THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 276, No. 44, Issue of November 2, pp. 41357–41364, 2001
Printed in U.S.A.
The Autoimmune Regulator (AIRE) Is a DNA-binding Protein*
Received for publication, May 29, 2001, and in revised form, August 30, 2001
Published, JBC Papers in Press, August 31, 2001, DOI 10.1074/jbc.M104898200
Pradeep G. Kumar, Malini Laloraya, Cong-Yi Wang, Quin-Guo Ruan,
Abdoreza Davoodi-Semiromi, Kuo-Jang Kao, and Jing-Xiong She‡
From the Department of Pathology, Immunology, and Laboratory Medicine, Center for Mammalian Genetics and Diabetes
Center of Excellence, College of Medicine, University of Florida, Gainesville, Florida 32610
Autoimmune polyendocrinopathy candidiasis ectodermal
dystrophy (APECED),1 also known as autoimmune polyglandular syndrome type 1 (APS1), is a rare autosomal recessive
disorder common in isolated populations such as Finns, Sardinians, and Iranian Jews (1). This syndrome is characterized by
destructive autoimmune diseases of the endocrine organs,
chronic candidiasis of mucous membranes, and ectodermal disorders. APECED is caused by mutations in the autoimmune
regulator (AIRE) gene on chromosome 21q22.3 (2– 4). The
AIRE gene has recently been cloned by two independent groups
of investigators (5, 6). The AIRE gene consists of 14 exons
coding for a 2445-base pair mRNA transcript, and the translated product is expected to have 545 amino acids with a
predicted molecular mass of 57.5 kDa. The predicted AIRE
protein has several domains indicative of a transcriptional
regulator protein (6). AIRE harbors two zinc fingers of plant
* This work was supported by National Institutes of Health Grants
1R24DK58778 (NIDDK), 1RO1HD37800 (NICHD), and 1P01AI-42288
(NIAID) and by a grant from the Juvenile Diabetes Research Foundation. The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
‡ To whom correspondence should be addressed: Dept. of Pathology,
Immunology, and Laboratory Medicine, Box 100275, University of Florida, Gainesville, FL 32610. Tel.: 352-392-0667; Fax: 352-392-3053; Email: she@ufl.edu.
1
The abbreviations used are: APECED, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy; PHD, plant homeodomain;
HSR, homogeneously staining region; CREB, cAMP-response elementbinding protein; CBP, CREB-binding protein; PAGE, polyacrylamide
gel electrophoresis; rhAIRE, recombinant human AIRE; PVDF, polyvinylidene difluoride; MES, 4-morpholineethanesulfonic acid; PCR, polymerase chain reaction; PKA, protein kinase A; PKC, protein kinase C;
EGR, early growth response factor.
This paper is available on line at http://www.jbc.org
homeodomain (PHD) type. A putative DNA binding domain
named SAND as well as four nuclear receptor binding LXXLL
motifs, an inverted LXXLL domain, and a variant of the latter
(FXXLL) hint that this protein functions as a transcription
coactivator (5–7). Furthermore, a highly conserved N-terminal
100-amino acid domain in AIRE has a significant homology to
the homogenously staining (HSR) domain of Sp100 and Sp140
proteins (7). This domain has been shown to function as a
dimerization domain in several Sp-100 related proteins (8). At
the subcellular level, AIRE can be found in the cell nucleus in
a speckled pattern in domains resembling promyelocytic leukemia nuclear bodies, also known as ND10, nuclear dots, or
potential oncogenic domains, associated with the AIRE homologous nuclear proteins Sp100, Sp140, and Lysp100 (9).
Interestingly, it has recently been shown that AIRE can
activate transcription from a reporter gene when fused to a
heterologous DNA binding domain. This activation required
the full-length protein or the presence of more than one activation domain. A glutathione S-transferase pull-down assay
showed that AIRE formed homodimers in vitro, probably
through the N-terminal domain (amino acids 1–207) or through
the minimal 1–100 amino acid domain resembling Sp-100. It
has also been shown that AIRE interacts in vitro with CREBbinding protein (CBP) through the CH1 and CH3 conserved
domains, which has led the investigators to suggest that the
transcriptional activities of AIRE might be mediated through its
physical interaction with the common coactivator CBP (10). CBP
is a key coactivator that modulates the transcriptional regulation
dependent on adenylate cyclase-signaling pathway in eukaryotes. The signal processing is mediated by a family of cyclic
AMP-responsive nuclear factors, including CREB, cAMP response element modulator (CREM), and activating transcription
factor 1 (ATF-1). These factors contain the basic domain/leucine
zipper motifs and bind as dimers to cAMP-responsive elements
(CREs). The activation function of CRE-binding proteins in turn
is modulated by several kinase-dependent phosphorylations and
is mediated by coactivators such as CBP and p300 (11).
Although it is important to note that the interaction of AIRE
with CBP might influence signal transduction mediated by
cAMP-responsive nuclear factors, some of the structural properties of AIRE are suggestive of its function beyond the suspected transactivation. The presence of two PHD zinc fingers
and a leucine zipper was the focus of our attention. The objectives of the present study were 1) to verify whether recombinant human AIRE oligomerizes in vitro and 2) to examine the
possibility of AIRE possessing DNA binding properties. In this
paper, we report that AIRE forms homodimers and homotetramers in vitro and that both the dimer and tetramer forms of
AIRE possess DNA binding activity. We further demonstrate
that AIRE exists in oligomerized forms in vivo and that phosphorylation of AIRE by cAMP-dependent PKA and/or PKC
could trigger its dimerization.
41357
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The autoimmune regulator (AIRE) protein is a putative transcription regulator with two plant homeodomain-type zinc fingers, a putative DNA-binding domain
(SAND), and four nuclear receptor binding LXXLL motifs. We have shown here that in vitro, recombinant
AIRE can form homodimers and homotetramers that
were also detected in thymic protein extracts. Recombinant AIRE also oligomerizes spontaneously upon phosphorylation by cAMP dependent protein kinase A or
protein kinase C. Similarly, thymic AIRE protein is
phosphorylated at the tyrosine and serine/threonine
residues. AIRE dimers and tetramers, but not the monomers, can bind to G-doublets with the ATTGGTTA motif
and the TTATTA-box. Competition assays revealed that
sequences with one TTATTA motif and two tandem repeats of ATTGGTTA had the highest binding affinity.
These findings demonstrate that AIRE is an important
DNA binding molecule involved in immune regulation.
41358
DNA Binding Activity of Human AIRE Protein
MATERIALS AND METHODS
immunocomplex was batch-eluted using a 100 –500 mM glycine gradient
and buffer-exchanged with phosphate-buffered saline.
