Characterization of Regulatory Elements and Methylation Pattern of the Autoimmune Regulator (AIRE) Promoter*

Astrid Murumägi {ddagger} §, Perttu Vähämurto {ddagger} and Pärt Peterson {ddagger} 

From the {ddagger}Institute of Medical Technology, University of Tampere and Department of Pathology, Tampere University Hospital, Lenkkeilijänkatu 6, Tampere 33014, Finland and the §Department of Biotechnology, University of Tartu, Tartu 51010, Estonia

Received for publication, October 11, 2002 , and in revised form, February 26, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Defects in the AIRE gene cause a monogenic autoimmune syndrome APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy), which is characterized by loss of self-tolerance to multiple organs. In concordance with its role in immune tolerance, AIRE is most strongly expressed in thymic epithelial cells and in cells of monocytic-dendritic lineage. The AIRE protein has been shown to function as a transcriptional regulator, however, the mechanisms regulating AIRE gene expression are not known. Here we have characterized the AIRE promoter region by identifying a minimal promoter region within 350 bp of the translation initiation codon. Electrophoretic mobility shift assays and transient transfections with mutated promoter constructs revealed a functional TATA box (-163 to -153) and binding sites for transcription complexes AP-1 (-307 to -296), NF-Y (-213 to -202), and Sp1 (-202 to -189). The presence of a 390-bp CpG island within the proximal promoter suggested that cytosine methylation has a role in transcriptional regulation of AIRE, which was supported by in vitro methylation experiments of promoter constructs. Sodium bisulfite sequencing showed a less methylated status of AIRE promoter in the thymic epithelial cell line TEC1A3 compared with HeLa and monocytic cells U937 and THP-1. Real-time PCR analysis showed that treatment with 5-aza-2'-deoxycytidine (5-azaCdR), a DNA methyltransferase inhibitor, up-regulated AIRE transcript levels in TEC1A3, U937, and HeLa cells and that even greater activations in TEC1A3 and U937 cells were observed using combined treatments with deacetylase inhibitor trichostatin A. These results suggest that AIRE gene expression is modulated through modifications in chromatin methylation and acetylation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in the autoimmune regulator (AIRE)1 gene cause an autosomal recessive disease called APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy) (1, 2). The syndrome, occurring as a result of defective tolerance to self-antigens, is characterized by endocrine organ-specific autoimmunity and chronic mucocutaneous candidiasis and ectodermal disorders (3).

AIRE encodes a 58-kDa protein with structural motifs indicative of a transcriptional regulator having a conserved nuclear localization signal, two plant homeodomain zinc-finger motifs, four LXXLL or nuclear receptor box motifs, a proline-rich region, a SAND domain, and an HSR domain (1, 2, 4). The protein has a tissue-specific expression pattern, being mainly expressed in the thymus, lymph nodes, the spleen, and fetal liver (1, 2, 5, 6). In the thymus, AIRE is expressed in medullary epithelial cells and cells of monocyte-dendritic lineage (5, 7). Subcellularly, AIRE is located in the cell nucleus and in nuclear dots resembling promyelocytic leukemia bodies (7, 8). Supporting AIRE subcellular location and its structural features, AIRE has been shown to act as a transcriptional activator, the process that is mediated through the plant homeodomain zinc fingers (8). AIRE also interacts with the common transcriptional coactivator CREB-binding protein (CBP) through the CH1 and CH3 conserved domains (9). Recent findings in AIRE-deficient mice suggest that the protein influences expression of peripheral self-antigens in the thymus, explaining the mechanism through which immune tolerance can be broken in multiorgan endocrine autoimmune diseases (10, 11).

The role of AIRE in tolerance mechanisms led us to investigate the transcriptional regulation of the AIRE gene. Here we have characterized the human AIRE gene promoter structure identifying a minimal promoter and its transcriptional control elements. We describe the methylation status of the AIRE promoter in several cell lines and demonstrate the up-regulation of AIRE mRNA as a response to 5-aza-2'-deoxycytidine (5-azaCdR) and trichostatin A (TSA) treatments.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—COS-7 cells were maintained in Dulbecco's modified Eagle's medium. HeLa and TEC 1A3 (human thymic epithelial cell line) cells were maintained in Eagle's minimum essential medium. Human monocytoid cell lines, THP-1 and U937, were cultured in RPMI 1640. Media was supplemented with 10% fetal calf serum and a 100 units/ml penicillin-streptomycin mixture (BioWhittaker, Europe).

Isolation of AIRE Promoter—A 1.2-kb genomic fragment containing the 5'-end region of the AIRE gene was isolated by PCR amplification from human genomic DNA. Nucleotide sequences for primers are listed in Table I. The 5'-flanking region of AIRE promoter was subcloned into the pHIV-LTR-luc plasmid (Prof. Kalle Saksela, University of Tampere, Finland) encoding firefly luciferase reporter gene and named as pAP1235. The promoter region in the pAP1235 construct was verified by sequencing.


