Characterization of regulatory elements and methylation pattern of the autoimmune regulator (AIRE) promoter.
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.
Murumägi
Astrid
A
Institute of Medical Technology, University of Tampere and Department of Pathology, Tampere University Hospital, Lenkkeilijänkatu 6, Finland.
Vähämurto
Perttu
P
Peterson
Pärt
P
D001483:Base Sequence; D002460:Cell Line; D018899:CpG Islands; D019175:DNA Methylation; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D016385:TATA Box; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D000096927:AIRE Protein
D017931:DNA Primers; D006877:Hydroxamic Acids; D014157:Transcription Factors; D000096927:AIRE Protein; C012589:trichostatin A