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INTRODUCTION |
The gene defective in the disease autoimmune polyendocrinopathy
candidiasis ectodermal dystrophy
(APECED),1 the autoimmune
regulator (AIRE), encodes a protein of 57.5 kDa (1, 2). The
predicted protein domains of AIRE suggest a role in transcriptional
regulation: a conserved nuclear localization signal, two PHD-type zinc
fingers, four LXXLL or nuclear receptor interaction motifs,
and the SAND and HSR domains (1-3). In agreement, we and others
reported recently that AIRE can activate transcription from a reporter
gene when fused to a heterologous DNA binding domain (4, 5) and that it
interacts with the co-activator CREB-binding protein (CBP) (4). We also
showed that the HSR domain, similar to Sp100, mediates AIRE-AIRE
homodimerization and that it is needed for the activating function
(4).
The disease caused by the defects in AIRE, or APECED, is a rare
autosomal recessive autoimmune disease characterized by defective tolerance to certain self-antigens (1, 2, 6). Based on its monogenic
etiology, APECED can be thought of as a model of organ-specific
autoimmune diseases and thus can provide insights into the pathogenesis
of autoimmunity.
The expression of AIRE in human tissues is found mostly in the thymus,
spleen, fetal liver, and lymph nodes (1, 2, 7, 8). In the thymus, AIRE
is seen in two types of antigen-presenting cells: medullary epithelial
cells and cells of monocyte-dendritic cell lineage that are central in
the negative selection of self-reactive T cells (7). The subcellular
localization of AIRE consists of several distinct patterns. In the
cytoplasm of transiently transfected cells, AIRE forms a pattern
resembling intermediate filaments or microtubules (7, 9, 10).
Accordingly, colocalization with vimentin has been reported (9, 10). In
the nucleus, AIRE forms discrete nuclear dots, bearing similarity to
PML nuclear bodies (7, 9, 10). The nuclear dot pattern has also been demonstrated in human tissue sections (7).
In view of data on the subcellular localization of AIRE, we have
determined the protein domains responsible for its targeting to
specific cellular locations. We have also further studied the function
of AIRE on a minimal promoter in a transfection assay and determined
the activation domain. Finally, we have demonstrated a potential
nuclear export signal in the N-terminal HSR domain of AIRE.
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EXPERIMENTAL PROCEDURES |
Cell Culture, Transfections, and Immunofluorescence--
U937
and COS-1 cells were maintained as monolayers in Dulbecco's modified
Eagle's medium supplemented with 100 units/ml penicillin-streptomycin and 10% bovine calf serum (Life Technologies, Inc.).
The transfections were performed using calcium phosphate precipitation
for COS-1 cells (11) or electroporation for U937 cells. For COS-1
transfections, 5 × 105 cells were grown on coverslips
in 6-well plates and transfected with 1 µg of the appropriate
expression vector. For U937 transfections, 1 × 106
cells were transfected with 1 µg of vector DNA. Immunofluorescence staining was performed 24 h post-transfection as described using a
monoclonal anti-AIRE antibody (7).
In nuclear export studies, leptomycin B (LMB; from M. Yoshida,
University of Tokyo) was added 24 h post-transfection initially at
5, 25, or 30 ng/ml, the cells were grown for 3 or 24 h and stained
as above. In subsequent experiments, 10 ng/ml LMB was used. The
proportion of cells showing either nuclear, combined nuclear and
cytoplasmic, and just cytoplasmic staining was calculated.
Immunofluorescence data were acquired using an Olympus BX50 microscope.
Images were captured with a Photometrics Imagepoint Cooled CCD video
camera and IPLab Spectrum 3.1a software for the Macintosh.