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TABLE I
Primer sequences used in this study

 

Computer Analysis—Transcription factor binding sites were predicted by using the MatchTM program, which uses TRANSFAC 5.0 matrices (core similarity 1.0 and matrix similarity 0.9). The presence of CpG islands was analyzed with the EMBOSS program CpGplot using the algorithms of Gardiner-Garden and Frommer (12). According to this analysis, a CpG-rich region is defined as stretches of DNA in which both the moving average of percentage of G plus C nucleotides is greater than 50 and the moving average of observed/expected CpG is greater than 0.6.

Construction of Deletion and Mutant Reporter Plasmids—The pAP1235 plasmid construct containing the 5'-flanking region was used as a template to synthesize a series of deletion reporter gene constructs. The forward and reverse primers containing NotI and BamHI restriction sites, respectively, were used in cloning of deletion constructs (Table I and Fig. 1). Site-directed mutagenesis of the pAP1235 construct was performed using the GeneEditor in vitro site-directed mutagenesis system (Promega). Mutations were introduced into putative DNA binding sites for the AP-1, NF-Y, and Sp1 transcription factors, as well as into the putative TATA box. Primer sequences used in site-directed mutagenesis are given below in Table I. All mutations were confirmed by DNA sequencing.



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FIG. 1.
Deletion analysis of the human AIRE promoter. A series of AIRE promoter 5'-deletion constructs were transiently transfected into COS-7 (black bars) and TEC1A3 (light bars) to identify the minimal promoter region. The left side of the figure shows the plasmid constructs with sizes indicated in base pairs; the right side shows promoter activity measured in relative luciferase units obtained from each transfection. Luciferase activity is relative to a pBL-KS promoterless control. Results shown are a mean ± S.D. of three experiments performed in triplicate.

 

Transient Transfections and Luciferase Reporter Assays—Cells (1.5 x 105) were transiently transfected with 2.5 µg of DNA (2 µg of luciferase reporter plasmid, 0.5 µg of {beta}-galactosidase expression vector pEF-BOS (Prof. Kalle Saksela, University of Tampere, Finland)) using ExGen 500 transfection reagent (Fermentas) following the manufacturer's instructions. After 24 h, the cells were lysed to measure the luciferase activity using the Luciferase Assay System (Promega). The luciferase activities were normalized for transfection efficiency according to {beta}-galactosidase activities. The transfections were performed in triplicate.

Electrophoretic Mobility Gel Shift Assay—Nuclear extracts were made from HeLa and TEC1A3 cells with the variation of the method of Dignam et al. (13). Briefly, 3 x 107 cells were washed twice with ice-cold phosphate-buffered saline and resuspended in buffer A (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 2 µg/ml aprotinin) and left on ice for 15 min. The lysates were passed several times through a 25-gauge needle. The nuclei were recovered by brief centrifugation at full speed and resuspended in buffer C (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, 1 µg/ml leupeptin, and 2 µg/ml aprotinin), followed by sonication and incubation on ice for 30 min. The reaction mix was centrifuged for 15 min at full speed, and the supernatant was collected and stored at -70 °C until use. Protein concentrations were determined using the Bio-Rad Dc protein assay (Bio-Rad Laboratories, Hercules, CA). The oligonucleotides used in EMSA are listed in Table I. Pre-binding of nuclear extract to poly(dI-dC) (Roche Applied Science) was carried out in a 10-µl reaction volume at 30 °C for 30 min in a buffer containing 10 mM Tris-HCl, pH 7.5, 25 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 1 mM MgCl2, 4% glycerol, and 0.05 mg/ml poly(dI-dC)·poly(dI-dC). For competition experiments, unlabeled competing oligonucleotides at 100-fold molecular excess were included in preincubation mixture. For antibody supershift experiments, corresponding antibody (2 µg) was included in the preincubation mixture. Purified 32P-end-labeled, double-stranded oligonucleotide was then added to the reaction mix and incubated at 30 °C for 30 min. The reaction mixtures were subjected to native 4.5% polyacrylamide gel electrophoresis. Following the electrophoresis, the gel was dried and exposed for autoradiography (Kodak Biomax MS-1, Sigma). The Sp1 and AP-1 consensus oligonucleotides were purchased from Promega. The Sp1, pan-Jun, and pan-Fos antibodies were purchased from Santa Cruz Biotechnology. Anti-NF-YA and anti-NF-YB antibodies were a gift from Dr. Roberto Mantovani (University of Milano, Italy).

