From the
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of AIRE PromoterA 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.
|
Computer AnalysisTranscription 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 PlasmidsThe 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.
|
Transient Transfections and Luciferase Reporter AssaysCells (1.5 x 105) were transiently transfected with 2.5 µg of DNA (2 µg of luciferase reporter plasmid, 0.5 µg of -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
-galactosidase activities. The transfections were performed in triplicate.
Electrophoretic Mobility Gel Shift AssayNuclear 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 MethylationSssI 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 IsolationGenomic 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 SequencingBisulfite 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 TreatmentDNA 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 PCRReal-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 PromoterAnalysis 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).
|
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 ElementsIn 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.
|
AIRE Promoter Contains CpG Island and the Activity Is Blocked by in Vitro MethylationWe 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).
|
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 LineThe 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.
|
5-AzaCdR and TSA Treatments Up-regulate AIRE ExpressionTo 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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
¶ 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.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
All ASBMB Journals | Molecular and Cellular Proteomics |
Journal of Lipid Research | Biochemistry and Molecular Biology Education |