Plasmid Constructs--
Wild-type AIRE cDNA was cloned into
the pSI mammalian expression vector (Promega) by polymerase chain
reaction using primers with EcoRI and SalI
restriction sites in the 5'- and 3'-ends, respectively. The truncated
AIRE fragments AIRE-(1-256), -(1-293), -(1-348), -(1-207),
-(292-545), -(175-545), and -(84-545) and the missense
mutation-containing cDNAs AIRE L28P, AIRE L28P/K83E, and AIRE C437P
were subcloned into pSI from various constructs described earlier (4)
using standard techniques. The mutation C302P was engineered into the
pSI-AIRE (wild type) vector to disrupt the first PHD zinc finger using
the GeneEditor site-directed mutagenesis system (Promega). The patient
mutation K83E was similarly engineered into wild-type pSI-AIRE. To
create pSI-AIRE C302P/C437P, an EcoRI-SacI digest
from pSI-AIRE C302P was cloned into pSI-AIRE C437P. For pGFP-AIRE NLS
the consensus nuclear localization signal plus six flanking amino acids
on either terminus (amino acids 101-141) were cloned by polymerase
chain reaction into pEGFP-C3 (CLONTECH). The
different AIRE plasmid constructs used in the experiments are
summarized in Fig. 1.
Protein Interaction Assays--
Expression and purification of
GST fusion proteins from GST·AIRE C302P and GST·AIRE C437P,
in vitro translation from pSI-AIRE C302P and pSI-AIRE C437P,
and GST pull-down assays were performed as described (4).
Promoter Assays--
In assays that look at the effect of AIRE
on the interferon
(IFN
) minimal promoter, the
reporter plasmid pIFN
-LUC (from Kalle Saksela, University of
Tampere) was used, which contains the IFN
minimal
promoter (nucleotides
55 to +19) upstream of the luciferase gene. The
assays were performed as follows. 2 µg of the reporter was
cotransfected with 2 µg of the appropriate pSI-AIRE construct by
electroporation into 1 × 106 COS-1 cells. The cells
were harvested and lysed 48 h after transfection, and luciferase
activity was measured using the luciferase assay system (Promega). The
luciferase values were normalized with respect to total protein
concentration. All experiments were confirmed twice.
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RESULTS |
The N-terminal HSR Domain of AIRE Is Required for Tubular
Localization--
Earlier reports by us and others have found
wild-type AIRE to be partially localized in tubular structures in the
cytoplasm of transfected cells (7, 9, 10). To identify the domain in
AIRE responsible for this particular localization, we performed transfections with several AIRE deletion constructs (Table
I, Fig.
1).
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Table I
Subcellular localization of various AIRE constructs
Cyto, cytoplasmic localization; nuc, nuclear localization; dot, nuclear
dot staining; fibr, microtubular cytoplasmic staining are shown.
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Fig. 1.
Schematic representation of the AIRE protein
and its different deletion constructs. Mutations
indicate the sites of the missense mutations in the constructs AIRE
L28P, AIRE L28P/K83E, AIRE K83E, AIRE C302P, AIRE C437P, and AIRE
C302P/C437P. The asterisk denotes mutations found in APECED
patients (R257X, L28P, and K83E).
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Only AIRE fragments containing an intact N terminus formed the tubular
cytoplasmic staining seen with wild-type AIRE. The required minimal
domain probably lies in the HSR domain, within at least the first 207 amino acids because none of the N-terminal deletions, AIRE-(84-545),
-(175-545), or -(292-545), exhibited a tubular pattern but were
diffusely localized in the cytoplasm, whereas AIRE-(1-207) localized
effectively into tubular structures (Table I). To further confirm this,
we tested the missense mutation AIRE L28P, found in APECED patients,
and a double mutant construct, AIRE L28P/K83E. We found that both
constructs lacked the tubular cytoplasmic pattern. However, the patient
mutant construct AIRE K83E showed a cytoplasmic tubular pattern similar
to wild type (Table I and Fig. 2). The
results were essentially the same in COS-1 and U937 cells. Thus, the
N-terminal HSR domain of AIRE is required for tubular localization.

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Fig. 2.
