The mouse Aire gene: comparative genomic sequencing, gene organization, and expression

Abstract

Mutations in the human AIRE gene (hAIRE) result in the development of an autoimmune disease named APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy; OMIM 240300). Previously, we have cloned hAIRE and shown that it codes for a putative transcription-associated factor. Here we report the cloning and characterization of Aire, the murine ortholog of hAIRE. Comparative genomic sequencing revealed that the structure of the AIRE gene is highly conserved between human and mouse. The conceptual proteins share 73% homology and feature the same typical functional domains in both species. RT-PCR analysis detected three splice variant isoforms in various mouse tissues, and interestingly one isoform was conserved in human, suggesting potential biological relevance of this product. In situ hybridization on mouse and human histological sections showed that AIRE expression pattern was mainly restricted to a few cells in the thymus, calling for a tissue-specific function of the gene product.

Figure 1

Comparative genomic organization of the AIRE locus. Exons are represented by solid boxes numbered from 1 to 14. Repetitive elements are depicted by arrowheads. CpG islands are represented by solid boxes. Putative TATA box promoter and conserved region are indicated by arrows.

Figure 2

(A) Dot-matrix comparative analysis of hAIRE and mouse Aire genomic sequences: HSAJ9610 is represented on the x-axis and AF073797 on the y-axis. Arrows denote exons; the arrowhead points to a conserved region located ∼3 kb upstream of the gene. (B) Alignment of the conserved nucleotide sequence identified in A. Numbers at the end of lines indicate nucleotide positions. Consensus sequence is drawn below the alignment. Box corresponds to the core conserved sequence.

Figure 2

(A) Dot-matrix comparative analysis of hAIRE and mouse Aire genomic sequences: HSAJ9610 is represented on the x-axis and AF073797 on the y-axis. Arrows denote exons; the arrowhead points to a conserved region located ∼3 kb upstream of the gene. (B) Alignment of the conserved nucleotide sequence identified in A. Numbers at the end of lines indicate nucleotide positions. Consensus sequence is drawn below the alignment. Box corresponds to the core conserved sequence.

Figure 3

Mapping of Aire to mouse Chromosome 10. Electrophoresis of PCR amplification of mouse DNAs with primers Mforw2 and Mrev32.(Lane 1) Hybrid SN17C3 (Chr. 10), (Lane 2) SN11CS3 (chr. 3); (Lane 3) EJ167 (chr. 3+17); (Lane 4) mouse genomic DNA; (Lane 5) human genomic DNA; (Lane 6) no DNA. (M) 100-bp ladder (Life Technologies).

Figure 4

Amino acid aligment of hAIRE and mouse AIRE proteins. The LXXLL motif is shown by an open box; the nuclear localization signal is underlined; the SAND domain is shown by a broken line. Shaded boxes indicate the PHD zinc fingers.

Figure 5

Differential splicing of Aire transcripts. (A) Deletion of exon 10. Sequence is reversed as indicated 3′ → 5′; (B) deletion of 3 nucleotides at the start of exon 8; (C) deletion of 12 nucleotides at the end of exon 6. Sequence is reversed as indicated 3′ → 5′.

Figure 5

Differential splicing of Aire transcripts. (A) Deletion of exon 10. Sequence is reversed as indicated 3′ → 5′; (B) deletion of 3 nucleotides at the start of exon 8; (C) deletion of 12 nucleotides at the end of exon 6. Sequence is reversed as indicated 3′ → 5′.

Figure 5

Differential splicing of Aire transcripts. (A) Deletion of exon 10. Sequence is reversed as indicated 3′ → 5′; (B) deletion of 3 nucleotides at the start of exon 8; (C) deletion of 12 nucleotides at the end of exon 6. Sequence is reversed as indicated 3′ → 5′.

Figure 6

Expression of Aire at 14.5 dpc is restricted to few cells in the thymus. RNA in situ hybridization with Aire antisense riboprobe recognizing exons 1–7. No signal was detected upon hybridization with a sense probe. (A) Sagittal section through 14.5 dpc. mouse embryo, counterstained with eosin. (B) Transverse section of thymic lobes of a 14.5 dpc. embryo. (C) Sagittal section of 14.5 dpc. thymus, counterstained with eosin. (D) Sagittal section of 14.5 dpc. thymus, counterstained with hematoxylin and eosin, at higher magnification. Arrows point to single cells or cell groups expressing Aire.

Figure 7

Expression of hAIRE in human juvenile thymus sections counterstained with Giemsa. Expression is restricted to a few cells in the medulla of the thymic lobule. (Top) The antisense probe; (middle) the sense probe; (bottom) the control β-actin probe. (Left) Bright field; (right) dark field. (A) Magnification, 100×; (B) another section with magnification, 500×.

Figure 7

Expression of hAIRE in human juvenile thymus sections counterstained with Giemsa. Expression is restricted to a few cells in the medulla of the thymic lobule. (Top) The antisense probe; (middle) the sense probe; (bottom) the control β-actin probe. (Left) Bright field; (right) dark field. (A) Magnification, 100×; (B) another section with magnification, 500×.