Native PAGE, SDS-PAGE, Electroblotting, and Western Blot Development—For native PAGE, the protein samples were diluted 1:4 with
native gel loading buffer (63 mM Tris-HCl (pH 6.8), 10% glycerol, and
0.005% bromphenol blue). Approximately 10 g of protein/well was
loaded onto a native discontinuous gel (4% stacking gel (pH 6.8) and
12% running gel, pH 8.3). Neither the gel nor the running buffer
contained SDS. For denaturing gel electrophoresis, the protein samples
were diluted 1:4 with SDS reducing buffer (63 mM Tris-HCl (pH 6.8), 2%
SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.005% bromphenol
blue). The samples were boiled for 5 min at 95 °C and loaded onto
discontinuous SDS gels containing 8 M urea (4% stacking gel (pH 6.8)
and 12% running gel (pH 8.3)). The gels were run in TGS buffer (192 mM
Tris, 25 mM glycine, and 0.1% SDS) on a MiniProtean III vertical
electrophoresis system (Bio-Rad) at 100 V for 3 h. The separated proteins were either stained with Coomassie Blue or were further processed for transfer onto Sequiblot PVDF (0.2 m, Bio-Rad).
For electroblotting, the gels were recovered and introduced into the
transfer module. The proteins were transferred onto a PVDF membrane
pre-wet in methanol and equilibrated in the transfer buffer using a
Mini Transblot cell (Bio-Rad). The transfer buffer had the following
composition: 40% methanol (v/v), 25 mM Tris, and 190 mM glycine (pH
8.2). The transfer was performed at 30 mA of constant current for 12–16
h, after which the PVDF membrane was recovered and air-dried.
For the development of blots, the membranes were pre-wet in methanol and then incubated in the Opti-4CN blocking reagent (Bio-Rad) for
1 h at 37 °C followed by extensive washing in phosphate-buffered saline
with 0.1% Tween 20 at room temperature. The membranes were incubated with appropriate primary antibodies for 1 h at room temperature.
After incubation, the membranes were washed with three changes of
phosphate-buffered saline, 0.1% Tween 20 and then incubated with
goat anti-rabbit IgG-horseradish peroxidase at a dilution of 1:5000. The
membranes were washed extensively after incubation and then developed with amplified Opti-4CN reagents (Bio-Rad) following the recommendations of the manufacturer.
N-terminal Microsequencing—N-terminal microsequencing of the
54-, 110-, and 220-kDa bands affinity-purified from mouse thymus on
anti-AIRE columns was performed as follows. 100 –300 mM glycine (pH
2.5) eluates from immobilized anti-AIRE immunocomplexes were
pooled and separated on native-PAGE polymerized for 18 h after the
protocol described in the earlier section. The separated proteins were
transferred onto a PVDF membrane (Millipore pSQ) pre-wet in methanol and equilibrated in the transfer buffer (0.1 M MES buffer (pH 6.0)
containing 40% v/v methanol). The transfer was performed at 30 mA of
constant current for 12–16 h, after which the PVDF membrane was
recovered and washed extensively in deionized water. The membranes
were briefly stained in Coomassie Brilliant Blue R-250 for 1 min and
de-stained in two changes of de-staining solution. The membranes were
dried, and bands were excised. The excised bands were subjected to
Edman degradation and the PTH derivatives were detected on a microbore liquid chromatography system.
Labeling of Oligonucleotides—The oligonucleotides used in this
study are listed in Table I. The oligonucleotides were end-labeled with
[␥-32P]ATP after a standard T4 polynucleotide kinase reaction. The
phosphorylation reaction was continued for 30 min at 37 °C, after which
the reaction was stopped by the addition of 1 l of 0.5 M EDTA/10 l of
total reaction volume. The unincorporated label was removed using
QIAQUICK nucleotide removal kit (Qiagen) following the instructions
of the manufacturer. The labeled oligonucleotides were eluted in 100 l
of TE buffer. The specific activities of the labeled oligonucleotides were
measured and were confirmed to be close to 20,000 cpm/100 fmol before
they were used in gel-shift assays.
Gel Shift Assays—Gel shift assays were performed with purified
AIRE (100 pM/reaction), and radiolabeled double-stranded oligonucleotides were prepared as stated above. The oligonucleotides used in this
study are listed in Table I. The protein preparations were preincubated
in binding buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM
dithiothreitol, 0.5 mM EDTA, 1 mM MgCl2, 4% glycerol) in the presence
of 0.05 mg/ml poly(dI-dC)䡠poly(dI-dC). Competition studies were performed by the addition of 1.75 pmol of unlabeled double-stranded oligonucleotide into the preincubation step. DNA-protein complexes were
formed by the addition of 0.175 pmol (40 –100,000 cpm/liter) of probe
and resolved by separation from unbound radiolabeled oligonucleotides
through 6% non-denaturing polyacrylamide gels (acrylamide:bisacrylamide ratio, 80:1) containing 2.5% glycerol in 0.5⫻ TBE (44.5 mM Tris,
44.5 mM boric acid, 1 mM EDTA) using a Protean II electrophoresis
system (Bio-Rad). The gels were pre-run at 100 V for 30 min before
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Plasmid Constructs Protein Expression—The full-length AIRE (amino acids 1–545) was amplified by polymerase chain reaction. The fragment was cloned into pET32 containing a thioredoxin tag (Novagen) for
6⫻His fusion protein expression. Escherichia coli BL21 (DE3) were
transformed and selected on ampicillin. Starting with a single colony,
500 ml of an overnight culture of a positive clone was set up. The
expression of the AIRE gene was induced with 10 M isopropyl-1-thio-D-galactopyranoside, and the culture was continued for 3 h. The cells
were harvested, and the recombinant protein was purified using the
B-PER 6⫻ histidine fusion protein purification system (Pierce) following the manufacturer’s instructions with slight modifications. Briefly,
the cells harvested from fresh induction were resuspended in 20 ml of
B-PER reagent by pipetting up and down until the cell suspension was
homogenous. The homogenous suspension was shaken gently at room
temperature for 10 min. The soluble proteins were separated from the
insoluble fraction by centrifugation at 27,000 ⫻ g for 15 min at 4 °C.
The supernatant was separated into two aliquots of 10 ml each and
loaded onto two nickel-chelated columns equilibrated with B-PER reagent. The columns were washed three times with the wash buffers
following the instructions of the manufacturer. The elution was performed on an imidazole gradient (0.1–1⫻), and 20 fractions of 0.5 ml
each were collected. The fractions were analyzed on SDS-PAGE, the
fractions containing the recombinant AIRE were pooled, and the protein concentration was adjusted to 10 g/l. Protein concentrations
were determined by BCA protein assay reagent (Pierce) using the
standard protocol. This preparation is designated as AIRE monomer.
Oxidation and Refolding of AIRE—A 20-ml fraction of the eluate pool
containing AIRE was resuspended in 5 volumes of ice-cold water containing 10 mM benzamidine hydrochloride. Guanidine hydrochloride
and sodium phosphate (pH 7.0) were added such that the final concentrations of 6 M guanidine and 10 mM phosphate were obtained, and the
mixture was allowed to stir at room temperature for 2 h. The denatured
soluble fraction was then diluted 10-fold over the course of 2 h into 1
liter of ice-cold oxidation, refold buffer containing Tris-HCl (pH 9.0), 1
M urea, and a redox couple (3 mM L-cysteine, 0.3 mM cystamine dihydrochloride). The solution was kept at 4 °C for 20 h. The solution was
then concentrated, and buffer was exchanged against 10 mM sodium
phosphate (pH 7.0) containing 2 M urea using Centricon centrifugal
filtration device (Millipore) with a molecular weight cut-off of 10,000.