The tubular staining pattern of wild-type
AIRE is abolished by the L28P patient mutation. Immunofluorescence
of COS-1 cells transiently transfected with either wild-type AIRE, AIRE
L28P mutant, or AIRE-(84-545). In contrast to the microtubular
staining of wild-type AIRE (A) the L28P mutant is located
diffusely both in the cytoplasm and nucleus (B), indicating
that the N terminus of AIRE is needed for the tubular localization. The
AIRE-(84-545) construct (C) also lacks the tubular
localization and moreover is almost exclusively located in the nucleus,
indicating that a nuclear export signal resides in the N terminus of
AIRE.
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The Nuclear Localization Signal of AIRE Is Functional--
Upon
studying nuclear localization we observed that C-terminal deletion
constructs lacking both (AIRE-(1-256), -(1-207), and -(1-293)) or
one (AIRE-(1-348)) PHD zinc finger but containing the nuclear
localization signal also localized in the nucleus, suggesting that the
nuclear localization signal is functional. The proportion of cells
showing nuclear localization was comparable with wild-type AIRE with
AIRE-(1-207) and AIRE-(1-256) but was smaller with AIRE-(1-293) and
AIRE-(1-348) (not shown).
The presence of a consensus nuclear localization signal in AIRE has
been reported based on the amino acid sequence (1, 2). It has not,
however, been shown to be functional. To address this question, a short
fragment containing the AIRE NLS (amino acids 101-141) was cloned
downstream of the green fluorescent protein (pEGFP-C3,
CLONTECH) and transiently expressed in COS-1 cells.
We found that the minimal nuclear localization signal effectively transported GFP into the nucleus (Fig.
3).

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Fig. 3.
The AIRE consensus nuclear localization
signal is functional. Cloned downstream of GFP, a short fragment
containing the nuclear localization signal (amino acids 101-141)
confers nuclear localization to GFP (B), which normally is
located both in the cytoplasm and nucleus (A). Nuclei were
stained blue with DAPI.
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The C-terminal Region Is Involved in Nuclear
Transport--
Recently, we and others reported that AIRE can function
as a transcriptional activator and that the PHD zinc fingers are
important for that function (4, 5). We set out to determine the role of
the PHD zinc finger-containing C terminus in the subcellular localization of AIRE.
N-terminal AIRE deletion constructs lacking the nuclear localization
signal (AIRE-(175-545) and AIRE-(292-545)) were still transported
into the nucleus, approximately as efficiently as wild-type AIRE (Table
I). To investigate the role of the PHD zinc fingers in this nuclear
transport we tested the missense mutants AIRE C302P and AIRE C437P.
Both mutants, however, were effectively transported into the nucleus in
a pattern indistinguishable from wild type (Table I). This would seem
to indicate that the PHD zinc fingers are not directly implicated as
another nuclear targeting signal. Apart from the PHD fingers, the C
terminus has the proline-rich region between the zinc fingers and a
70-amino acid region distal to the second PHD finger (Fig. 1). We
conclude that the C terminus alone is sufficient to transport AIRE into the nucleus. Taken together with the results presented above, this
suggests that AIRE has two functional nuclear localization signals, the
consensus NLS in the N terminus and another thus far undefined in the C terminus.
Full-length AIRE Protein Is Needed for the Formation of Nuclear
Dot-like Staining--
In addition to the tubular cytoplasmic
localization, AIRE has been reported to exhibit a nuclear-staining
pattern resembling PML nuclear bodies, also known as ND10, nuclear
dots, or potential oncogenic domains (PODs) (7, 9, 10, 12, 13). We
performed transfections with wild-type AIRE and several deletion
constructs to determine the domain(s) needed to produce the nuclear
dot-like structures.
