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Human Molecular Genetics Pages 277-290  

AIRE encodes a nuclear protein co-localizing with cytoskeletal filaments: altered sub-cellular distribution of mutants lacking the PHD zinc fingers
Introduction
Results
   Transient expression of AIRE and characterization of polyclonal and monoclonal antibodies
   Sub-cellular localization of wild-type AIRE
   Altered cellular localization of truncated AIRE products
   Endogenous AIRE
Discussion
   AIRE sub-nuclear localization
   AIRE co-localizes with cytoskeletal filaments
Materials And Methods
   Recombinant AIRE expression in E.coli and purification of the protein
   AIRE expression plasmids for transient transfection
   Antibody production and purification
   Cell culture and transfection experiments
   Stimulation experiments
   Isolation of peripheral blood lymphocytes
   Indirect immunofluorescence
   Western blot analysis
Acknowledgements
References

AIRE encodes a nuclear protein co-localizing with cytoskeletal filaments: altered sub-cellular distribution of mutants lacking the PHD zinc fingers

AIRE encodes a nuclear protein co-localizing with cytoskeletal filaments: altered sub-cellular distribution of mutants lacking the PHD zinc fingers

Cornelia Rinderle, Hoang-My Christensen, Susann Schweiger, Hans Lehrach and Marie-Laure Yaspo*

Max Planck Institute for Molecular Genetics, Ihnestrasse 73, D-14195 Berlin, Germany

Received September 2, 1998; Revised and Accepted November 18, 1998

The gene responsible for autoimmune polyendocrino-pathy candidiasis ectodermal dystrophy (APECED) recently has been positionally cloned to 21q22.3. This novel gene, AIRE, encodes for a predicted 57.7 kDa protein featuring two PHD-type zinc fingers shared by other proteins involved in chromatin-mediated tran-scriptional regulation. APECED is an autosomal recessive condition characterized by multiple polyendocrinopathies, and the typical triad of APECED symptoms includes hypoparathyroidism, primary adrenocortical failure and chronic mucocutaneous candidiasis. The aetiology of APECED is linked directly to mutations within the coding region of AIRE. These mutations are predicted to lead to truncated forms of the protein lacking at least one of the PHD zinc fingers. In this study, we have investigated the sub-cellular localization of AIRE expressed transiently in COS cells and fibroblasts. We found that AIRE has a dual nuclear and cytoplasmic localization. The wild-type protein is directed to speckled domains in the nucleus and also shows co-localization with cytoskeletal filaments. N-terminal AIRE fragments deleted for the PHD domain show altered nuclear localization, suggesting that the APECED mutations may elicit their primary effects in the nucleus.

INTRODUCTION

We recently have positionally cloned the AIRE gene, a novel putative transcription-associated factor found mutated in a rare autoimmune disease affecting mainly the endocrine glands, and named autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED; OMIM 240300) (1,2). The typical triad of APECED symptoms is represented by the occurrence of hypoparathyroidism, primary adrenocortical failure and chronic mucocutaneous candidiasis. However, the APECED phenotype is characterized by a variable combination of signs reflecting the autoimmune destruction of different endocrine and non-endocrine target organs: (i) hypoparathyroidism, adrenocortical failure, insulin-dependent diabetes mellitus (IDDM), gonadal failure, hypothyroidism, pernicious anemia and hepatitis; and (ii) ectodermal dystrophies such as vitiligo, keratopathy and dystrophy of dental enamel (3). APECED is an autosomal recessive condition and is the only described human autoimmune disease with a Mendelian mode of inheritance that maps outside the MHC (4,5). The disease is reported worldwide, albeit being exceptionally prevalent among the Finnish population, Sardinians and Iranian Jews (6,7).

The etiology of APECED is directly associated with mutations in the coding region of AIRE. The AIRE gene encodes a predicted 57.7 kDa protein harboring a nuclear localization signal (NLS) and two PHD zinc finger motifs. The majority of the mutations described so far are predicted to lead to a premature termination of the gene product, deleting at least one of the PHD motifs, suggesting that mutations in AIRE elicit their effects through a loss-of-function mechanism (1,2,8). The PHD finger is a modular domain existing as a single or multiple unit in a number of nuclear proteins (9,10). These include a leukocyte-specific component of the nuclear body, Sp140 (11), and proteins involved in chromatin-mediated transcriptional modulation such as Mi-2 autoantigen (12), ALL-1 (13), ATRX (14), TIF1 (15) or KRIP-1 (16). Thus, it was postulated that AIRE may encode a nuclear protein playing a role in the regulation of transcription on the basis of its remarkable structural features. Whereas homology of AIRE with known proteins was generally restricted to this PHD domain, extended structural similarity could be observed with the group of human Sp100 proteins. Besides a 100 residue N-terminal region, AIRE and Sp140 share a putative DNA-binding segment referred as the SAND domain, located upstream of the PHD fingers and spanning 80 residues (17). An LXXLL nuclear receptor motif (18) and an NLS are also common to those proteins, suggesting that AIRE and Sp100 proteins may derive from a common ancestor (17). Sp100 is a component of the nuclear body previously shown to be a target of autoantibodies in patients with primary biliary cirrhosis (19). However, the functional significance of either the N-terminal homologous sequence motif, the PHD or SAND domain is not yet understood, and the basic function of the AIRE protein so far remains elusive.

To shed light on the understanding of AIRE function, we have here determined the sub-cellular localization of wild-type AIRE and two mutant forms expressed as truncated N-terminal fragments of the protein. We have found that AIRE has the ability to be translocated into the nucleus while showing also a characteristic cytoskeletal distribution pattern in COS cells, human fibroblasts and HeLa cells. The two AIRE mutants lacking the PHD fingers showed altered sub-cellular distribution, indicating that these truncated forms of the protein are not localized correctly within the nucleoplasm.

