in our system of analysis(Figs. 5,6 and 8). The highest level of transactivation
activity of MafA was obtained with 500 ng of eﬀector
plasmid (data not shown), and that of MafB was obtained with 100 ng of eﬀector. Since we carried out thetransfection assay using 100 ng of eﬀector, MafA activity
was not maximal, but that of MafB was. Except for thispoint, MafA does not contain ample transactivation
activity. We also demonstrated that the amount of MafA
protein was regulated by glucose concentration (Figs. 9C
and D). There are several possible explanations for thisresult. The most likely possibility is that MafA protein isunstable in the cells, whereas other Mafs are stable innormal glucose condition (Fig. 9A). Recently, it has been
published that chicken L-Maf, which is the homologueof mouse MafA, is phosphorylated by FGF/ERK signaling pathways and that phosphorylation of L-Maf
induces its degradation [52]. In addition, it has been reported that quail MafA is phosphorylated and its
phosphorylation is important for maximum transactivation [53]. These data suggest that mouse MafA mustalso be phosphorylated and that its phosphorylation
may regulate the transcriptional activity and/or stabilityof MafA by extracellular signaling, glucose. We are now
trying to analyze the relationship between the phosphorylation and protein stability of MafA.
In conclusion, we isolated mouse MafA as a homologue of zebraﬁsh SMaf1 and isolated MafA was expressed in several tissues including pancreatic beta cells.
MafA was able to activate transcription from RIPII, but
not RIPI, through the RIPE3b element. Further analysis
concerning the protein modiﬁcation and stability of
MafA may open up new insights into understanding
insulin production and one of the causes of diabetes
mellitus.

Acknowledgments
We thank Dr. T. O’connor for helpful discussions. We are gratefulto Drs. T. Moriguchi, S. Kawauchi, and H. Ogino for providing
plasmids, Dr. T. Terunuma for technical advice, and M. Hamada and
Ms. A. Godo for their excellent assistances. This work was supported
in part by Grant-in-Aid from the Ministry of Education, Science,Sports and Culture, the Japanese Society for Promotion of Sciences(RFTF), and Program for Promotion of Basic Research Activities forInnovative Biosciences (PROBRAIN).

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