This preparation was then adjusted to pH 5.2 with acetic acid and
allowed to stir at 4 °C overnight. The solution was clarified by centrifugation, adjusted to pH 8.0, and concentrated, and the buffer was
exchanged against Tris buffer (pH 8.5) using Millipore centrifugal filtration device. As a final refolding step, the pH of the preparation was
adjusted to pH 5.0 with acetic acid, and buffer exchange was performed
with 10 mM sodium phosphate buffer containing 150 mM NaCl (pH 7.2).
The final volume of the preparation was adjusted so as to have a protein
concentration of 10 g/l. This preparation is designated as refolded
AIRE.
Immobilization of Anti-AIRE Antibody on CarbolinkTM Gel—Total
IgG from the sera of rabbits immunized with recombinant human AIRE
(rhAIRE) was purified using IgG purification kit (Pierce) with manufacturer’s instructions. The IgG was oriented on a CarbolinkTM gel
employing an immobilization chemistry that used sodium periodate to
oxidize carbohydrates located on the Fc portion of antibody molecules,
converting vicinal hydroxyl groups to reactive aldehyde groups. These
aldehyde groups were allowed to react with the hydrazide groups of the
resin to form hydrazone bonds. The resin is designated as immobilized
anti-AIRE hereafter. IgG recovered from non-immunized animals were
also immobilized in a similar fashion, which is designated as immobilized normal rabbit IgG.
Immunoprecipitation of AIRE from Mouse Thymus—Thymus tissues
from four immature B6 mice were excised and homogenized in radioimmune precipitation buffer (50 mM Tris, pH 8.0, 1 mM EDTA, 150 mM
NaCl, 0.1% SDS, 1% Nonidet P-40, and 0.5% sodium deoxycholate)
containing 0.1% phenylmethylsulfonyl fluoride (1 ml of buffer/mg of
fresh tissue weight). The tissue homogenate was cleared by centrifugation at 20,000 ⫻ g for 20 min at 4 °C. The supernatant was recovered
and pre-cleared with immobilized normal rabbit IgG (60 l of hydrated
beads/1 ml of lysate). The mixture was incubated for 1 h at 4 °C on a
rocker. The supernatant was recovered after centrifugation at 16,000 ⫻
g (5 min) and mixed with immobilized anti-AIRE antibody. The mixture
was incubated overnight at 4 °C on a rocker. After incubation, the
preparation was centrifuged at 16,000 ⫻ g for 5 min to recover the
beads. The beads were washed five times with 1 ml of fresh radioimmune precipitation buffer each time. Bound AIRE recovered from the
41359
DNA Binding Activity of Human AIRE Protein
FIG. 1. Expression, purification and Western blot analysis of
human AIRE. A, expression levels of AIRE in E. coli before (lane 2) and
after (lane 3) induction with isopropyl-1-thio--D-galactopyranoside.
The band appearing at 56 kDa is rhAIRE. Lane 4 shows AIRE fraction
purified from a Ni2⫹-chelating affinity column on an imidazole gradient. In vitro refolding of AIRE produced distinct dimer and tetramer
bands on native PAGE (lane 5). Denaturation of the refolded product
yielded a 56-kDa band (lane 6). Lane 1 shows the positions of molecular
weight markers. B, AIRE immunoprecipitated from mouse thymus
extracts using immobilized anti-rhAIRE antibodies were separated by
native PAGE and electroblotted onto PVDF as described under “Materials and Methods.” These blots showed three bands on Western blots
when probed with the same antibody (lane 1). This indicated that AIRE
exists in dimeric and tetrameric forms in human thymus. N-terminal
microsequencing of these three bands confirmed that all these bands
possessed the same N termini as that of AIRE. Identical strips were
probed with anti-Ser(P)/Thr(P) (lane2) and anti-Tyr(P) (lane 3) antibodies. The anti-AIRE-positive bands stained positive with both antiTyr(P) and anti-Ser(P)/Thr(P) antibodies used in this study. C, in vitro
phosphorylation of rhAIRE with Abl protein kinase (lane 2), CK-1 (lane
3), CK-2 (lane 4), cAMP-dependent PKA (lane 5), and PKC (lane 6).
Lane 1 shows the relative positions of molecular weight markers. M,
monomer; D, dimer; T, tetramer.
further use in homology modeling. The templates used include 11GSA,
1PYDA, 1OTCA, 1PRCH, 2RAMB, and 2YHX. Alignments were performed using the program package GCG, and the model was built using
Insight II and Discover (Molecular Simulations Inc., San Diego, CA) and
evaluated with Swissmodel program (12).
RESULTS
AIRE Purification, Antibody Production, and Refolding Assays—The full-length construct of human AIRE (hAIRE) in
pet32 showed a very low basal level of expression of AIRE,
which was augmented severalfold after induction with isopropyl-1-thio--D-galactopyranoside (Fig. 1A, lanes 2 and 3). Because B-Per reagent could extract AIRE in the soluble fraction,
as shown in lane 3, we discarded the pellet after confirming the
efficiency of the recovery. The chelated nickel affinity chromatography followed by imidazole gradient elution produced a
protein fraction at an estimated molecular size of 56 kDa, as
represented in lane 4 (Fig. 1A), which shows a purified fraction
of AIRE. This fraction is termed AIRE monomer for descriptive
purposes.
The processing of the rhAIRE under oxidation and refolding
conditions brought about a major shift in the pattern of migration of this molecule (Fig. 1A, lane 5). Although a minor fraction
of AIRE still appears at the 56-kDa position, the major bands
appear at estimated molecular sizes of 110 and 220 kDa when
the refolded rhAIRE was analyzed on native PAGE. Both these
bands migrated to the 56-kDa position on SDS-PAGE in the
presence of 8 M urea (Fig. 1A, lane 6), suggesting the formation
of dimers and tetramers of AIRE during the refolding process.
AIRE Forms Dimers and Tetramers in Vivo—Polyclonal antibodies raised in rabbit in our laboratory against the full-
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loading of the binding reaction. After loading the samples, the DNAprotein complexes were separated by electrophoresis at 150 V for 3.5 h.
All procedures were performed at room temperature. After electrophoresis, the gels were exposed to x-ray film (Fuji Film).
Gel shift competition assays were performed by the introduction of a
synthetic zinc finger peptide (ZIF 268) in the reaction. The basic reaction was set up as stated above, with a modification involving the
addition of ZIF 268 at a concentration of 100 pM. This reaction was
allowed to incubate for 15 min at room temperature, after which AIRE
was introduced at concentrations ranging from 100 pM through 800 pM.