RESULTS

Transient expression of AIRE and characterization of polyclonal and monoclonal antibodies

In order to investigate the sub-cellular localization of wild-type and deletion mutants of the AIRE protein in mammalian cells, we designed the constructs shown in Figure 1. The full-length construct contains a cDNA encoding for the 545 residues AIRE protein (AIRE-B1-1pA). Two AIRE mutants truncated at amino acid residues 306 and 209 were designated AIRE-[Delta]SacI and AIRE-[Delta]BamHI, respectively. AIRE-[Delta]SacI is truncated within PHD1, whereas AIRE-[Delta]BamHI is lacking a larger protein segment encompassing the SAND domain and both PHD zinc fingers. Full-length or truncated AIRE were expressed transiently in monkey COS cells, HeLa cells and human primary fibroblasts using an SV40 promoter. For immunodetection of the AIRE protein, two polyclonal antisera were raised against synthetic peptides corresponding to the N-terminal region and to the nuclear targeting signal (sp97179 and sp97181; see Materials and Methods). Affinity-purified antibodies were tested on western blots containing the 6×His-tagged recombinant AIRE fusion protein expressed in Escherichia coli. Both sp97179 and sp97181 antisera selectively recognized the His-tagged full-length AIRE (data not shown). Figure 2 shows a western blot analysis of the expression of the AIRE constructs in transfected COS1 cells using antibody sp97181. The immunoblot revealed one strong immunoreactive band corresponding to the gene product of each construct. The size of the full-length AIRE protein expressed in transfected cells was calculated at 58.8 kDa, which is in agreement with the predicted mol. wt of 57.7 kDa. When cells were transfected with the truncated constructs AIRE-[Delta]SacI and AIRE-[Delta]BamHI, appropriate size bands were seen at 34.7 and 23.5 kDa, respectively. No immunoreactivity was found in mock transfection nor in cells transfected with empty pSG5 vector. Similar results were obtained with sp97179 antiserum. Monoclonal antibodies were produced by immunizing mice with AIRE recombinant protein. Two monoclonal antibodies (Mab155 and Mab137) reacted specifically with expressed AIRE constructs as tested by immunocytofluorescence and western blot analysis (data not shown).


Figure 1. Schematic diagram of the AIRE constructs. The full-length protein is 545 amino acids. Gray boxes indicate the PHD zinc fingers, the hatched box the nuclear localization signal, the filled box the nuclear receptor-binding domain and the dark hatched box the SAND domain. The AIRE-[Delta]SacI mutant is truncated after 306 amino acids, the AIRE-[Delta]BamHI mutant after 209 amino acids.


Figure 2. Western blot analysis of cell extracts from transiently transfected COS1 cells. Cells were transfected with the indicated plasmids. The blot was probed with sp97181 antiserum. Expression of the full-length protein (lanes 3 and 4) is compared with mock- (lane 1) or pSG5-only-transfected cells (lane 2). Expression of the mutant proteins is shown in lane 5 (AIRE-[Delta]SacI) andlane 6 (AIRE-[Delta]BamHI). Arrows indicate the detected proteins for AIRE, AIRE-[Delta]SacI and AIRE-[Delta]BamHI constructs.

Immunocytofluorescence detection of the AIRE constructs expressed in COS cells was investigated 24 and 48 h post-transfection by confocal laser microscopy and serial optical sections, after staining with antibodies sp97179 and sp97181. The staining pattern obtained with sp97181 antiserum was essentially similar to that of sp97179, Mab137 or Mab155. Only transfected cells showed a labeling with either of these antibodies, indicating that COS1 cells are not expressing detectable endogenous AIRE. Mock- or pSG5-only-transfected cells showed no evident staining with either antiserum. Immunofluorescence labeling as well as western blot-specific detection were blocked by pre-incubation of the antibodies with AIRE recombinant protein, further confirming the specificity of the antibodies (data not shown). All experiments were performed in parallel with both polyclonal antibodies, and we will describe here data obtained using sp97181 antiserum. Endogenous AIRE expression was investigated using sp97181 and Mab155 antibodies.

Sub-cellular localization of wild-type AIRE

COS1 cells transfected with the full-length construct showed two populations of stained cells, one with a punctuate granular staining strictly restricted to the nucleus, as defined by YOYO-1 labeling of DNA, and a second one showing also a cytoplasmic expression of AIRE (Fig. 3). Transfection experiments carried out with either 2, 5, 10 or 20 µg of AIRE B1-1pA cDNA led to similar observations. When >300 transfected cells were analyzed, cytoplasmic staining was observed in ~70% of the cells, whereas the AIRE expression was confined to the nucleus in the remaining 30%. In all of the cells where the staining was exclusively nuclear, the antibody reacted with punctuate structures. AIRE localized into small distinct micro-speckles uniformly distributed in a given optical section of the nucleoplasm but excluded from the nucleoli (Figs 3I and 4AI). Serial optical sections and confocal imaging showed that the nuclear labeling was present in domains representing ~5-8 µm of the nucleoplasm depth and thus localized within at least two-thirds of the nuclear volume. Since AIRE shares several functional domains with Sp100, we investigated whether these proteins localize to the same nuclear sub-domains. Double-staining revealing AIRE and Sp100 did not show any coincidental pattern. Moreover, the AIRE speckles were different from the dots containing Sp100 protein which defined larger and far less numerous dots in COS and HeLa cells (data not shown).