Interestingly enough, none of the tested deletion constructs showed the
dot-like nuclear staining (Table I). When nuclear localization was
seen, the pattern was diffuse. This was also true for the patient
mutation L28P and the double mutant L28P/K83E, both of which have
diminished transcriptional activating activity (10% and 3%
respectively) compared with wild-type AIRE (4). The APECED patient
mutation K83E also failed to produce the nuclear dot staining, although
it is fully functional in transcriptional activation (see below). On
the other hand, the PHD zinc finger mutations C302P and C437P, which
had no discernible effect on subcellular localization, caused severely
decreased transcriptional activation (see below). As shown previously
(4), the L28P and L28P/K83E mutants were unable to homodimerize. We
thus tested the C302P and C437P mutants for homodimerization by GST
pull-down and found that both could form homodimers as efficiently as
the wild-type protein (not shown).
Leptomycin B Leads to Increased Nuclear Localization of
AIRE--
Leptomycin B (LMB) is a specific inhibitor of CRM-1-mediated
nuclear export (14-16), a process that has recently been shown to be
important for the function of several proteins, including transcription
factors (17). Different nuclear export signal sequences have been
described, many of which are rich in leucine (18, 19). The presence of
several putative export sequences on N- and C-terminal regions and the
dual subcellular localization of AIRE in the cytoplasm and nucleus
suggested that it might, in addition to being transported into the
nucleus, also be actively exported from it. To test this hypothesis,
cells transfected with AIRE were subjected to 5, 25, or 35 ng/ml LMB
for 3 or 24 h. The proportion of cells exhibiting either a
nuclear, cytoplasmic, or combined nuclear and cytoplasmic staining
pattern was calculated. We found that treatment of cells with LMB at 15 ng/ml for 3 h increased the proportion of cells showing nuclear
localization ~2-fold, whereas the proportion of cells with only
cytoplasmic staining decreased accordingly (Fig.
4). Increasing the LMB concentration or
treatment time had little effect on the outcome (not shown). To
delineate the nuclear export signal we first tested N and C-terminal deletion constructs as putative signals were found in both termini. We
found that AIRE-(1-207) showed a response to LMB indistinguishable from wild type, whereas the C-terminal constructs were unaffected (Fig.
4). In accordance, the AIRE-(84-545) deletion construct was localized
almost exclusively in the nucleus (Fig. 2 and Table I). We suggest that
AIRE is shuttled between the nucleus and cytoplasm, possibly to
regulate its function.

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Fig. 4.
LMB treatment of AIRE-transfected cells leads
to nuclear accumulation of AIRE. Percentage of cells showing
either nuclear (nuc., cells showing only nuclear or nuclear
and cytoplasmic staining) or cytoplasmic (cyto., cells
showing cytoplasmic staining only) localization are given.
A, with wild-type AIRE the proportion of cells showing
nuclear localization is approximately 2-fold after LMB treatment.
B, AIRE-(1-207) shows a similar response to LMB suggesting
that the N terminus harbors a nuclear export signal.
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AIRE Activates Transcription from the Interferon
Minimal
Promoter--
To assess the function of wild-type AIRE and the effect
of patient mutations on that function, we devised an in vivo
reporter assay. The reporter used contains the IFN
minimal promoter (nucleotides
55 to +19) upstream of the luciferase
gene. Wild-type or mutated AIRE was cotransfected with the appropriate
reporter, and luciferase activity was measured 48 h
post-transfection.
The reporter construct expressed small baseline amounts of luciferase,
and the addition of wild-type AIRE increased the transcription of the
reporter gene ~5.5-fold (Fig. 5). To
determine whether an activation domain could be demonstrated, we tested
our deletion/mutation constructs (Fig. 1). The C-terminal constructs
AIRE-(84-545) and AIRE-(292-545) showed an activation comparable with
wild-type AIRE and the AIRE-(175-545) construct a slightly lower
activation. In accordance, the constructs carrying mutations in the PHD
fingers, AIRE C302P, AIRE C437P, and AIRE C302P/C437P, had reduced
transcriptional activating capabilities. Thus, wild-type AIRE can
activate the IFN
minimal promoter, and the PHD fingers
are directly implicated as the activation domain.