Gel shift assays of these reactions were performed in the same fashion
as discussed above.
Oligo Library Screening—We determined the ability of purified recombinant AIRE to bind DNA employing two oligo libraries. The first
library (N26 oligo library) had randomized a 26-mer stretch flanked by
arms for PCR amplification. The second library (G2N7G2 library) had
N9GGN7GGN6 sequence flanked by adapter sequence for PCR amplification. The libraries were PCR-amplified under standard conditions
(melting at 94 °C for 2 min followed by cycling at 94 °C for 30 s,
annealing at 63 °C for 30 s, and extension at 72 °C for 1 min; a final hold
at 72 °C for 10 min was given for completion of chain elongation). After
amplification, the PCR product was suspended in 1⫻ binding buffer and
incubated with purified and refolded rhAIRE for 1 h at room temperature with constant shaking. After this, anti-AIRE antibody (1:100 dilution) was added to the mixture, and the incubation was continued for an
additional 1 h. Protein A-agarose with preblocked nonspecific binding
sites (1:10 dilution) was added to the mixture, and the incubation was
allowed to continue for 1 h. At the end of this final incubation, the
suspension was centrifuged at 16,000 rpm on an Eppendorf centrifuge.
The supernatant was carefully removed, and the pellet was washed 5
times with 1.5 ml of 1⫻ binding buffer each time. The final pellet was
resuspended in 1⫻ TE. A 2-l aliquot of the resuspended pellet was
PCR-amplified under the settings mentioned above. The whole procedure was repeated 5 times as discussed earlier. After the last amplification, the PCR product was separated on a 3% agarose gel, and the
band was eluted and cloned using pCR4-TOPO cloning kit for sequencing (Invitrogen). TOPO10 cells were transformed adopting a one-shot
chemical transformation protocol.
Thirty-two clones each were selected and sequenced from the AIREselected N26 library and the G2N7G2 library. Plasmid minipreps were
made from the selected colonies and sequenced using M13 forward
primer. The insert sequences were aligned. Two different controls were
included in this experimentation. The first control involved the PCR
amplification of G2N7G2 library (before selection by AIRE) and its
cloning into pCR4-TOPO vector. Forty-four clones were sequenced, and
the sequences were analyzed to evaluate the bias in the random areas
of the oligonucleotides in this library (control 1). The second control
involved the cloning of oligonucleotides selected by reagents excluding
AIRE from the experimental reaction set-up (control 2).
Relative Binding Affinity Assays—Relative binding affinity assays
were performed in a manner similar to that outlined for gel-shift assays. In this assay, we constructed four independent sets of four reactions, each using hot oligonucleotides oligo-TGG, oligo-TG, oligo-GG,
and oligo-G, respectively. Each set had a gel shift with the given
oligonucleotide and competitions with the other three oligonucleotides.
The concentrations of the protein, hot oligonucleotide, and competitors
were the same as those in the gel-shift assays detailed earlier in this
section.
In Vitro Phosphorylation Assay—rhAIRE monomer was resuspended
in kinase buffer containing 40 mM HEPES (pH 7.4), 10 mM MgCl2, and
3 mM MnCl2. 100 g/ml ATP was added into the suspension. The kinase
reaction was initiated by the introduction of the corresponding protein
kinases. We tested the phosphorylation of AIRE by Abl protein kinase
(New England Biolabs), casein kinase I, casein kinase II (Promega
Corp.), cAMP-dependent protein kinase A and PKC (Cell Signaling
technology). The reactions were incubated for 30 min at 27 °C, after
which AIRE was immunoprecipitated with anti-AIRE-agarose. The immunocomplexes were dissociated with 100 mM glycine buffer (pH 2.6),
and the anti-AIRE-agarose conjugate was separated by centrifugation.
The supernatant was neutralized with 100 mM Tris buffer (pH 10.0).
The eluates were separated on native PAGE and transferred onto
PVDF membranes. The membrane strips were probed with the corresponding anti-Tyr(P) or anti-Ser(P)/Thr(P) antibodies. One of the strips
was probed with anti-AIRE antibody to verify the position of AIRE.
Homology Modeling—The full-length sequence of human AIRE (GenBankTM accession number NM 000383.1) was subjected to a global
threading, and significant threading matches were filtered out considering high global and local Z scores. This process yielded a few templates for
41360
DNA Binding Activity of Human AIRE Protein
TABLE I
Oligonucleotides used for gel shift assays detailed in this study
Name
Sequence of consensus oligonucleotides
Mutation in nonconsensus
AP-1
ATF/CREB
NF-E2
SP1
EGR
YY1
GATA
TGG
TG
GG
G
CGCTTGATGACTCAGCCGGAA
AGAGATTGCCTGACGTCAGAGAGCTAG
TGGGGAACCTGTGCTGAGTCACTGGAG
ATTCGATCGGGGCGGGGCGAGC
GAATTCAGCGGGGGCGAGCGGGGGCGA
GGTCTCCATTTTGAAGCGGAA
CACTTGATAACAGAAAGTGATAACTCT
TTATTAATTGGTTATATTGGTTA
TTATTAATTGGTTA
ATTGGTTATATTGGTTA
ATTGGTTA
CA 3 TG
AC 3 TG
GA 3 AG
GG 3 TT
GG 3 TA
CCA 3 TTG
GA 3 CT
producing two bands corresponding to the respective dimer and
tetramer positions of AIRE (Fig. 2A, lane 2). The addition of a
10 M excess of cold consensus oligonucleotide brought about
significant reduction in the intensity of the gel-shifted bands,
signifying the specificity of the interaction (Fig. 2A, lane 3).
Furthermore, the use of a mutant EGR consensus with base
replacements at the underlined positions (GAATTCAGCGGGGGCGAGCGGGGGCGA, GG 3 TA in mutant) did not bring
about competitive removal of the gel-shifted bands (Fig. 2A,
lane 4). Moreover, hot mutant EGR consensus also failed to
show a gel-shifted band, confirming this observation (Fig. 2A,
lane 5). This indicated the possible requirement of those underlined guanine residues in this oligonucleotide for its binding
to AIRE.
To further confirm that the zinc finger domain of AIRE is
involved in this interaction, we performed a competition assay
where AIRE was made to interact with EGR consensus in the
presence of a known synthetic zinc finger peptide ZIF 268
showing strong affinity toward the EGR consensus. In the
absence of the competitor peptide, AIRE brought about a gel
shift as shown in Fig. 2A, lane 2. In the presence of 100 pM ZIF
268, AIRE could not bring about visible mobility shifts over a
concentration range of 100 –300 pM (Fig. 2B, lanes 3–5), but a
4-fold excess of AIRE (400 pM) restored the mobility shift (Fig.
2B, lane 6). A further increase in the concentration of AIRE
resulted in the appearance of the lower gel-shifted band (corresponding to AIRE dimer) as well (Fig. 2B, lanes 7–10).