In cells where AIRE was expressed in the cytoplasm, the antibody decorated cytoplasmic fibers stretching from the nuclear envelope to the plasma membrane. Fibers were spanning 4-8 µm of the cell depth and appeared arranged in a scaffold-like structure often forming bundles around the nuclear envelope (Fig. 3II), reminiscent of intermediate filaments or microtubules (20,21). This AIRE filamentous staining pattern was generally observed in conjunction with the characteristic nuclear speckles, albeit that the nuclear staining sometimes consisted of fibrils spanning the nucleoplasm. Also, a few of the transfected cells were devoid of detectable labeling in the nucleus. No remarkable difference in the AIRE localization pattern could be noted between cells analyzed 24 or 48 h after transfection. The AIRE localization pattern was reproducible when comparing independent transfection experiments and was similar with either fixation method (see Materials and Methods). To authenticate further the identity of the cytoskeletal filaments revealed by sp97181, transfected COS cells were double-stained with either sp97181 and anti-vimentin (Fig. 4AI and II), or with sp97181 and anti-[alpha] tubulin (Fig. 4AIII). Figure 4AI shows a cell expressing AIRE mainly in the nucleus, where the characteristic pattern appears composed of 50-100 speckles. In contrast, no evident punctuate nuclear staining could be observed in the cell shown in Figure 4AII, where AIRE and vimentin appear as co-localizing onto a similar fibrous network. Nonetheless, it should be mentioned that AIRE and vimentin fibers are only partially overlapping in some of the transfected cells. In the complex cytoskeletal dynamic architecture, vimentin can be found associated with microtubules (21,22), sometimes co-localizing onto identical fibers (23). Figure 4AIII represents a confocal imaging of a transfected cell double-stained for AIRE and microtubules. AIRE (labeled in green) defines nuclear speckles and a cytoskeletal pattern co-localizing with microtubules (labeled in red). The data indicate that overexpressed AIRE protein has the ability to co-localize with the cytoskeletal network, microtubules and vimentin. The coincidental pattern with microtubules was found to be more striking than that with vimentin, although the exact nature of this association remains to be addressed formally by complementary biochemical methods.


Figure 3. Sub-cellular distribution of the AIRE protein. COS1 cells were transfected with 5 µg of pSG5-AIRE and stained for AIRE with antibody sp97181 (red). Nuclei were stained with YOYO-1 (green). Images were scanned using a confocal laser microscope scanner. I: nuclear localization; Nu, nucleoli. II: cytoplasmic and nuclear localization of AIRE. (a) Red and green images merged; overlapping signals appear yellow. (b) Red image. (c) Green image.

We investigated AIRE cellular localization in two other cell types, human primary fibroblasts and HeLa cells. A similar dual cytoplasmic and/or nuclear AIRE staining pattern was observed in transfected primary fibroblasts (Fig. 4B), showing here discontinuous AIRE cytoplasmic fibers. As described for COS cells, a fine nuclear speckled pattern was observed in transfected fibroblasts and HeLa cells (data not shown). No endogenous AIRE was detected in these cells.

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   B

Figure 4. (A) Co-localization of cytoplasmic AIRE with vimentin and microtubuli in COS7 cells. I and II: COS7 cells were transfected with pSG5-AIRE and co-stained for AIRE (sp97181, red) and vimentin (green). Images were analyzed with an epifluorescence microscope. (a) Red and green images merged; co-localization appears yellow. (b) Red image. (c) Green image. III: note that the staining color is inverted. COS7 cells were transfected with pSG5-AIRE and co-stained for AIRE (sp97181, green) and microtubuli (red). Images were analyzed with a confocal laser microscope scanner. (a) Red and green images merged; co-localization appears yellow. (b) Green image. (c) Red image. (B) Co-localization of cytoplasmic AIRE with vimentin in human primary fibroblasts. Human primary fibroblasts were transfected with pSG5-AIRE and co-stained for AIRE (sp97181, red) and vimentin (green). Images were analyzed with an epifluorescence microscope. (a) Red and green images merged; co-localization appears yellow. (b) Red image. (c) Green image.

Altered cellular localization of truncated AIRE products

The two N-terminal AIRE protein fragments expressed in COS cells or fibroblasts showed dramatic changes in their cellular distribution as compared with wild-type AIRE. The AIRE-[Delta]SacI construct expressing a 35 kDa protein truncated within the PHD1 domain was also found localized in both cytoplasmic and nuclear compartments. In contrast to wild-type, AIRE-[Delta]SacI protein showed a drastically altered nuclear sub-localization pattern. At 24 h post-transfection, the mutant protein systematically localized in discrete nuclear domains consisting of intensely labeled foci, whereas no speckled pattern organization could be distinguished (Fig. 5AI). These intense nuclear dots were heterogeneous in size but often appeared as lipid-like round structures found as pairs but also as three, four or multiple inclusions in the nucleoplasm, sometimes seen in the immediate vicinity of the nucleoli. Importantly, those dots were significantly larger than the speckles described for wild-type AIRE (Fig. 4AI), and were observed systematically in independent transfection assays. In some of the cells analyzed 48 h post-transfection, these inclusions were set against a very faint diffuse staining in the nucleoplasm and excluding nucleoli (data not shown). In COS cells, cytoplasmic AIRE-[Delta]SacI showed at least in part co-localization with vimentin, and often revealed fiber bundles around the nuclear envelope which occasionally were associated with small aggregates (Fig. 5AII). When compared with microtubule filaments, AIRE-[Delta]SacI pattern was found coincident with anti-tubulin staining as seen in Figure 5AIII.

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   B
   C

Figure 5. (A) AIRE-[Delta]SacI forms nuclear inclusions and co-localizes with vimentin and microtubuli in COS7 cells. I and II: COS7 cells were transfected with pSG5-AIRE-[Delta]SacI and co-stained for AIRE (sp97181, red) and vimentin (green). Nuclei in I were stained with DAPI. (a) Images merged; co-localization appears orange. White arrowheads indicate nuclear AIRE-[Delta]SacI. (b) Red image. (c) Green image. III: note that the staining color is inverted. COS7 cells were transfected with pSG5-AIRE-[Delta]SacI and co-stained for AIRE (sp97181, green) and microtubuli (red). (a) Images merged; co-localization appears orange. (b) Green image. (c) Red image. (B) AIRE-[Delta]Sac and p80-coilin do not co-localize in COS1 cells. COS1 cells were transfected with pSG5-AIRE-[Delta]SacI and co-stained for AIRE (Mab137, green) and p80-coilin (red). (a) Images merged. (b) Green image. (c) Red image. (C) Sub-cellular localization of AIRE-[Delta]SacI and co-localization with vimentin in human primary fibroblasts. Fibroblasts were transfected with pSG5-AIRE-[Delta]SacI and co-stained for AIRE (sp97181, red) and vimentin (green). I: nuclear localization of AIRE-[Delta]SacI. White arrowheads indicate nuclear AIRE-[Delta]SacI. II: cytoplasmic co-localization of AIRE-[Delta]SacI with vimentin. (a) Red and green images merged; co-localization appears yellow. (b) Red image. (c) Green image.