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Fig. 5.
AIRE activates transcription from the
interferon minimal promoter. Wild-type
AIRE (wt) leads to an activation ~5.5-fold. The C-terminal
constructs AIRE-(84-545) and AIRE-(292-545) show a similar activation
whereas AIRE-(175-545) activates slightly less efficiently. The PHD
zinc finger-disrupting mutations C302P and C437P lead to a severely
decreased activation. Interestingly, the patient mutation K83E has no
effect on the activation.
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DISCUSSION |
Microtubular Localization of AIRE Is Induced by the N-terminal HSR
Domain--
As indicated by the results of transfection experiments
with the different AIRE deletion and mutation constructs, the domain responsible for the localization into the cytoskeleton is the N
terminus (Table I). We narrowed the minimal domain down to the first
207 amino acids. The most likely candidate and the only intact domain
in this sequence is the HSR domain, comprising amino acids 1-100 of
AIRE.
The exact identity of the tubular fibers AIRE colocalizes with is not
entirely clear. Rinderle et al. (10) have shown that AIRE
colocalizes with vimentin and microtubules in transfected COS and
primary fibroblast cells. They also observed that the N-terminal
construct (amino acids 1-209) localized in microtubules in primary
fibroblasts but did not do so in COS cells. The results of another
group are in concordance with our findings as to the common Finnish
APECED mutation R257X: they found it to be localized in
filamentous structures in the cytoplasm (5) (Table I). They also showed
colocalization of AIRE with vimentin (9).
We have shown earlier that the AIRE HSR domain has a predicted
four-helix bundle structure and that it mediates AIRE-AIRE homodimerization (4). Interestingly, the L28P patient mutation not only
abolishes homodimerization and transcriptional activation properties
(4) but is also unable to localize in the microtubular structures of
the cytoskeleton. This might indicate that homodimerization through the
HSR domain is a prerequisite for the tubular localization.
All but two AIRE missense mutations found so far in APECED patients are
in the HSR domain (1, 2, 5, 7), which indicates that it is sensitive to
conformational changes (4) and likely to be important in the function
of AIRE. Further evidence for the importance of the HSR domain is
offered because we report here that it mediates the localization of
AIRE to microtubular structures. It has been shown that the I
B
protein, a key molecular target in the regulation of NF
B activity,
is localized in the microtubule network through its interaction with a
cytoskeleton-associated protein (20). Although it is not yet known
whether AIRE interacts with the microtubular network directly or via
some other protein-protein interaction, we suggest that the deposition
of AIRE in these structures plays an important part in the regulation
of its function.
The Consensus Nuclear Localization Signal Is Functional--
To
address the functionality of the nuclear localization signal found in
the N terminus of AIRE, we studied its ability to transport GFP into
nuclei. A polypeptide containing the bipartite AIRE NLS (amino acids
101-141) effectively transported the GFP·NLS fusion into the nuclei
of transiently transfected COS-1 cells.
The C Terminus of AIRE Contains a Nuclear Transport Signal--
In
studying the role of the PHD finger-containing C terminus in the
subcellular localization of AIRE we rather surprisingly found that the
C terminus alone can function as a mediator of nuclear transport
irrespective of the consensus nuclear localization signal in the N
terminus. The PHD zinc fingers, however, are not likely to be
responsible for the nuclear transport as missense mutations (C302P,
C437P, and C302P/C437P) fail to alter the subcellular localization of
the protein. It can be hypothesized that AIRE, through the C terminus,
binds some protein in the cytoplasm, thus inducing transport into the
nucleus and subsequent transcriptional regulating activity. We conclude
that the C terminus of AIRE is involved in its nuclear transport,
possibly as a means of regulating its activity as well as subcellular
distribution. Thus, AIRE has two functional nuclear localization
signals, the consensus NLS of the N terminus and another as yet
undefined in the C terminus.