Consensus Sequence Recognized by AIRE Is Partly Different
from That of EGR—We screened an oligo library with preselected guanine doublets with a random seven-nucleotide
spacer (G2N7G2 library) and a random oligo library (N26 library) to identify the AIRE binding consensus sequence. The
libraries were subjected to five generations of pull-down assays, and the products after the fifth amplification were cloned
and sequenced.
We analyzed 59 clones selected out by AIRE from the
G2N7G2 library by direct sequencing using M13 forward
primer. The sequences were aligned and shaded using Genedoc
software (Fig. 3A). Alignment of the sequences from 32 clones is
presented because of software limitations. A quantity-based
generation of consensus from the aligned sequences was performed using Genedoc software, selecting the base that shows
the highest frequency of representation at every given position.
The consensus line appears at the bottom of Fig. 3A. The
pre-selected G-doublets are shaded gray in the consensus line
and shaded black in the alignment. It is clear that AIRE preferred an A/T-rich neighborhood around GG for its binding. A
careful examination of the consensus line showed that this
sequence contained two ATTGGTTA motifs (underlined in double lines) and a weak TATA-box (TTATTA) (underlined in a
single line).
To verify whether this observation was because of a bias in
the library used, we cloned the unselected library into TA-
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length rhAIRE recognized the AIRE in mouse thymus extracts
on Western blots (Fig. 1B, lane 1). Along with the expected
56-kDa band, we also observed the presence of anti-rhAIREpositive bands at molecular weight positions of 110 and 220
kDa, suggesting the possible oligomerization of AIRE in vivo
(Fig. 1B, lane 1). Both these high molecular weight bands were
microsequenced, confirming that they are derivatives of AIRE
(data not shown).
AIRE Is Phosphorylated in Vivo—A computer-based prediction (www.cbs.dtu.dk/services/NetPhos) of the properties of
AIRE had indicated that this molecule is quite possibly phosphorylated at serine and/or at threonine at several locations,
with one possible tyrosine phosphorylation site at Tyr-394. To
verify this prediction, we immunoprecipitated AIRE from total
protein extract of mouse thymus using anti-AIRE antibody
immobilized on CarbolinkTM resin using a CarbolinkTM IgG
Immobilization Kit (Pierce). Immunoprecipitated AIRE was
recovered using a glycine gradient. The fractions were neutralized, separated on native PAGE, and analyzed on Western
blots. Native immunoblots of the immunoprecipitated fractions
probed with anti-AIRE antibody detected bands at 56, 110, and
220 kDa, indicating the presence of monomers, dimers, and
tetramers of AIRE in tissue extracts (Fig. 1B, lane 1). Identical
lanes were probed with anti-Ser(P)/Thr(P) (Fig. 1B, lane 2) and
anti-Tyr(P) (Fig. 1B, lane 3) antibodies, which indicated that
AIRE existed in a phosphorylated condition in mammalian
tissues.
AIRE Can Be Phosphorylated in Vitro—Five different kinases (Abl protein kinase, CK1, CK2, cAMP-dependent PKA
and PKC) were selected considering the predicted ability
(www.cbs.dtu.dk/services/NetPhos) of one or all of them to
phosphorylate recombinant human AIRE produced by prokaryotic expression. Abl protein kinase and the casein kinases used
in this experiment could not bring about phosphorylation of
rhAIRE in vitro (Fig. 1C, lanes 2– 4). Interestingly, cAMP-dependent PKA brought about phosphorylation of rhAIRE (Fig.
1C, lane 5). To our surprise, we observed that the rhAIRE band
shifted toward the 100-kDa position, indicating a possibility of
dimerization of this molecule in response to phosphorylation.
Protein kinase C also brought about phosphorylation of
rhAIRE. Again, rhAIRE appeared to dimerize in response to
phosphorylation although the monomer form of rhAIRE was
also visible after the termination of the phosphorylation reaction (Fig. 1, lane 6).
AIRE Can Bind to a Zinc Finger Binding Consensus—We
screened seven consensus sequences known to be recognized by
zinc finger/leucine zipper proteins (Table I) to examine the
possibility of AIRE interacting with DNA through its zinc finger domains. In our initial attempts, we conducted gel shift
assays with monomeric AIRE. None of the oligonucleotides
brought about a gel shift when we used AIRE monomer in the
assays (data not shown). Interestingly, the refolded recombinant protein showed a strong gel shift with EGR consensus,
DNA Binding Activity of Human AIRE Protein
cloning vector, and 44 clones were sequenced (control 1). Fig.
3B represents an alignment of the sequences from the unselected clones analyzed. The consensus sequence was generated as described above. The library had a G/C-bias, as could be
interpreted from Fig. 3B. This observation strengthens the
conclusions derived from Fig. 3A that rhAIRE selected A/T-rich
motifs around the G doublets. In the screening experiment in
the absence of AIRE (control 2), no PCR amplification occurred
after the second generation of immunoprecipitation, indicating
the specificity of the complex formation and the screening
experiments.
The sequences of the clones derived from the N26 library
were aligned as shown in Fig. 3C. AIRE showed clear selection
of two different motifs from the oligo library pool, the first one
being a T-box (TTATTA) (underlined in single line in the consensus line) and the second a G-box (ATTGGTTA) (underlined
in double lines). Three clones (clones 6, 32, and 25 in Fig. 3C)
possessed only a T-box, and three (clones 8, 9, and 10) did not
show any clear consensus motif. The majority of the selected
clones had either a TG-box (12 of 32) or a GG-box (11 of 32)
combination. A minor population (3 of 32) had TGG (clones 15,
18, and 22 of Fig. 3C). This observation is in perfect agreement
with the interpretations that we deduced from our G2N7G2
library-screening experiments.
Relative Binding Affinity of AIRE to TTATTA and to ATTGGTTA Motifs—To address the relative binding affinities of
AIRE to the TTATTA (T-box) motif and to the ATTGGTTA
motif (G-box), we performed gel shift assays and competitions
with various combinations of four oligonucleotides: oligo-TGG,
oligo-TG, oligo-GG, and oligo-G (Table I). Two major gel shift
patterns emerged from these analyses. When the labeled
oligo-GG and oligo-G are used, two shifted bands were observed
(Fig. 4 lanes 9 –16). The upper band corresponds to the AIREtetramer position, and the lower band corresponds to the dimer
position. Interestingly, a higher band was observed when both
labeled oligos with a T-box (oligo-TGG and oligo-TG) were
analyzed (Fig. 4, lanes 1– 8).
AIRE showed strong binding with hot oligo-TGG (Fig. 4, lane
4), which could be competed out substantially with cold
oligo-TG (Fig. 4, lane 1). Cold oligo-GG brought about partial
inhibition in gel shift (Fig. 4, lane 2), but cold oligo-G did not
reduce the shifted band of hot oligo-TGG (Fig. 4, lane 3).