The peculiar nuclear localization of AIRE-[Delta]SacI evoked structures referred to as nuclear bodies, in particular coiled bodies (24). Transfected COS cells were double-stained with sp97181 and an antibody against p80-coilin, a protein used as a marker for coiled bodies (25). In fact, the structures formed by AIRE-[Delta]SacI are not overlapping with coiled bodies revealed by anti-p80-coilin, which defines larger nuclear inclusions (Fig. 5B). In transfected human fibroblasts and HeLa cells, similar observations were noted for AIRE-[Delta]SacI, though the nuclear inclusions were often significantly larger than in COS cells (Fig. 5C).

The AIRE-[Delta]BamHI construct showed a strikingly different sub-cellular localization compared with full-length AIRE and AIRE-[Delta]SacI. This truncated protein of 23.5 kDa presented a drastically impaired cytoplasmic distribution pattern where fibers could never be observed in any of the COS cells expressing AIRE-[Delta]BamHI. Instead, large cytoplasmic aggregates commonly were concentrated in the perinuclear region (Fig. 6AI) or at one pole of the nucleus (Fig. 6AII), albeit sometimes dispersed in the cytoplasm (Fig. 6AIII and IV), disturbing to some extent the ability to co-localize with either vimentin or microtubules. The same construct expressed in fibroblasts could also form cytoplasmic aggregates (Fig. 6BI) but, interestingly, the mutant protein retained the ability to co-localize along vimentin intermediate filaments in this cell type. Nonetheless, AIRE-[Delta]BamHI and vimentin staining revealed unusual wavy filaments that were never observed otherwise (Fig. 6BII). Cells containing large aggregates of the AIRE-[Delta]BamHI protein generally presented a dramatically altered distribution of the vimentin intermediate filaments (Fig. 6AIII). This is particularly exemplified in the fibroblast cell shown in Figure 6BI where vimentin appears trapped within AIRE aggregates. This evokes the hypothesis that protein-protein interactions involved in maintaining the shape and integrity of intermediate filaments are impaired in cells overexpressing AIRE-[Delta]BamHI. AIRE-[Delta]BamHI aggregates may promote a steric mechanical adverse effect on the cytosketal dynamic structure. The nuclear staining showed a confined pattern comparable with that of the AIRE-[Delta]SacI truncated protein. Intensely labeled discrete foci appearing as pairs or as multiple dots with a typical diameter of ~1 µm were observed at 24 or 48 h post-transfection (Fig. 6AI and BI). Orthogonal sections of such nuclear inclusions indicate rod-like structures spanning 2-5 µm in the nucleoplasm depth (data not shown). However, no speckled nuclear staining could be seen at 24 or 48 h post-transfection.

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Figure 6. (A) AIRE-[Delta]BamHI forms cytoplasmic aggregates and nuclear inclusions in COS7 cells. I: COS7 cells were transfected with pSG5-AIRE-[Delta]BamHI and stained for AIRE (sp97181, red). Nuclei were stained with DAPI. (a) Red and blue images merged, white arrowheads indicate nuclear AIRE-[Delta]BamHI. (b) Red image. II and III: COS7 cells were transfected with pSG5-AIRE-[Delta]BamHI and stained for AIRE (sp97181, red) and vimentin (green). Nuclei in II were stained with DAPI. (a) Images merged; co-localization appears yellow. (b) Red image. (c) Green image. IV: note that the staining color is inverted. COS7 cells were transfected with pSG5-AIRE-[Delta]BamHI and stained for AIRE (sp97181, green) and microtubuli (red). (a) Images merged; co-localization appears yellow. (b) Green image. (c) Red image. (B) Sub-cellular localization of AIRE-[Delta]BamHI and co-localization with vimentin in human primary fibroblasts. Fibroblasts were transfected with pSG5-AIRE-[Delta]BamHI and co-stained for AIRE (sp97181, red) and vimentin (green). Nuclei in I were stained with DAPI. I: cytoplasmic aggregates and nuclear AIRE-[Delta]BamHI. White arrowheads indicate nuclear AIRE-[Delta]BamHI. II: cytoplasmic filamentous localization of AIRE-[Delta]BamHI. (a) Images merged; co-localization appears yellow. (b) Red image. (c) Green image.

Importantly, in the three cell types analyzed, our data show that deletion of the C-terminal third of AIRE containing the PHD motifs abolished the normal nuclear distribution. We do not address here whether the PHD zinc fingers directly mediate the correct protein localization to specific nuclear domains. The truncated proteins still have the ability to be targeted to the nucleus, which is compatible with the fact that they retain the NLS domain. However, the two deletion mutants appear mislocalized in the nucleus when lacking an element conferring the speckled punctuate pattern, which would be located between residue 306 and the protein C-terminus.

Endogenous AIRE

Several lines of evidence point to a very restricted expression pattern of the AIRE protein. Albeit detectable by RT-PCR, endogenous AIRE expression could not be revealed by immuno-cytofluorescence in human primary fibroblasts nor in HeLa, Jurkat or Daudi cell lines (data not shown). However, fine nuclear dots were observed in fibroblasts with sp97181, but these were considered unspecific since none of the other antisera could reproduce this pattern. By western blot analysis, neither nuclear nor cytoplasmic cell fractions obtained from HeLa cells and monocytic cell line U937 reacted significantly with antibodies sp97179 or sp97181 (data not shown). Immunoblots prepared from human adult liver, thyroid and parathyroid tissues did not show any reaction with anti-AIRE sera (data not shown). Taken together, these data confirm that the AIRE protein is not or is seldom expressed in these tissues and cell types. Attempts to induce AIRE expression in stimulated non-expressing cells did not allow the detection of AIRE, as investigated by immunocytofluorescence. Since AIRE shares several functional domains with the interferon-inducible Sp100 protein (26), we tested whether the AIRE gene was interferon responsive. HeLa cells stimulated with interferon-[gamma] (IFN-[gamma]), IFN-[beta] or a combination of both showed negative staining for AIRE, whereas Sp100 dots increased in size and number in these experiments (data not shown). Stimulation of Daudi cells with IFN-[alpha], or Jurkat cells with anti-CD3 or phorbol ester/Ca2+ ionophore, also did not promote AIRE expression (data not shown).