In Vitro DNA Methylation—SssI methylase (New England BioLabs) was used to methylate AIRE promoter luciferase reporter construct pAP1235. Briefly, plasmid DNA was incubated with 1 unit of methylase per 1 µg of DNA in 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9, supplemented with 160 µM S-adenosylmethionine. Reactions were carried out at 37 °C overnight. The methylated plasmid DNA was purified through a Wizard DNA Clean-Up system (Promega) and transfected into TEC1A3 and COS-7 cells in parallel with the unmethylated pAP1235 construct.

Genomic DNA Isolation—Genomic DNA was extracted from TEC1A3, HeLa, THP-1, and U937 cells according to Sambrook et al. (14). Briefly, the genomic DNA was isolated by cell lysis with proteinase K (Promega) digestion and extraction with phenol/chloroform. After the precipitation, the genomic DNA was digested with restriction enzymes SmaI and SacI, separated on a 1.5% agarose gel with 1x TAE buffer (40 mM Tris-HCl, 1 mM EDTA, pH 8), blotted on nylon membrane, and hybridized with the radioactive probe corresponding to the genomic sequence (-568 to -195 bp). After hybridization and subsequent washing, the filters were exposed to autoradiography film.

Sodium Bisulfite Genomic DNA Sequencing—Bisulfite genomic sequencing was performed as described previously (15, 16). The genomic DNA (2 µg) was denatured in 0.3 M NaOH at 37 °C for 15 min. After the addition of 3 M sodium bisulfite (Sigma) and 10 mM hydroquinone (Sigma), samples were mixed, overlaid with mineral oil, and incubated at 50 °C for 16 h. The modified DNA was purified through the Wizard DNA Clean-Up system (Promega) and denatured by addition of 0.3 M NaOH at 37 °C for 15 min. The bisulfite-reacted DNA was precipitated and resuspended in 1 mM Tris-HCl, pH 8, and used immediately or stored at -20 °C. The sequence of interest in the bisulfite-reacted DNA was amplified by PCR in a reaction mixture containing 200 ng of DNA, 0.5 µM primers, 200 µM dNTPs, 1x buffer, 5% Me2SO, and 1 unit of Hercules polymerase (Stratagene). Each amplification reaction consisted of a 5-min incubation at 95 °C followed by 40 cycles of 1 min at 94 °C, 1 min at 56 °C, 1.5 min at 72 °C, and a final elongation step for 7 min at 72 °C. Primer sequences are listed in Table I. The modified DNA was further amplified by nested amplification, DNA fragments were gel-purified with a SephaglasTM Bandprep kit (Amersham Biosciences) and cloned into pCRII-TOPO vector (Invitrogen). Approximately 10 clones were sequenced per cell line.

5-AzaCdr and TSA Treatment—DNA methyltransferase inhibitor, 5-azaCdr, was added to cells at final concentrations from 0.2 to 15 µM for 72 h. Deacetylase inhibitor, TSA (100 ng/ml), was added for 24 h alone or at the end of the 5-azaCdr treatment, where indicated. Subsequently, cells were used for RNA isolation using the Total RNA Isolation system (Promega), and 3 µg of total RNA was converted to cDNA using the First-Strand cDNA Synthesis kit (Amersham Biosciences).

Quantitative Real-time PCR—Real-time PCR was performed with the LightCycler instrument (Roche Applied Science) using a ready-to-use one-step QuantiTectTM SYBR® Green RT-PCR kit (Qiagen). Reactions were set up in 15 µl of final volume containing 2 µl of sample cDNA or standards, 0.5 µM primers, and 7.5 µl of 2x RT-PCR master mix. The amplification program for AIRE included an initial denaturation step at 95 °C for 15 min, followed by 55 cycles of denaturation at 94 °C for 15 s, annealing at 60 °C for 25 s, and extension at 72 °C for 20 s. SYBR® Green fluorescence was measured after each extension step. The specificity of amplification was subjected to melting curve analysis. The relative amount of the AIRE transcript was normalized to the amount of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript in each cDNA. The PCR conditions for GAPDH differed in that the annealing temperature was 58 °C and amplification consisted of 45 cycles. Cloned AIRE and GAPDH cDNAs were used as standards for quantification. Primer sequences to amplify the AIRE or GAPDH were as follows: AIRE-F, CCCTACTGTGTGTGGGTCCT; AIRE-R, ACGTCTCCTGAGCAGGATCT; GAPDH-F, CTGAGCTAGACGGGAAGCTC; and GAPDH-R, TCTGAGTGTGGCAGGGACT. Each of the PCR assays was run in triplicate, and the AIRE and GAPDH copy numbers were estimated from the threshold amplification cycle numbers using software supplied with the LightCycler thermal cycler.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
AIRE Proximal Promoter Contains Several Positive Regulatory Elements—We first isolated the human AIRE gene 5'-flanking region using PCR amplification of genomic DNA. The nucleotide sequence of the clone was found to be 100% identical with the genomic sequence in GenBankTM (accession number AB006684 [GenBank] ). The transcription initiation site, 128 bp upstream of the ATG codon, determined by rapid amplification of cDNA ends method, has been described earlier (1).