Oligo-TG also showed good binding with AIRE protein (Fig. 4,
lane 8). This binding could be totally abolished in competitions
with oligo-TGG (lane 5), oligo-GG (lane 6), or oligo-G (lane 7).
Furthermore, the binding of AIRE to oligo-GG (lane 12) could
be blocked significantly by oligo-TGG (lane 9) and to a lesser
extent by oligo-TG (lane 10) and oligo-G (lane 11). The binding
of AIRE to oligo-G showed a pattern similar to that of oligo-GG
(Fig. 4, lanes 13–16). It is interesting to note that the shifted
AIRE dimer is not detectable without competitor oligos (Fig. 4,
lanes 12 and 16) but appears in the presence of competitors.
This is because the labeled oligos only become available for
binding with the AIRE dimers when the competitor oligos
occupied the binding sites of the AIRE tetramers. These results
further suggest that the AIRE tetramers have higher binding
affinity than the AIRE dimers.
Homology Modeling—A threading of the sequence of the
full-length human AIRE protein returned several templates
that were used to generate an energy-minimized model for the
full-length protein (Fig. 5). The model depicts an important
conformational ingenuity in that both the PHD domains and
the SAND domain are arranged in a linear fashion, providing
maximal DNA binding surface. The positioning of the leucine
zipper well within the middle DNA binding domain (PHD1)
would permit the zip-locking of the protein dimer onto the
DNA. The leucine-zipper skeleton is shown in red in Fig. 5.
DISCUSSION
Because of the presence of very characteristic domains found
in classical DNA-binding proteins in AIRE, it was suspected
from the very day of its discovery that AIRE may function as a
DNA-binding protein. Though the DNA-binding activity of this
protein was not established, recent investigations by a few
groups were successful in unfolding the molecular basis of
AIRE action. Recently, Bjorses et al. (9,13) demonstrate that
wild type AIRE was translocated into the nucleus where it
forms distinct speckled domains throughout the nucleoplasm,
excluding the nucleoli. These authors further demonstrate the
transcriptional transactivation potential of this protein by
means of cotransfection experiments with Gal4 DBD-AIRE
fusion protein and the luciferase reporter genes under either
thymidine kinase promoter or the adenovirus E1b TATA sequence with upstream Gal4 response elements (13). Later,
Pitkanen et al. (10) performed a series of Gal4 system reporter
assays in HUH-7 and COS-1 cells, which showed that the
expression of full-length AIRE cDNA as the fusion protein with
GAL4 DNA binding domain caused ⬃30 – 40-fold activation of
the chloramphenicol transferase (CAT) reporter from the pG5CAT plasmid containing three GAL4 response elements upstream of the E1b minimal promoter (10).
The ability of AIRE to bind itself was recently assessed using
a glutathione S-transferase pull-down assay. It was found that
in vitro translated, labeled full-length AIRE bound specifically
to glutathione S-transferase-AIRE fusion but not to glutathione S-transferase alone, suggesting that AIRE homodimerizes
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FIG. 2. Electrophoretic mobility shift assays with rhAIRE. A,
incubation of rhAIRE with EGR-1 consensus brought about a weak
(corresponding to label D) and a strong (corresponding to label T)
gel-shifted bands (lane 2). The relative positions of the bands indicated
that the lower and the upper bands resulted from the binding of AIRE
dimer and tetramer to the oligonucleotide, respectively. The use of a
10-fold excess of cold consensus oligonucleotide brought about a total
reduction of the gel-shifted bands (lane 3). However, the use of a 50-fold
excess of non-competitor oligonucleotide (EGR-nonconsensus) did not
compete out the gel-shifted bands (lane 4). Use of a hot EGR nonconsensus (Table I) did now show gel shift (lane 5). Lane 1 shows the free
label. B, competition assays with zinc finger peptide ZIF 268. Though
ZIF 268 would bind EGR consensus, the complex moved along with the
free label, resulting in the absence of any distinct gel-shifted band (lane
2). In the presence of 100 pM ZIF 268, AIRE could not bring about visible
mobility shifts over a concentration range of 100 –300 pM (lanes 3–5),
but a 4-fold excess of AIRE (400 pM) restored the mobility shift (lane 6).
A further increase in the concentration of AIRE (500 – 800 pM) resulted
in the appearance both the upper and the lower gel-shifted bands (lanes
7–10). D, dimer; T, tetramer.
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41362
DNA Binding Activity of Human AIRE Protein
FIG. 4. Binding affinity of AIRE to
oligo-TGG, oligo-TG, oligo-GG, and
oligo-G, evaluated by gel shift competition assays. Gel-shifted bands with
labeled oligo-TGG (lanes 1– 4), oligo-TG
(lanes 5– 8), oligo-GG (lanes 9 –12), and
oligo-G (lanes 13–16) are shown. Lanes
4, 8, 12, and 16 are controls (no competition). Competitions were set up as follows: lanes 1–3, with oligonucleotides
TG, GG, and G; lanes 5–7, with oligonucleotides TGG, GG, and G; lanes 9 –11,
with TGG, TG, and G; and lanes 13–15,
with TGG, TG, and GG in the respective
order. D, dimer; T, tetramer.
in vitro very much like Sp100 (10). Recombinant full-length
hAIRE from the E. coli expression system did not dimerize,
probably because post-translational processing of AIRE may be
prerequisite for dimerization. Because it is known that proteins
could be unfolded and refolded successfully in vitro, our initial
attempts were to examine whether we could develop a strategy
to refold rhAIRE in vitro. Under the conditions stated in this
paper, AIRE refolded and dimerized as expected. At the same
time, we observed the formation of tetramers as well (Fig. 1). If
tetramer formation were not an artifact, it would imply that
AIRE has dimerization and tetramerization domains in its
structure. This appears to be the case if we combine the observation of Pitkanen et al. (10) that AIRE could dimerize utilizing
its N-terminal HSR domain and the fact that AIRE does have
a leucine zipper extending over amino acids 319 –341. At this
moment, the possibility of oligomerization of AIRE through its
leucine zipper is not experimentally proven. But it is established that leucine zipper proteins dimerize, engaging the zipper domains of the interacting partners. In that event, it is
quite likely that AIRE might dimerize using the leucine zipper
motif and would tetramerize using the HSR domain.
The functional significance of the formation of dimers and
tetramers of AIRE would depend on whether such molecular
interactions take place in vivo or not. To address this issue, we
performed a native PAGE followed by Western blot analysis of
human thymus extracts. Anti-rhAIRE antibody recognized
three bands at molecular weight positions of 56, 110, and 220
kDa. These are the expected positions of monomer, dimer, and
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FIG. 3. Sequences of clones selected by pull-down assay. A, alignment of sequences selected by AIRE from a G2N7G2 library. The line below
the aligned block is a consensus sequence generated as described “Materials and Methods.” The shaded G-doublets are the putative zinc finger
contact points. The sequence of the 26-nucleotide region after five cycles of selection and amplification are shown. In addition to the selection of
two repeats of ATTGGTTA (underlined in double lines), AIRE selected a weak TATA-box (TTATTA) (underlined in a single line) as well. B,
alignment of sequences from 44 clones originated from an unselected G2N7G2 library. A consensus line is shown at the bottom of the alignment
block. The G2N7G2 library showed a G/C-bias. C, alignment of sequences selected by rhAIRE from an N26 library. Thirty-two clones were sequenced
and aligned. G-doublets are shaded in gray. The T-box is underlined with single line. The G-box is underlined with double line. Clones are grouped
with vertical bars, indicating the consensus box(es) present in them.