Interestingly, recent data obtained on mouse embryonic sections suggest that the AIRE expression pattern is restricted to a limited number of cells in developing thymus (M.L. Yaspo, unpublished data). We investigated whether AIRE could be detected by immunocytofluorescence (using sp97181 and Mab155) in fresh human peripheral blood leukocytes. Very few cells were clearly stained, where AIRE appeared localized mainly in the nucleus, defining 10-15 dots distributed within the nucleoplasm (Fig. 7A). As an example, Figure 7B shows optical sections of a cell displaying both nuclear and cytoplasmic staining with Mab155, with a strong labeling in the perinuclear region. In blood cells, the AIRE pattern is consistent with the spatial distribution observed in transfected non-expressing cells, even though the dots were less numerous. We show data obtained with Mab155 monoclonal antibody, since sp97181 detection gave a slight nuclear background. However, observations were comparable for both antibodies.

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Figure 7. Endogenous expression of AIRE in peripheral blood lymphocytes. Freshly isolated peripheral blood lymphocytes were stained for AIRE with Mab155 (red). (A) Nuclei were stained with DAPI and images were analyzed with epifluorescence. (B) Nuclei were stained with YOYO-1 (green). I-VI: optical sections taken using a confocal laser microscope scanner. The depth of each section is indicated on the right. (a) Red and green images merged. (b) Red image. (c) Green image.

DISCUSSION

Mutations affecting transcriptional regulators have been reported in a number of human pathologies, and generally involve loss-of-function mutations and haploinsufficiency mechanisms (27). AIRE is a novel putative transcription-associated factor found mutated in APECED autoimmune disease. As a first step to uncover the pathways in which the AIRE protein could play a role, we have analyzed the sub-cellular distribution of full-length and truncated AIRE protein constructs expressed transiently in mammalian cells.

AIRE sub-nuclear localization

We demonstrated that the wild-type AIRE gene product has the ability to be targeted into the nucleus where it is found associated with distinct speckled sub-domains in the nucleoplasm. Nuclear factors have been shown to be localized in discrete macromolecular domains within the cell nucleus, often specifying a functional organization, such as sites of transcription or regions of pre-mRNA splicing (28). The distribution in punctuate domains has been described previously for many transcription factors or transcription-associated factors, where this pattern is likely to represent protein complexes on target sites of the chromatin. The speckled pattern is thought to reflect the transcriptional activity of the cell (28), and the number of speckles observed for AIRE is possibly indicative of multiple interaction sites on the chromatin. Thus, the sub-nuclear localization observed for the AIRE protein would be in agreement with its putative function as a transcriptional regulator. Overexpressed AIRE in non-expressing cells shows a higher number of dots than endogenous AIRE seen in peripheral blood cells. This is probably due to a quantitative effect as a consequence of protein overexpression but it could also reflect a qualitative difference in potential interacting factors that may be absent in non-expressing cells. The difference in the number of dots observed for transfected and endogenous protein has also been reported for other nuclear factors, such as for ALL-1 (13). Sp100, sharing several regions of homology with AIRE, defines nuclear dots, but these were not found to co-localize with AIRE in transfected cells. It is worth mentioning that, unlike Sp100, AIRE was not up-regulated by interferon in Daudi or HeLa cells, suggesting that the gene may be regulated by a different signal transduction pathway. In contrast to AIRE, Sp100 was expressed at a basal level in HeLa cells. Therefore, it would be important to investigate if AIRE gene expression can be up-regulated in cells naturally expressing this protein. Nonetheless, inspection of the AIRE genomic sequence (accession no. HSAJ9610) did not reveal any canonical interferon-responsive element (IRE) in the promoter region.

The AIRE-[Delta]BamHI and AIRE-[Delta]SacI truncated proteins have lost the capacity to be directed to speckled domains. Instead, they localized to nuclear inclusions that are very reminiscent of nuclear bodies (24). However, these large nuclear inclusions are not coincident with coiled bodies, as defined with p80-coilin. These inclusions may correspond to other undefined nuclear bodies or may simply reflect artifactual structures formed by aggregates of mutant AIRE proteins. One hypothesis which could explain the peculiar localization of the mutants is that the truncated proteins could be embedded in nuclear body structures by default, as a consequence of lacking a domain normally interacting with either a core DNA target or a chromatin-associated protein. AIRE-[Delta]BamHI lacks the two PHD motifs and the SAND domain, and AIRE-[Delta]SacI is truncated within PHD1, yet these two mutants show a similar nuclear mislocalization. The potential DNA-binding SAND domain (17) does not seem to play a role in the nuclear spatial distribution. The data indicate that a domain present in the C-terminal third of the protein potentially mediates the speckled sub-nuclear localization. However, it is unclear whether the PHD fingers themselves confer the ability of the AIRE protein to localize to speckles. The molecular target of the PHD motif remains to be identified. In the case of ALL-1 containing one PHD finger for instance, another N-terminal element of the protein directs speckled nuclear distribution (13). Moreover, it is worth noting that the nuclear speckled distribution is not an intrinsic feature of all PHD finger proteins. For example, Sp140 does not show speckles but co-localizes to nuclear bodies (11). Ectopic overexpression of AIRE in apparently non-expressing cells is compatible with endogenous AIRE expression in peripheral blood cells. It will be extremely important to determine the type of those few cells expressing AIRE.