To demonstrate the activity of the AIRE promoter, the fragment was subsequently cloned in front of a luciferase reporter gene and transfected transiently into COS-7 and TEC1A3 cell lines. The luciferase activity with the pAP1235 reporter construct was 146- and 44-fold in COS-7 and TEC1A3 cells, respectively, compared with a promoterless control plasmid pBL-KS (Fig. 1), indicating cis-acting elements in the AIRE promoter region. To further map the regions responsible for transcriptional control we made deletion constructs of the pAP1235 plasmid from the 5'-end of the promoter fragment. Luciferase assays with the deletion constructs pAP940, pAP583, and pAP350 gave comparably similar activities in COS-7 cells, whereas the pAP248 construct resulted in significant decrease of the activity to 19% in COS-7 cells when compared with the pAP1235 construct. An even further decrease of the luciferase activity (to ~3%) was seen with the pAP190 deletion construct. Essentially similar results were obtained using the TEC1A3 cell line. As a conclusion of deletion construct analysis, the results showed that the sequence elements required for the minimal AIRE promoter activity reside within the first 350 bp upstream of the translation start site.

Characterization of Factors Binding to the Promoter—Analysis of the 350-bp promoter region with the MatchTM program indicated the presence of high score potential binding sites for transcription factor complexes such as a GC box (at position -202 to -188), an inverted CCAAT box (position -212 to -200), and a binding site for transcription factor AP-1 (position -307 to -296).

To characterize the transcription factors that interact with putative binding sites in the AIRE promoter region, EMSA, coupled with supershift assays, was performed. A shifted band appeared with the AP-1 site double-stranded oligonucleotide (-313 to -291) as a probe (Fig. 2A). The complex disappeared by the addition of a 100-molar excess amount of the unlabeled probe but was not affected by addition of the same amount of unrelated oligonucleotide to the reaction mix as a competitor. The mutated oligonucleotide probe from the AP-1 site was unable to form the complex, further underlining the specificity of the AP-1 site. To further characterize the complex, we performed a supershift assay by using antibodies raised against pan-Jun and pan-Fos proteins. The shifted band that appeared with the AIRE AP-1 site was supershifted by the addition of pan-Jun antibody, however, not by pan-Fos antibody (Fig. 2A). Similar DNA-protein complexes and supershift results were observed in both cell lines. As a control probe, we used a previously described AP-1 consensus site in complex formation and supershift analysis (Fig. 2A).



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FIG. 2.
Electrophoretic mobility gel shift assay with AIRE promoter binding sites. A, AIRE promoter AP-1 binding site. Competition and supershift assay demonstrated AP-1 binding to its consensus site at the AIRE promoter. Incubation of nuclear extracts from HeLa or TEC1A3 cells with pan-Jun antibody resulted in a supershifted protein-DNA complex, whereas the pan-Fos antibody did not supershift the complex. As a control, the AP-1 consensus probe was used. B, AIRE promoter CCAAT box. Competition and supershift assay showed NF-Y-specific binding to the inverted CCAAT box in AIRE promoter in HeLa and TEC1A3 cells. As a control, the NF-Y consensus probe was used. C, AIRE promoter GC box. Competition and supershift assay demonstrated the Sp1 binding to GC box in AIRE promoter. Addition of antibody specific for the Sp1 antagonized the protein-DNA complex. The arrows indicate specific complexes, and arrowheads mark antibody supershifts.

 

The relevance of the CCAAT box in AIRE gene regulation was determined with probes coding for the CCAAT box sequence. DNA-binding reactions showed a shifted protein-DNA complex, which was fully abolished by addition of a 100-fold molar excess of cold oligonucleotide (Fig. 2B). Only a CCAAT box-specific oligonucleotide inhibited the formation of complex, because the protein-DNA complex was not competed away by a nonspecific competitor as a probe. In contrast, as an indication of protein binding specificity, mutations introduced into the AIRE CCAAT box consensus sequence abolished protein-DNA complex formation. To identify whether the protein within the specific protein-DNA complex was in fact NF-Y, supershift assays were performed with antibodies against A and B subunits of the NF-Y transcription complex. Both anti-NF-YA and anti-NF-YB antibodies were able to supershift the protein-DNA complexes, resembling the supershifted complex obtained with the CCAAT consensus sequence (Fig. 2B).

Furthermore, the importance of the GC box in the AIRE promoter was tested. The shifted complex obtained with the AIRE Sp1 probe was similar to the complex obtained with a control consensus Sp1 element (Fig. 2C). The complex disappeared with the addition of the unlabeled competitor but was not influenced by excess amount of unrelated oligonucleotide. The mutated AIRE Sp1 oligonucleotide used as probe resulted in no complex formation. The identity of the complex was further confirmed using an antibody specific for the Sp1 transcription factor. The Sp1 antibody was able to specifically interfere with the formation of the protein complex with the AIRE promoter element as well as with the consensus Sp1 element in supershift assays. Taken together, these results indicated that the AIRE promoter contains at least three specific regulatory elements, which form protein-DNA complexes with AP-1, NF-Y, and Sp1 transcription family members.

