The Autoimmune Regulator (AIRE) Is a DNA-binding Protein

Abstract

The autoimmune regulator (AIRE) protein is a putative transcription regulator with two plant homeodomain-type zinc fingers, a putative DNA-binding domain (SAND), and four nuclear receptor binding LXXLL motifs. We have shown here that in vitro, recombinant AIRE can form homodimers and homotetramers that were also detected in thymic protein extracts. Recombinant AIRE also oligomerizes spontaneously upon phosphorylation by cAMP dependent protein kinase A or protein kinase C. Similarly, thymic AIRE protein is phosphorylated at the tyrosine and serine/threonine residues. AIRE dimers and tetramers, but not the monomers, can bind to G-doublets with the ATTGGTTA motif and the TTATTA-box. Competition assays revealed that sequences with one TTATTA motif and two tandem repeats of ATTGGTTA had the highest binding affinity. These findings demonstrate that AIRE is an important DNA binding molecule involved in immune regulation.Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), 1 also known as autoimmune polyglandular syndrome type 1 (APS1), is a rare autosomal recessive disorder common in isolated populations such as Finns, Sardinians, and Iranian Jews (1). This syndrome is characterized by destructive autoimmune diseases of the endocrine organs, chronic candidiasis of mucous membranes, and ectodermal disorders. APECED is caused by mutations in the autoimmune regulator (AIRE) gene on chromosome 21q22.3 (2-4). The AIRE gene has recently been cloned by two independent groups of investigators (5, 6). The AIRE gene consists of 14 exons coding for a 2445-base pair mRNA transcript, and the translated product is expected to have 545 amino acids with a predicted molecular mass of 57.5 kDa. The predicted AIRE protein has several domains indicative of a transcriptional regulator protein (6). AIRE harbors two zinc fingers of plant homeodomain (PHD) type. A putative DNA binding domain named SAND as well as four nuclear receptor binding LXXLL motifs, an inverted LXXLL domain, and a variant of the latter (FXXLL) hint that this protein functions as a transcription coactivator (5-7). Furthermore, a highly conserved N-terminal 100-amino acid domain in AIRE has a significant homology to the homogenously staining (HSR) domain of Sp100 and Sp140 proteins (7). This domain has been shown to function as a dimerization domain in several Sp-100 related proteins (8). At the subcellular level, AIRE can be found in the cell nucleus in a speckled pattern in domains resembling promyelocytic leukemia nuclear bodies, also known as ND10, nuclear dots, or potential oncogenic domains, associated with the AIRE homologous nuclear proteins Sp100, Sp140, and Lysp100 (9).Interestingly, it has recently been shown that AIRE can activate transcription from a reporter gene when fused to a heterologous DNA binding domain. This activation required the full-length protein or the presence of more than one activation domain. A glutathione S-transferase pull-down assay showed that AIRE formed homodimers in vitro, probably through the N-terminal domain (...

Aire employs a histone-binding module to mediate immunological tolerance, linking chromatin regulation with organ-specific autoimmunity

“…S3A Lower). This effect of poly(dI-dC), and the absence of previously reported Aire-specific motifs (26,27) in the DNA used in our EMSAs (28), suggest a substantial non-sequencedependent component to DNA binding, and raise questions about the importance of sequence specificity to the overall binding. Although we describe here the direct interaction between Aire and nucleosomes, we turned to more sensitive assays to probe Aire-chromatin interactions in vivo, because EMSA analyses cannot resolve small micromolar differences potentially contributed by H3 tail binding.…”

AIRE Recruits P-TEFb for Transcriptional Elongation of Target Genes in Medullary Thymic Epithelial Cells

“…Of note, activators found on enhancers, such as CIITA, NF-B, steroid hormone receptors, and c-Myc, all bind and recruit P-TEFb to their transcription units (reviewed in reference 35). An important difference is that unlike these type IIB activators, AIRE cannot initiate transcription and its interactions with DNA appear more promiscuous (24,39). Thus, it is able to interact with more targets but has a greater requirement for a preassembled PIC.…”

“…Monomers of AIRE neither bind DNA in vitro (24,39) nor activate transcription in cells (14). Of interest, most mutations in patients disrupt this oligomerization of AIRE (14,32).…”

“…When fused to a heterologous DNA-binding domain, AIRE can activate transcription in transient-expression assays (37). Because it binds preferentially to GG repeats and AT-rich sequences (24,39), its interactions with DNA could be rather promiscuous (reviewed in references 28 and 36). The first PHD may also function as an E3 ubiquitin ligase (47).…”

Ovarian autoimmune disease: clinical concepts and animal models

“…74 Briefly, AIRE was originally considered a transcription factor based on its nuclear localization, it ability to bind DNA and the presence of a SAND (Sp100, AIRE-1, NucP41/75, DEAF-1) functional domain. [75][76][77] However, AIRE also contains two PHD (Plant HomeoDomain)-type zinc fingers that confer its ability to associate with numerous protein partners and that are essential for regulation of gene expression. [78][79][80] These PHD domains associate with histone 3 at unmethylated lysine 4 residues (H3K4me0), which are typically within transcriptionally inactive chromatin regions, and in combination with DNA-dependent protein kinase, appear to promote subsequent transcription.…”