Our data suggest that the pathological consequences of some of the mutations found in the AIRE gene may elicit their effects at least in part by affecting the spatial organization of AIRE in the cell nucleus. It is unlikely that this effect is due to a difference in protein expression level between wild-type and mutants, since identical observations were made when using different DNA concentrations and different cell types. Without mimicking strictly the mutations found in AIRE, the two mutants described in this study represent adequate models for predicting the cellular localization of APECED defective proteins. AIRE-[Delta]BamHI produces a 209 residue protein and AIRE-[Delta]SacI encodes a 306 residue protein, while the Finnish major APECED mutation is predicted to produce a truncated polypeptide of 259 amino acids (1). It is therefore reasonable to suggest that the defective peptide synthesized in the majority of APECED patients would show a comparable nuclear localization with that of AIRE-[Delta]SacI and AIRE-[Delta]BamHI. Fine dissection of AIRE protein domains will be necessary to identify the minimal domain conferring a functional nuclear speckled pattern.

AIRE co-localizes with cytoskeletal filaments

AIRE immunocytofluorescence detection revealed an unexpected filamentous system extending throughout the cytoplasm of transfected cells. AIRE does not contain coiled-coil domains as defined by the Lupa’s algorithm (29), which represent an essential condition for promoting protein-protein association as a building block to form bundles or filaments (30,31). These observations infer that wild-type AIRE interacts with structural components of the cytoplasmic compartment. Further investi-gations led us to confirm that the stained AIRE fibers are found partly co-localizing with the vimentin filamentous system but that coincidence was even more striking with microtubules. Although high protein concentration due to AIRE overexpression in COS cells expressing SV40 large T antigen can trigger protein aggregation and creation of cytosolic artificial structures, it is unlikely that the co-localization with vimentin or microtubules represents an artifactual interaction. Moreover, the same pattern was observed in human fibroblasts and HeLa cells. Vimentin is the major protein constituent of cytoplasmic intermediatefilaments maintaining cell shape and cytoskeletal integrity (32). Microtubules are highly dynamic structures that play a key role in cell morphogenesis in the formation of mitotic spindles (33). The occurrence of nuclear factors interacting with components of the cytoskeleton is not an unprecedented observation. An interes-ting example is the regulation of the function of the Gli zinc finger transcription factor, a vertebrate homolog of the Drosophila ci gene product (34). This transcription factor is targeted mainly to the cytoplasm where it is anchored to microtubules, whereas a truncated form of Gli processed by proteolytic cleavage is directed to the nucleus (35,36). Nuclear factors interacting with vimentin have also been described, such as a component of the nuclear matrix, NMP125, transiently stored along with vimentin during mitosis (37), or hnRNP S1 proteins co-localizing with vimentin filaments (38). It is conceivable that AIRE has the capacity to associate with vimentin, since the protein harbors a cluster of basic amino acid residues within the nuclear targeting signal. A similar motif was described previously as being essential for cytoplasmic plectin-vimentin junctions (39). Co-localization of AIRE with both vimentin and microtubules is not very surprising, since intermediate filaments and microtubules can be found associated with each other in the cell via cross-bridging proteins such as kinesin (22). Confocal analysis showed a better coincidence of AIRE with microtubules than with vimentin, but it is of paramount importance to determine in the near future the exact nature of this interaction. The temporal and spatial decoration of filament arrays and nuclear speckles by anti-AIRE antibodies appears variable, suggesting the existence of a dynamic or passive trafficking of AIRE in the cell. It is possible to envisage that AIRE resides on cytoskeletal fibers as part of a docking mechanism regulating nuclear translocation.

The cytoplasmic distribution of the two AIRE mutants engineered in our study shows that the potential domain indispensable for the co-localization with vimentin or microtubules should be located in the N-terminal half of the protein. Nevertheless, AIRE-[Delta]BamHI presents a strikingly altered cytoplasmic distribution, in particular in COS cells, where mutant protein aggregates can impair the formation of vimentin filaments. The AIRE-[Delta]BamHI mutant may form aggregates by loss of conformational structure, a feature commonly observed for other short truncated proteins. These aggregates may simply provoke a steric hindrance impairing the formation of cytoskeletal filaments. AIRE-[Delta]BamHI protein still co-localizes to vimentin fibers in fibroblasts where AIRE aggregates are seldom found. A hypothesis which may, in part, explain this phenomenon is that fibroblasts divide more slowly than COS cells so that the vimentin network may not have been yet rearranged (40), hence preventing AIRE from entrapping vimentin. The vimentin network appears more dense in fibroblasts, and vimentin involved in a higher order of polymerization, such as bundles, may be less accessible to interacting proteins than vimentin forming a loose network. In contrast, the AIRE-[Delta]SacI protein shows a cytoplasmic pattern similar to wild-type, suggesting that the altered cytoplasmic localization observed with one of the AIRE mutants may not be of fundamental relevance to APECED pathogenesis. Expression of AIRE in the cytoplasm has been observed in some blood cells, suggesting that the results corresponding to protein overexpression reflect at least in part the endogenous protein localization.

The data indicate that AIRE is seldom or not expressed in most tissues. The human AIRE cDNA was isolated originally from a thymus cDNA library, and data obtained from mouse embryonic sections analyzed at 14.5 d.p.c. indicate a pattern of expression restricted to a few cells in the thymus (M.L. Yaspo, unpublished data). Neither Daudi nor Jurkat cells express AIRE, even after stimulation. Peripheral blood lymphocytes showed only a few stained cells. Comparable data are reported by Bjorses et al., describing a nuclear pattern revealed by anti-AIRE serum in human histological sections from thymus, spleen, liver and lymph nodes (41). Typing those cells will prove extremely relevant to the understanding of the biological role of AIRE in the immune system. In contrast to other PHD proteins involved in autoimmunity, such as Mi-2 and Sp100, AIRE is not an autoantigen in APECED patients (8). We have shown here that two N-terminal AIRE protein fragments modeling APECED mutants were mislocalized within the cell nucleus. The data suggest that the nuclear AIRE function may first be abolished by a deleterious spatial organization of the mutant proteins. Correlation of AIRE normal function with downstream biochemical pathways leading to APECED symptoms will provide important insights into the mechanisms of transcriptional control involved in autoimmunity.