Functional Analysis of TATA Box, AP-1, NF-Y, and Sp1 Regulatory Elements—In addition to the AP-1, NF-Y, and Sp1 regulatory elements, the AIRE promoter contains a TATA box located at the position 33 bp upstream of the transcriptional start site. To study the functional significance of the TATA box and transcriptional complexes in the AIRE promoter, mutations were introduced into the pAP1235 luciferase reporter construct using site-directed oligonucleotide mutagenesis and transfected into the COS-7 and TEC1A3 cell lines (Fig. 3). The mutations introduced into the TATA box decreased AIRE promoter activity to 48% in COS-7 and to 69% in TEC1A3 cells, respectively. Mutations in the CCAAT box reduced the promoter activity to 44% in COS-7 cells, compared with 78% in TEC1A3 cells. Nucleotide substitutions within the GC box resulted in 75% activity in COS-7 and 52% in TEC1A3 cells compared with the full-length pAP1235 construct. Similarly, mutations in the AP-1 binding site decreased the promoter activity to 18% in COS-7 and to 35% in TEC1A3 cells. Thus, the results of these experiments showed that the TATA box, the AP-1 binding site, the inverted CCAAT box, and the GC box are functional. Moreover, the factors binding to these sites are essential for the AIRE gene regulation.



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FIG. 3.
Functional analysis of the AIRE-mutated regulatory sequences. Site-directed mutations were introduced into the binding sites of AP-1, NF-Y, and Sp1 transcription factors and to TATA box (black bars, COS-7; light bars, EC1A3). Crosses indicate the mutation sites. Results shown are mean ± S.D. of three experiments performed in triplicate.

 

AIRE Promoter Contains CpG Island and the Activity Is Blocked by in Vitro Methylation—We noted that the region surrounding the AIRE proximal promoter is GC-rich. The CpG dinucleotide distribution within the 1.2-kb genomic fragment was statistically analyzed as described by the method of Gardiner-Garden and Frommer (12). Correspondingly, a 390-bp CpG island was located at the AIRE promoter region, starting from about 300 bp upstream of the translational start site and encompassing the first exon (Fig. 4A).



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FIG. 4.
In vitro methylation inhibits AIRE promoter activity. A, location of the CpG island at the AIRE promoter. The CpG island is marked by a box, and numbers on the x-axis indicate the nucleotide positions relative to the translational start site. B, the pAP1235 AIRE promoter construct, either unmethylated, or methylated with SssI methylase, was transiently transfected into TEC1A3 cells. The transcription activity is shown in relative units of luciferase activity.

 

The effect of in vitro methylation on the promoter of the AIRE gene was tested in a transient expression assay using the pAP1235 promoter construct. After methylation with SssI methylase, which methylates cytosines residues within CpG dinucleotide, the methylated and unmethylated reporter constructs were transfected into TEC1A3 cells and assayed for expression. As a result in vitro methylation completely suppressed AIRE promoter activity compared with the unmethylated promoter construct (Fig. 4B). Similar results were obtained with experiments using the COS-7 cell line (data not shown).

AIRE Promoter Is Less Methylated in the Thymic Epithelial Cell Line—The methylation status of CpG sites in the AIRE promoter region was first studied in TEC1A3 and THP-1 cell line genomic DNA using the methyl-sensitive restriction enzyme SmaI inside of a methylation-insensitive SacI fragment spanning the AIRE promoter. Subsequent hybridization with the AIRE promoter-specific probe revealed uncleaved SacI fragments, suggesting methylated status of the SmaI sites (data not shown).

The DNA from HeLa, TEC1A3, and from two monocyte cell lines, THP-1 and U937, was further analyzed by bisulfite genomic sequencing. Genomic DNA was treated with sodium bisulfite under conditions where cytosines are converted to uracils, while methylated cytosines remain unmodified. The promoter region contained 47 CpG sites, covering a 390-bp fragment of the AIRE promoter. All tested CpG sites showed a heavy methylation pattern in monocytic cell lines (THP-1 and U937 cells, Fig. 5). Results obtained from HeLa cells were consistent with the results from THP-1 and U937 cells, although few CpG sites had a partial methylation pattern. In contrast, in thymus epithelial TEC1A3 cells, 9 CpG sites out of 47 were completely unmethylated and 11 CpG sites possessed a partial methylation pattern. The results indicated a different methylation pattern of AIRE promoter in TEC1A3 cells compared with the other three cell lines.