DNA Binding Activity of Human AIRE Protein
tetramer of AIRE in the respective order (Fig. 1). The identity
of the high molecular weight bands as derivatives of AIRE was
established by microsequencing analysis. Thus, the potential
presence of dimer and tetramer forms of AIRE in human thymus is established.
Homo- and/or hetero-oligomerization of proteins is important
in the modulation of their function. In vivo, proteins oligomerize in response to stimuli of intrinsic or extrinsic origin. Phosphorylation or dephosphorylation of proteins at specific targets
by respective kinases or phosphatases is a common pathway
through which biological signal transduction propagates. Because recombinant AIRE did not possess the capability to autodimerize, we suspected a possible activation of this molecule
by phosphorylation that might be required for its dimerization.
A computer-based prediction of the possible phosphorylation
sites in AIRE pointed toward a high chance of phosphorylation
of serine/threonine at multiple locations. In the light of this
prediction, we tried a series of kinases for their ability to
phosphorylate AIRE in vitro. Interestingly, we found that cAMPdependent protein kinase A and PKC could heavily phosphorylate AIRE (Fig. 1C). It is also important to state at this point
that the phosphorylation of AIRE by cAMP-dependent PKA
and PKC also led to the dimerization of this molecule. Although
the relevance of this in vitro demonstration cannot be applied
to the in vivo scenario, we feel that there is a strong possibility
that AIRE is a substrate for cAMP-dependent protein kinase A
and/or PKC. Furthermore, phosphorylation of AIRE appears to
be the trigger for its dimerization in vitro and possibly in vivo.
This observation, in conjunction with the report that AIRE
interacts with CREB-binding protein (10) suggest that AIRE
could act as a downstream modulator of cAMP-dependent signal transduction and transcription regulation. Because the human CREB protein contains the leucine zipper motif (14), it is
also possible that there is a direct interaction between CREB
and AIRE.
It is well known that zinc finger proteins represent an important class of regulatory DNA-binding proteins. Different
varieties of zinc finger proteins are all linked by the utilization
of zinc ions to add a structural element to the binding component. One characteristic of this motif is that it recognizes Grich DNA (15–19). Because AIRE contained both leucine zipper
and zinc finger domains, we decided to characterize the potential DNA binding activity of AIRE using oligonucleotides
known to be recognized by proteins having these distinct domains. Thus, we performed gel shift assays with AIRE and
consensus sequences recognized by a number of leucine zipper/
zinc finger proteins (Table I). The results showed that AIRE
introduced band shift with the EGR consensus but not with the
EGR nonconsensus oligonucleotide. The band shift could be
competed out with an excess of cold EGR oligonucleotide and
also with ZIF 268 synthetic peptide, indicating that the band
shift is introduced by the interaction of zinc finger motifs in
AIRE and EGR consensus (Fig. 2). Although AIRE monomer
failed to bring about band shift, the AIRE dimer and tetramer
forms brought about strong band shifts under our assay conditions, implicating the requirement of oligomerization of this
molecule for it to gain the DNA binding activity. Pull down
assays with an oligonucleotide library with sequence of
N9GGN7GGN6 indicated the selection of two motifs in the
oligonucleotides that would bind AIRE. The first motif is a
weak TATA-box (TTATTA), and the second motif is a tandem
repeat of ATTGGTTA (G-box). Gel shift competition assays
with oligonucleotides oligo-TGG, oligo-TG, oligo-GG, and
oligo-G showed that oligo-TGG exhibited the highest AIRE
binding capability that could not be abolished by oligo-GG or
oligo-G (Fig. 4, lanes 1– 4). The binding of AIRE to oligo-TG
appeared to be highly unstable, as all the three competitors
used in this study could abolish the gel shift (Fig. 4, lanes 5– 8).
The binding of AIRE to oligo-GG (Fig. 4, lanes 9 –12) or oligo-G
(Fig. 4, lanes 13–16) appeared to be comparable. Based on the
binding affinity of AIRE with these oligonucleotides under the
given conditions of competition, we conclude that we could
arrange them in the following descending order: oligo-TGG ⬎
oligo-GG ⬎ oligo-G ⬎ oligo-TG. It is noteworthy to state that
the complexes between AIRE and oligo-TGG and oligo-TG migrated to a larger molecular mass position compared with the
expected positions of AIRE dimer and AIRE tetramer. It is
likely that high affinity binding between AIRE and the TGG or
TG oligonucleotides produce complexes with more than one
tetramer unit of AIRE per oligonucleotide molecule in our
assay system.
The construction of a three-dimensional model for the fulllength human AIRE protein with the available templates gives
us an insight into the structural architecture of this molecule in
space. As could be interpreted from this model, this protein has
the following visible domains: SAND (197–262), PHD (298 –341
and 433– 476), the N-terminal -helix-loop-helix (HSR), and
the leucine zipper (319 –341). In the folded form, both the HSR
and the leucine zipper domains are available for molecular
interaction. A recent determination of the three-dimensional
structure of the SAND domain from Sp100b represented a
novel ␣/-fold, in which a conserved KDWK sequence motif is
found within an ␣-helical, positively charged surface patch (20).
In our model for AIRE, the proposed DNA binding SAND and
the known DNA binding zinc finger domains are also exposed
for DNA-protein interactions. These interpretations match well
with our experimental observations.
The importance of the leucine zipper domain and the zinc
finger domains in deciding the functional competence of AIRE
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FIG. 5. Molecular modeling of human AIRE. Templates 11GSA,
1PYDA, 1OCTA, 1PRCH, 2RAMB, and 2YHX were the result of a
threading of AIRE sequence against the available Protein Data Bank
entries followed by a filtering considering the local and global Z scores.
All the three putative DNA binding domains (PHD1, PHD2, and SAND)
are surface-positioned in this molecule, making them available for
DNA-protein interactions. The leucine zipper (drawn in a red skeleton)
is within the PHD1. The SP-100 HSR is free on the surface. From this
model, we interpret that AIRE could dimerize using the leucine zipper
motif and could tetramerize using the HSR domain.