MATERIALS AND METHODS

Recombinant AIRE expression in E.coli and purification of the protein

The QIA expression method (Qiagen, Germany) was used for bacterial expression and purification of the 6×His-taggedrecombinant AIRE protein. A 1.8 kb SalI-NotI cDNA fragment derived from clone B1-1pA and containing the complete AIRE coding sequence was cloned into the pQE32N vector (pQE32N-AIRE). The correct cloning orientation and the reading frame were verified by sequencing. Escherichia coli strain SCS1 pSE III was transformed with pQE32N-AIRE and protein expression was induced for 4 h with 1 mM isopropyl-[beta]-d-thiogalactopyranoside (IPTG). The His-tagged protein was purified under denaturing conditions on an Ni-NTA agarose column according to the manufacturer’s recommendations (Qiagen), and analyzed by SDS-PAGE and western blotting.

AIRE expression plasmids for transient transfection

For expression of the full-length 545 amino acids protein in mammalian cells, the 1.8 kb EcoRI insert from B1-1pA AIRE cDNA was cloned into the expression vector pSG5 (Invitrogen, San Diego, CA) and named pSG5-AIRE. The correct orientation was verified by restriction digest and sequencing. AIRE deletion mutants were generated by restriction digests using unique restriction sites in the cDNA. The pSG5-AIRE-[Delta]BamHI construct was generated by deleting a 1.1 kb BamHI 3[prime]-terminal fragment from pSG5-AIRE cDNA, producing a protein that is truncated at residue 209. In this construct, a stop codon is provided by the pSG5 vector sequence after coding for 17 nonsense amino acids at the AIRE-[Delta]BamHI C-terminus. The pSG5-AIRE-[Delta]SacI construct was generated by deleting a 0.8 kb SacI-BglII fragment from pSG5-AIRE cDNA and religation of the DNA molecule after generating blunt ends by T4 DNA polymerase and Klenow fragment. This construct encodes a protein truncated at amino acid 306; a stop codon is provided by the vector sequence after coding for two nonsense amino acids at the C-terminus of AIRE-[Delta]SacI.

Antibody production and purification

Polyclonal antibodies against the AIRE protein were obtained by injecting rabbits with the synthetic peptides MATDA-ALRRLLRLHR (corresponding to amino acids 1-15) and SQPRKGRKPPAVPK (corresponding to amino acids 107-120), respectively. The resulting immune sera sp97179 (for amino acids 1-15) and sp97181 (for amino acids 107-120) were affinity purified against their corresponding synthetic peptides immobilized on a HiTrap NHS-activated 1 ml column (Pharmacia, Freiburg, Germany) according to the manufacturer’s recommendations.

Monoclonal antibodies were obtained by immunizing mice against the recombinant His-tagged AIRE protein. Splenocytes were fused to the myeloma cell line SP2/0-Ag14, and hybridomas were screened by reactivity to the His-tagged AIRE protein in an enzyme-linked immunosorbent assay (ELISA). The specificity of the hybridomas Mab137 and Mab155 was confirmed by immunofluorescence and western blot analysis of COS7 cells transfected with pSG5-AIRE.

Cell culture and transfection experiments

COS1 cells were maintained at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 1000 mg/l glucose, 10% fetal calf serum (FCS), 10 U/ml penicillin and 10 µg/ml streptomycin. Transfections were performed by electroporation as follows: 106 cells grown at 80-90% confluence were centrifuged, washed twice in ice-cold phosphate-buffered saline (PBS) containing 2 mM HEPES (HeBS) and resuspended in 800 µl of HeBS. DNA was diluted in 130 µl of HeBS before being added to the cells (either 2, 5, 10 or 20 µg of DNA). After 10 min incubation on ice, cells were pulsed with a field strength of 3 kV/cm (capacitance 25 µF) using a Gene Pulser (Bio-Rad, Hercules, CA). Cells were allowed to recover on ice for 10 min before being transferred in 10 ml of pre-equilibrated DMEM containing 25 mM HEPES. Transfected cells were seeded in Leighton tubes (Costar, Cambridge, CA) for immunofluorescence studies (1.5 × 105 cells/Leighton) and in 10 cm Petri dishes(4 × 105 cells/dish) for cell extract preparations, and incubated at 37°C and 5% CO2 for 24 or 48 h. COS7 cells and fibroblasts were maintained at 37°C and 5% CO2 in DMEM/F12 medium containing 1000 mg/l glucose, 10% FCS, 10 U/ml penicillin and 10 µg/ml streptomycin. Cells were transfected using theLipofectACE method according to the manufacturer’s recommendations (Gibco Life Technologies, Karlsruhe, Germany). Cells were seeded into a six-well plate (4 × 105 cells/well) containing glass coverslips and allowed to grow for 24 h before transfection. Transfections were performed using 3 µg DNA/well and cells were incubated in the LipofectACE/DNA mix for 6 h. Cells were analyzed by indirect immunofluorescence 48 h post-transfection.

Stimulation experiments

Jurkat and Daudi cells were stimulated in suspension. Jurkat cells were either stimulated with 100 ng/ml phorbol myristate-13-acetate (PMA; Sigma, Deisenhofen, Germany) + 1 µM ionomycin (Sigma) for 4, 8, 12, 16 or 24 h or with 80 µg/ml anti-mouse-CD3 antibody (Chemicon International, Temecula, CA) for 16 or 24 h. Daudi cells were stimulated with 1000 U/ml IFN-[alpha] (murine recombinant, kindly provided by Thorsten Buch, University of Cologne) for 18 or 36 h. A total of 5 × 105 HeLa cells/well were seeded into six-well plates containing glass coverslips. HeLa cells were starved overnight in serum-free DMEM and stimulated with 1000 U/ml IFN-[gamma] (human recombinant; Boehringer Mannheim, Mannheim, Germany) for 24 and 48 h or with a combination of 1000 U/ml IFN-[beta] (from human fibroblasts; ICN, Costa Mesa, CA) for 42 h and 100 U/ml IFN-[gamma] for the last 18 h.

Isolation of peripheral blood lymphocytes

Human blood was collected on heparin and fractionated by centrifugation on a ficoll gradient. The fraction corresponding to the peripheral blood lymphocytes (PBLs) was collected and washed twice in PBS before being processed for indirect immunofluorescence.