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FIG. 5.
CpG methylation in the AIRE promoter region (-315 to +65) after bisulfite treatment of genomic DNA. The methylation status of 47 CpG sites in TEC1A3, HeLa, THP-1, and U937 cell lines is shown. Binding sites for AP-1 (-307 to -296), NF-Y (-213 to -202), Sp1 (-202 to -189), and TATA box (-163 to -153) are underlined, and the translation start site (atg) is in boldface.

 

5-AzaCdR and TSA Treatments Up-regulate AIRE Expression—To test the potential role of DNA methylation and chromatin modification in the control of AIRE expression, a demethylating agent 5-azaCdR and a histone deacetylase specific inhibitor, TSA, were used. TEC1A3 cells were grown in the presence of various 5-azaCdR concentrations, and purified RNA was analyzed by quantitative real-time PCR for AIRE expression in three cell lines (TEC1A3, U937, and HeLa). The 5-azaCdR treatment alone with the highest concentration (15 µM) was able to up-regulate AIRE expression levels 4.5 (TEC1A3)-, 26 (U937)-, and 16.5 (HeLa)-fold when compared with the untreated cells (Fig. 6). Lower 5-azaCdR concentrations also yielded lower activation of the AIRE expression. However, the combination of 0.5 µM 5-azaCdR and TSA was even able to further increase the expression of AIRE in TEC1A3 (6.5-fold) and U937 (49.5-fold) cells, although this was not observed in HeLa cells. The activation of AIRE mRNA, although by a lesser extent, was also seen by TSA treatment alone. The results showed that methylation and deacetylase activity inhibition treatments are able to activate the AIRE promoter and that, in TEC1A3 and U937 cells, the combination of the two treatments resulted in higher AIRE transcript expression levels suggesting that both methylation and histone deacetylation are responsible for the silencing of the AIRE gene.



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FIG. 6.
Quantitative analysis of AIRE mRNA levels following treatment with 5-azaCdR and TSA. Real-time quantitative PCR analyses of total RNA isolated from TEC1A3, U937, and HeLa cells treated with various concentrations of 5-azaCdR (0.2, 0.5, 5, and 5 µM) and TSA (100 ng/ml) or in combination. The expression levels of the AIRE transcript were normalized for the expression levels of the GAPDH transcripts in the same cDNA samples.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The highest AIRE expression is seen in thymic epithelial cells (5, 6). Mittaz et al. (17) concluded from human and mouse AIRE promoter sequence analysis that both human and mouse genes contain conserved binding sites for thymus- and hema-topoiesis-specific transcription factors. Analysis of the human AIRE promoter sequence undertaken here did not detect high score binding sites for tissue-specific transcription factors. However, the possibility that tissue-specific transcription factors participate in AIRE transcriptional regulation cannot be excluded, because these factors may interact with other protein complexes binding to AIRE promoter or some transcription factors may bind to the upstream regulatory region. Considering that AIRE controls the expression of other genes, we wanted to elucidate the mechanisms behind the AIRE gene transcriptional control.

The 1235-bp fragment upstream of the initiation codon showed strong promoter activity in COS-7 and in the thymic epithelial cell line TEC1A3 for the expression of reporter gene. The deletional analysis of the 5'-flanking AIRE promoter region demonstrated that the proximal 350-bp region contained all necessary elements for minimal promoter activity, because the pAP350 construct gave comparably similar luciferase results with pAP583, pAP940, and pAP1235 deletion constructs. Strong promoter activity within a relatively short 350-bp upstream region suggested the presence of specific transcription factor binding sites, which was also supported by computer analysis giving high score potential binding sites for transcription factors such as AP-1, NF-Y, and Sp1. We also found that the luciferase expression level in TEC1A3 was consistently lower, being one-third of the expression seen in COS-7 cells. As one explanation, this could be due to the higher metabolic activity of COS-7 cells. Interestingly, however, the luciferase expression of the 248-bp proximal fragment was almost similar in two cell lines suggesting one critical DNA binding element within the -248 to -350 region. The highest scoring element in this region was the AP-1 site suggesting that higher expression of this transcription complex in COS-7 cells might be the reason for the luciferase expression discrepancy in the two cell lines. The further deletion to -190, excluding the CCAAT box, almost abolished promoter activity indicating the role of this site in AIRE promoter regulation.