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DNA Binding Activity of Human AIRE Protein
REFERENCES
1. Ahonen, P. (1985) Clin. Genet. 27, 535–542
2. Aaltonen, J., Bjorses, P., Sandkuijl, L., Perheentupa, J., and Peltonen, L.
(1994) Nat. Genet. 8, 83– 87
3. Bjorses, P., Aaltonen, J., Vikman, A., Perheentupa, J., Ben Zion, G.,
Chiumello, G., Dahl, N., Heideman, P., Hoorweg-Nijman, J. J., Mathivon,
L., Mullis, P. E., Pohl, M., Ritzen, M., Romeo, G., Shapiro, M. S., Smith,
C. S., Solyom, J., Zlotogora, J., and Peltonen, L. (1996) Am. J. Hum. Genet.
59, 879 – 886
4. Heino, M., Scott, H. S., Chen, Q., Peterson, P., Maebpaa, U., Papasavvas,
M. P., Mittaz, L., Barras, C., Rossier, C., Chrousos, G. P., Stratakis, C. A.,
Nagamine, K., Kudoh, J., Shimizu, N., Maclaren, N., Antonarakis, S. E.,
and Krohn, K. (1999) Hum. Mutat. 13, 69 –74
5. The Finnish-German Autoimmune Polyendocrinopathy Candidiasis Ectodermal Dystrophy Consortium (1997) Nat. Genet. 17, 399 – 403
6. Nagamine, K., Peterson, P., Scott, H. S., Kudoh, J., Minoshima, S., Heino, M.,
Krohn, K. J., Lalioti, M. D., Mullis, P. E., Antonarakis, S. E., Kawasaki, K.,
Asakawa, S., Ito, F., and Shimizu, N. (1997) Nat. Genet. 17, 393–398
7. Gibson, T. J., Ramu, C., Gemund, C., and Aasland, R. (1998) Trends Biochem.
Sci. 23, 242–244
8. Seeler, J. S., Marchio, A., Sitterlin, D., Transy, C., and Dejean, A. (1998) Proc.
Natl. Acad. Sci. U. S. A. 95, 7316 –7321
9. Bjorses, P., Pelto-Huikko, M., Kaukonen, J., Aaltonen, J., Peltonen, L., and
Ulmanen, I. (1999) Hum. Mol. Genet. 8, 259 –266
10. Pitkanen, J., Doucas, V., Sternsdorf, T., Nakajima, T., Aratani, S., Jensen, K.,
Will, H., Vahamurto, P., Ollila, J., Vihinen, M., Scott, H. S., Antonarakis,
S. E., Kudoh, J., Shimizu, N., Krohn, K., and Peterson, P. (2000) J. Biol.
Chem. 275, 16802–16809
11. De Cesare, D., and Sassone-Corsi, P. (2000) Prog. Nucleic Acid Res. Mol. Biol.
64, 343–369
12. Schwede, T., Diemand, A., Guex, N., and Peitsch, M. C. (2000) Res. Microbiol.
151, 107–112
13. Bjorses, P., Halonen, M., Palvimo, J. J., Kolmer, M., Aaltonen, J., Ellonen, P.,
Perheentupa, J., Ulmanen, I., and Peltonen, L. (2000) Am. J. Hum. Genet.
66, 378 –392
14. Dwarki, V. J., Montminy, M., and Verma, I. M. (1990) EMBO J. 9, 225–232
15. Desjarlais, J. R., and Berg, J. M. (1992) Proteins 12, 101–104
16. Desjarlais, J. R., and Berg, J. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,
7345–7349
17. Desjarlais, J. R., and Berg, J. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,
2256 –2260
18. Desjarlais, J. R., and Berg, J. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,
11099 –11103
19. Pavletich, N. P., and Pabo, C. O. (1991) Science 252, 809 – 817
20. Bottomley, M. J., Collard, M. W., Huggenvik, J. I., Liu, Z., Gibson, T. J., and
Sattler, M. (2001) Nat. Struct. Biol. 8, 568 –570
21. Ishii, T., Suzuki, Y., Ando, N., Matsuo, N., and Ogata, T. (2000) J. Clin.
Endocrinol. Metab. 85, 2922–2926
22. Wang, C. Y., Davoodi-Semiromi, A., Huang, W., Connor, E., Shi, J. D., and She,
J. X. (1998) Hum. Genet. 103, 681– 685
23. Heino, M., Peterson, P., Sillanpaa, N., Guerin, S., Wu, L., Anderson, G., Scott,
H. S., Antonarakis, S. E., Kudoh, J., Shimizu, N., Jenkinson, E. J., Naquet,
P., and Krohn, K. J. (2000) Eur. J. Immunol. 30, 1884 –1893
Downloaded from http://www.jbc.org/ by guest on June 12, 2020
becomes evident if we correlate the known AIRE mutations
with the APECED disease. So far more than 20 different mutations of the AIRE gene have been identified in APECED
patients (21). The PHD domains in AIRE are located at amino
acids 299 –340 (PHD1) and 434 – 475 (PHD2). The leucine zipper is located at position 319 –341. The major mutations identified so far include the Finnish mutation R257X and the 13-bp
deletion in exon 8 (nucleotides 1094 –1106), producing truncated proteins of 28 and 47 kDa, both lacking either both or the
second PHD zinc finger domains (10). Two other potential
pathogenic mutations detected are a 2-base pair insertion in
exon 11, resulting in a premature termination codon at amino
acid 479 and a protein that is 66 amino acids shorter and a T 3
C transition at ⫹2 of intron 3 (22). A recent report revealed
novel frameshift mutations of the AIRE gene. These include an
insertion of a cytosine at nucleotide 29,635 at exon 10
(29635insC), which should lead to a premature termination at
the codon 371, producing a truncated protein missing the second plant homeodomain-type zinc finger motif and the third
LXXLL motif, and a deletion of a guanine at nucleotide 33,031
at exon 13 (33031delG), which should result in a premature
termination at the codon 520, yielding a truncated protein
missing the third LXXLL motif (21). The majority of the mutations listed above does affect the PHD domain(s) and/or the
leucine zipper domain, although two of the mutations affect
only the last LXXLL motif. In addition to what was thought
about the implications of these mutations to date, our data
strongly indicate that mutations affecting the leucine zipper
domain and/or the zinc finger domain would make AIRE incompetent to bind DNA. This would affect the expression of
gene(s) under the regulatory influence of AIRE.
With the identification of AIRE as a DNA-binding protein,
two relevant points to be addressed are the factors influencing
AIRE expression and the gene(s) under the regulatory influence of AIRE. It appears that the first question has already
been addressed, since a recent report states that AIRE was
absent in RelB-deficient mouse (23). The target genes of AIRE
are yet to be identified.
The Autoimmune Regulator (AIRE) Is a DNA-binding Protein
Pradeep G. Kumar, Malini Laloraya, Cong-Yi Wang, Quin-Guo Ruan, Abdoreza
Davoodi-Semiromi, Kuo-Jang Kao and Jing-Xiong She
J. Biol. Chem. 2001, 276:41357-41364.
doi: 10.1074/jbc.M104898200 originally published online August 31, 2001
Access the most updated version of this article at doi: 10.1074/jbc.M104898200
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This article cites 23 references, 6 of which can be accessed free at
http://www.jbc.org/content/276/44/41357.full.html#ref-list-1