Indirect immunofluorescence

PBLs, Jurkat or Daudi cells (106/well) were centrifuged in 6-well plates onto poly-l-lysine-coated glass coverslips. Other cell types were grown directly on untreated glass coverslips.

Cells were rinsed briefly in PBS and then either fixed in1:1 methanol/acetone for 10 min at -20°C and air dried for 15 min or fixed in 3.7% parafomaldehyde (PFA) in PBS for 10 min followed by a brief rinse in PBS. Cells were permeabilized for 10 min in PBS/0.2% Triton X-100, washed three times in PBS and then incubated in PBS containing 3% bovine serum albumin (BSA) for 1 h at room temperature or at 4°C overnight. After a brief rinse in PBS, cells were incubated with the polyclonal antibodies diluted 1:200 in PBS/0.1% Triton X-100 (PBS-T) or with the hybridoma supernatants diluted 1:2 in PBS/0.2% Triton X-100 for 1 h at room temperature. Cells were washed three times in PBS-T for 10 min followed by 1 h incubation with a Cy3-labeled anti-rabbit or anti-mouse antibody diluted 1:200 in PBS. Cells were washed twice in PBS-T and once in PBS for 10 min and stained with 12 nM YOYO-1 iodide in PBS (Molecular Probes, Leiden, The Netherlands) for 15 min, washed in PBS three times for 5 min and mounted in 75% glycerol/PBS or Vectashield (Vector Laboratories, Burlingame, CA).

Simultaneous detection of AIRE and vimentin was performed by co-staining cells with sp97179 (or sp97181) and anti-vimentin antibodies. Vimentin polyclonal antibody raised in goats (kindly provided by P. Traub, Max Planck Institute for Cell Biology, Ladenburg, Germany) was diluted 1:400 and incubated for 1 h, followed by incubation with a fluorescein isothiocyanate (FITC)-conjugated anti-goat secondary antibody diluted 1:200 in PBS. Coverslips were mounted in Vectashield containing 5 µg/ml 4[prime],6-diamidino-2-phenylindole (DAPI).

Simultaneous detection of AIRE and microtubules wasperformed by rinsing the cells briefly in 1.2× PEM (120 mM PIPES, 6 mM EGTA, 2.4 mM magnesium chloride) before fixation in 3.7% PFA/1.2× PEM. After permeabilization as described above, cells were washed three times in PBS and blocked in 0.5% BSA/PBS for 20 min. Co-staining was performed with sp97181 (diluted 1:200) and two anti-tubulin-antibodies (MCA77s and MCA78s, each diluted 1:30; Serotec, Oxford, UK) in 0.5% BSA/PBS. After three washes in PBS, cells were again permeabilized and blocked in 0.5% BSA/PBS for 20 min. AIRE was detected by incubation with an FITC-conjugated anti-rabbit antibody, and microtubules by a Cy3-conjugated anti-rat antibody for 1 h. Cells were washed three times in PBS and post-fixed in 3.7% formaldehyde for 10 min. After one wash in PBS, cells were treated with 50 mM ammonium chloride for 15 min, washed in PBS and mounted in Vectashield.

Co-staining of AIRE and p80-coilin was performed as described for co-staining with vimentin. The anti-p80-coilin antibody [kindly provided by A. Lamond (University of Dundee, Dundee, UK) and E. Chan (The Scripps Research Institute, La Jolla, CA)] was diluted 1:500 and detected by a Cy3-conjugated anti-rabbit secondary antibody. AIRE was stained with supernatant Mab137 diluted 1:2 and detected by a FITC-conjugated anti-mouse antibody.

Cells were either visualized and scanned with a confocal laser microscope (LSM 510-axioplan2; Zeiss, Jena, Germany) or analyzed with an epifluorescence microscope (Axioskop 50; Zeiss). Photographs were taken with a CCD camera.

Western blot analysis

Harvested cells were lysed in a buffer containing: 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8, 1 mM EDTA, and supplemented with 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM [beta]-mercaptoethanol, 10 µg/ml leupeptin and 10 µg/ml pepstatin. A total of 20 µg of total protein extracts was separated by 12% SDS-PAGE and blotted on a PVDF membrane. The membrane was blocked for 2 h in TBS-T (20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) containing 3% BSA followed by incubation with the polyclonal antiserum (sp97179, sp97181) diluted 1:1000 in TBS-T for 1 h. After washing the membrane three times for 5 min in TBS-T, the membrane was incubated for 1 h with an anti-rabbit IgG alkaline phosphatase conjugate (Calbiochem, La Jolla, CA) diluted 1:5000 in PBS-T. The membrane was then washed three times for 5 min in TBS-T, briefly rinsed twice in TBS and incubated in Western Blue Stabilized Substrate (Promega, Madison, WI) for 6 min. The reaction was stopped by rinsing the membrane with H2O.

In order to demonstrate the specificity of the antibodies in immunofluorescence and western blot detection, experiments were repeated after pre-incubation of the antisera with an excess of His-tagged AIRE recombinant protein in PBS-T for 1 h at room temperature.

ACKNOWLEDGEMENTS

The authors wish to thank Professor P. Traub for discussion and for the generous gift of the anti-vimentin antibody, Drs K.L. Chan and A. Lamond for providing anti-p80 coilin antibodies andDr T. Sternsdorf (Hamburg University, Germany) for the anti-sp100 antibodies. We also thank Drs K. Wertz (MPI Immunobiologie, Freiburg, Germany) and A. Sittler for discussions. We wish to thank Dr Vas Ponnambalam (University of Dundee, UK) for providing cell fraction lysates. We are grateful to Dr T. Buch (University of Cologne, Germany) for advice and for providing mouse interferon. We thank Dr T. Vogel (University Klinikum, Dusseldorf, Germany) for providing tissue samples for western blot analysis. This work was supported by the Deutsches Human Genome Projekt (BMBF grant 01KW9608).

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*To whom correspondence should be addressed. Tel: +49 30 8413 1356; Fax: +49 30 8413 1380; Email: yaspo@mpimg-berlin-dahlem.mpg.de

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