The EMSA experiments and mutated construct analysis confirmed four important regions for AIRE activity. From these promoter elements, the mutation inside of the AP-1 element had the most dramatic reduction of the promoter activity by decreasing it to 18% (COS-7) and 35% (TEC1A3), clearly demonstrating the responsiveness of AIRE expression from AP-1 complex. The AP-1 complex consists of distinct homodimers or heterodimers composed of various members of the Fos, Jun, and ATF subfamilies (18). Using pan-Jun and pan-Fos antibodies in EMSA, we could show that proteins within the AP-1 complex, binding to the AIRE promoter, belong to the Jun subfamily but not to the Fos transcription factor family. Second, we showed that the CCAAT and GC boxes, located at -212 to -200 bp and -202 to -188 bp, respectively, are important in AIRE activation. The NF-Y transcription factor complex binding to the CCAAT box consists of three subunits, NF-YA, NF-YB, and NF-YC, and all of them are required for efficient DNA binding (19). The interaction between NF-YB and NF-YC allows association of this complex further with NF-YA and subsequent binding to their consensus sequence in DNA (20). Supershift assays with two members of this complex, NF-YA and NF-YB, pointed to the formation of a complete NF-Y complex on the AIRE promoter. Third, AIRE promoter activity is also mediated by the Sp1 transcription complex, because the Sp1-specific antibody interfered with the GC box-shifted complex and because the mutated Sp1 binding site construct had strongly decreased transcriptional activity in the COS-7 and TEC1A3 cell lines. It should be mentioned that two more GC boxes were predicted by computer analysis, albeit with a lower score, at nucleotides -37 to -24 and -131 to -109. Finally, the AIRE promoter contains a canonical vertebrate TATA box allowing for recruitment of a complex containing the TATA-binding protein and RNA polymerase II holoenzyme (21, 22). As expected, the mutations created within the TATA site had significant effect on AIRE promoter activity consistent with its predicted functional role.

The presence of a CpG island suggested the possibility that the AIRE gene might be regulated through changes in methylation status, which was first tested by in vitro methylation of the pAP1235 promoter construct. The four cell lines studied exhibited a methylated CpG pattern by bisulfite sequencing, and this was particularly obvious in DNA from HeLa and monocytic cell lines THP-1 and U937. In TEC1A3 cell DNA, several CpG sites appeared unmethylated or partially methylated. Methylation of the Sp1 site has been shown to cause gene silencing (23, 24), and three CpG sites within the Sp1 binding GC box appeared methylated except in TEC1A3 cells where one of the sites was only partially methylated (Fig. 5). AIRE expression level is highest in thymus epithelial cells (5); therefore, the thymic epithelial cell line represents the closest cell culture model for studying AIRE expression. Treatment with 5-azaCdR alone was able to induce higher AIRE mRNA expression levels in TEC1A3 and U937 cells, and the activation was further enhanced when the cells were treated in combination with deacetylase inhibitor TSA. A similar increase in mRNA level after 5-aza-CdR treatment was also observed in the HeLa cell line; however, the TSA treatment did not influence the expression level, suggesting that chromatin modification in the AIRE promoter region may vary between cell types. Synergistic effect of 5-azaCdR and TSA in enhanced re-expression of several hypermethylated genes was first reported by Cameron et al. (25). The association of CpG methylation and chromatin modifications has been established in experiments showing that the Sin3-histone deacetylase complex is recruited to chromatin by methylated CpG-binding protein, MeCP2 (26, 27).

This is the first study to reveal the functional control of the AIRE promoter. In conclusion, our results demonstrate that the AIRE promoter contains binding sites for three transcription factor complexes, AP-1, NF-Y, and Sp1, and a functional TATA box. In addition, results of methylation analyses show a highly methylated CpG island inside the AIRE promoter in several cell lines, albeit at lower level of methylation in thymic epithelial cell line DNA. Furthermore, an increase of AIRE mRNA was seen when the cells were treated with 5-azaCdR and TSA, indicating that chromatin methylation modifications, most likely combined with histone acetylation state, may have an important role in AIRE transcriptional regulation.


    FOOTNOTES
 
* The work was supported by grants from the Academy of Finland, the Sigrid Juselius Foundation, the Medical Fund of Tampere University Hospital, The Centre for International Mobility, and the World Federation of Scientists. 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. Back

To whom correspondence should be addressed. Tel.: 358-3-215-7754; Fax: 358-3-215-7710; E-mail: part.peterson{at}uta.fi.

1 The abbreviations used are: AIRE, autoimmune regulator promoter; APECED, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy; 5-azaCdR, 5-aza-2'-deoxycytidine; TSA, trichostatin A; EMSA, electrophoretic mobility shift assay; DTT, dithiothreitol; RT, real-time; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back


