Abstract
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.
Highlights
Mutations in the autoimmune regulator (AIRE)1 gene cause an autosomal recessive disease called APECED [1, 2]
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
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
Summary
HeLa and TEC 1A3 (human thymic epithelial cell line) cells were maintained in Eagle’s minimum essential medium. Isolation of AIRE Promoter—A 1.2-kb genomic fragment containing the 5Ј-end region of the AIRE gene was isolated by PCR amplification from human genomic DNA. The promoter region in the pAP1235 construct was verified by sequencing. 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. A CpG-rich region is defined as stretches of DNA in which both
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