AIRE Harbors a Nuclear Export Signal--
The dual subcellular
localization of AIRE and the presence of putative nuclear export
signals prompted us to consider the possibility of nuclear export of
AIRE. Indeed, we found that leptomycin B, an inhibitor of
CRM-1-mediated nuclear export, increased ~2-fold the proportion of
cells showing a nuclear localization whereas the number of cells
showing only cytoplasmic distribution decreased accordingly (Fig. 4).
The nuclear export signal was found to most likely reside in the N
terminus of AIRE because AIRE-(1-207) showed a response to LMB
indistinguishable from wild type. Further, AIRE-(84-545), lacking a
putative nuclear export signal and the microtubular localization-mediating HSR domain was almost exclusively located in the
nucleus. LMB treatment of AIRE-expressing cells did not lead to
complete nuclear accumulation. In the case of AIRE, the effect is seen
as an increase in the number of cells showing nuclear staining. We
suggest that this is partially because of the localization of AIRE in
the microtubular network of the cytoplasm. Binding to these structures
in the cytoplasm could inhibit nuclear transport to some extent. Thus,
a tempting hypothesis is that AIRE is shuttled between the nucleus and
cytoplasm to regulate its activity, the microtubular network
functioning as a cytoplasmic storage compartment and the nuclear dots
as the actual site of AIRE function.
AIRE Activates the IFN
Minimal Promoter--
To study the
transcriptional regulating properties of AIRE, we devised a functional
assay utilizing the IFN
minimal promoter upstream of the
luciferase gene. We found that wild-type AIRE activated the
IFN
minimal promoter 5.5-fold (Fig. 5). This finding is
in agreement with previous studies where AIRE was found to activate
transcription when fused to a heterologous DNA binding domain (4, 5).
However, the functional significance of the activation requires further
study. Furthermore, we showed that the AIRE-(292-545) construct,
containing the PHD fingers and the proline-rich region inbetween, was
also fully functional (Fig. 5). The AIRE-(84-545) construct,
containing in addition the SAND domain, was equally active. We also saw
activation by AIRE-(175-545), albeit slightly lower than wild type,
which was absent in a GAL4-based reporter assay (4), probably
indicating that the minimal promoter assay used here is the more
sensitive. The PHD fingers of the C terminus are thus implicated as an
activation domain.
To summarize, we show here that different domains of the AIRE protein
serve distinct roles in its subcellular localization and function. The
N terminus confers microtubular localization via the HSR domain. A
nuclear export signal is also likely to reside in the same domain. As
we have earlier shown that the HSR domain is required for the
homodimerization of AIRE, its importance becomes evident. The N
terminus of AIRE is also the domain most conserved in mouse Aire,
exhibiting almost 100% homology (21, 22). The C terminus contains an
additional nuclear localization signal, and the PHD zinc fingers
function as an activation domain.
The present study adds to increasing evidence that define AIRE as a
transcriptional regulator. The exact mechanism of how AIRE exerts
transcriptional activation and influences its protein partners, though,
remains unresolved. The manifesting symptoms of APECED indicate a
defect in the mechanisms inducing tolerance to self-antigens. The lack
of tolerance seen in APECED might be considered to arise from impaired
negative selection of T-cells. Although the mechanisms of tolerance
induction in the thymus medulla are also largely unknown, the
importance of medullary epithelial cells and dendritic
antigen-presenting cells has been established (23). In light of the
limited expression of AIRE in these very cells and because it is a
transcriptional regulator, we suggest that AIRE may regulate genes
acting in negative selection in the thymus medulla. The data presented
here give a firm basis for further study on the functional significance
of the alternating subcellular localization of AIRE, and the specific
mechanisms of its action. Especially, the recognition of the proteins
AIRE is involved with in each subcellular location will help elucidate this. We believe that the resolution of the aforementioned issues as
well as the results shown in this study will eventually give important
information on the induction and maintenance of immune tolerance.