    ACKNOWLEDGMENTS
 
We acknowledge the excellent technical assistance of Ulla Kiiskinen and support of Prof. Andres Metspalu during the study. We also thank Jukka Pitkänen and John Rowell for their careful reading of the manuscript.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. 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[Medline] [Order article via Infotrieve]
  2. The Finnish-German APECED Consortium (1997) Nat. Genet. 17, 399–403[Medline] [Order article via Infotrieve]
  3. Ahonen, P., Myllärniemi, S., Sipilä, I., and Perheentupa, J. (1990) N. Engl. J. Med. 322, 1829–1836[Abstract]
  4. Gibson, T. J., Ramu, C., Gemund, C., and Aasland, R. (1998) Trends Biochem. Sci. 23, 242–244[CrossRef][Medline] [Order article via Infotrieve]
  5. Heino, M., Peterson, P., Kudoh, J., Nagamine, K., Lagerstedt, A., Ovod, V., Ranki, A., Rantala, I., Nieminen, M., Tuukkanen, J., Scott, H. S., Antonarakis, S. E., Shimizu, N., and Krohn, K. (1999) Biochem. Biophys. Res. Commun. 257, 821–825[CrossRef][Medline] [Order article via Infotrieve]
  6. Heino, M., Peterson, P., Sillanpää, 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[CrossRef][Medline] [Order article via Infotrieve]
  7. Björses, P., Pelto-Huikko, M., Kaukonen, J., Aaltonen, J., Peltonen, L., and Ulmanen, I. (1999) Hum. Mol. Genet. 8, 259–266[Abstract/Free Full Text]
  8. Pitkänen, J., Vähämurto, P., Krohn, K., and Peterson, P. (2001) J. Biol. Chem. 276, 19597–19602[Abstract/Free Full Text]
  9. Pitkänen, J., Doucas, V., Sternsdorf, T., Nakajima, T., Aratani, S., Jensen, K., Will, H., Vähämurto, 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[Abstract/Free Full Text]
  10. Anderson, M. S., Venanzi, E. S., Klein, L., Chen, Z., Berzins, S. P., Turley, S. J., von Boehmer, H., Bronson, R., Dierich, A., Benoist, C., and Mathis, D. (2002) Science 298, 1395–1401[Abstract/Free Full Text]
  11. Ramsey, C., Winqvist, O., Puhakka, L., Halonen, M., Moro, A., Kampe, O., Eskelin, P., Pelto-Huikko, M., and Peltonen, L. (2002) Hum. Mol. Genet. 11, 397–409[CrossRef][Medline] [Order article via Infotrieve]
  12. Gardiner-Garden, M., and Frommer, M. (1987) J. Mol. Biol. 196, 261–282[Medline] [Order article via Infotrieve]
  13. Dignam, J. D., Martin, P. L., Shastry, B. S., and Roeder, R. G. (1983) Methods Enzymol. 101, 582–598[Medline] [Order article via Infotrieve]
  14. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  15. Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W., Molloy, P. L., and Paul, C. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1827–1831[Abstract]
  16. Grunau, C., Clark, S. J., and Rosenthal, A. (2001) Nucleic Acids Res. 29, e65–e77[Abstract/Free Full Text]
  17. Mittaz, L., Rossier, C., Heino, M., Peterson, P., Krohn, K. J., Gos, A., Morris, M. A., Kudoh, J., Shimizu, N., Antonarakis, S. E., and Scott, H. S. (1999) Biochem. Biophys. Res. Commun. 255, 483–490[CrossRef][Medline] [Order article via Infotrieve]
  18. Karin, M., Liu, Z., and Zandi, E. (1997) Curr. Opin. Cell Biol. 9, 240–246[CrossRef][Medline] [Order article via Infotrieve]
  19. Mantovani, R. (1999) Gene (Amst.) 239, 15–27[CrossRef][Medline] [Order article via Infotrieve]
  20. Bi, W., Wu, L., Coustry, F., de Crombrugghe, B., and Maity, S. N. (1997) J. Biol. Chem. 272, 26562–26572[Abstract/Free Full Text]
  21. Kuras, L., and Struhl, K. (1999) Nature 399, 609–613[CrossRef][Medline] [Order article via Infotrieve]
  22. Pugh, B. F. (2000) Gene (Amst.) 255, 1–14[CrossRef][Medline] [Order article via Infotrieve]
  23. Cao, Y. X., Jean, J. C., and Williams, M. C. (2000) Biochem. J. 350, 883–890[CrossRef][Medline] [Order article via Infotrieve]
  24. Kitazawa, S., Kitazawa, R., and Maeda, S. (1999) J. Biol. Chem. 274, 28787–28793[Abstract/Free Full Text]
  25. Cameron, E. E., Bachman, K. E., Myohanen, S., Herman, J. G., and Baylin, S. B. (1999) Nat. Genet. 21, 103–107[CrossRef][Medline] [Order article via Infotrieve]
  26. Jones, P. L., Veenstra, G. J., Wade, P. A., Vermaak, D., Kass, S. U., Landsberger, N., Strouboulis, J., and Wolffe, A. P. (1998) Nat. Genet. 19, 187–191[CrossRef][Medline] [Order article via Infotrieve]
  27. Nan, X., Ng, H. H., Johnson, C. A., Laherty, C. D., Turner, B. M., Eisenman, R. N., and Bird, A. (1998) Nature 393, 386–389[CrossRef][Medline] [Order article via Infotrieve]




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