Functional Domains of Autoimmune Regulator (AIRE) Modulate INS-VNTR Transcription in Human Thymic Epithelial Cells

Abstract

INS-VNTR (insulin-variable number of tandem repeats) and AIRE (autoimmune regulator) have been associated with the modulation of insulin gene expression in thymus, which is essential to induce either insulin tolerance or the development of insulin autoimmunity and type 1 diabetes. We sought to analyze whether each functional domain of AIRE is critical for the activation of INS-VNTR in human thymic epithelial cells. Twelve missense or nonsense mutations in AIRE and two chimeric AIRE constructs were generated. A luciferase reporter assay and a pulldown assay using biotinylated INS-class I VNTR probe were performed to examine the transactivation and binding activities of WT, mutant, and chimeric AIREs on the INS-VNTR promoter. Confocal microscopy analysis was performed for WT or mutant AIRE cellular localization. We found that all of the AIRE mutations resulted in loss of transcriptional activation of INS-VNTR except mutant P252L. Using WT/mutant AIRE heterozygous forms to modulate the INS-VNTR target revealed five mutations (R257X, G228W, C311fsX376, L397fsX478, and R433fsX502) that functioned in a dominant negative fashion. The LXXLL-3 motif is identified for the first time to be essential for DNA binding to INS-VNTR, whereas the intact PHD1, PHD2, LXXLL-3, and LXXLL-4 motifs were important for successful transcriptional activation. AIRE nuclear localization in the human thymic epithelial cell line was disrupted by mutations in the homogenously staining region domain and the R257X mutation in the PHD1 domain. This study supports the notion that AIRE mutation could specifically affect human insulin gene expression in thymic epithelial cells through INS-VNTR and subsequently induce either insulin tolerance or autoimmunity.

Keywords: AIRE; VNTR; gene regulation; insulin; insulin autoimmunity; protein domain; thymus; transcription; type 1 diabetes.

FIGURE 1.

Twelve AIRE mutants derived from APS-1 patients. The location of 12 mutations (above the chart) scattered around all four functional domains of AIRE protein was constructed in a mammalian expression vector. The chart displays each mutation that corresponds to the functional domain. The transfected and expressed WT and mutant AIREs in HEK-293 cells were shown by anti-AIRE Western blot analysis. Actin was shown as the loading control. The AIRE mRNA expression was shown by RT-PCR with GAPDH as an internal control.

FIGURE 2.

Transcriptional activities of INS-class III-VNTR-luciferase assay using WT or mutant AIRE in hTEC. A, each mutant or WT AIRE cDNA (as homozygous AIRE) was co-transfected with the INS-class III-VNTR reporter vector into hTEC cells. TK-Renilla was used as an internal control. The 12 mutants' transcriptional activities were normalized with TK-Renilla for transfection efficiency and calculated relative to the WT AIRE (as 100%). The relative activity versus WT AIRE showed high significance with a p value of <0.01 for P252L and P326Q and a p value of <0.001 for the remaining mutants. B, equal amount of each mutant mixed with WT AIRE (as heterozygous AIRE) were co-transfected with INS-class III-VNTR reporter vector as above and presented as calculated relative luciferase activity to WT/WT (as 100%). The p value shows: *, <0.05; **, <0.01: ***, <0.001. C, the hTECs were infected with either Ad-LacZ or Ad-AIRE or transfected with CMV-INS cDNA for 3 days. The AIRE and the endogenous human insulin gene expression were measured using RT-PCR and real time PCR.

FIGURE 3.

Biotin-labeled INS-VNTR probe pulldown assay. A, Western blot of WT or mutant AIRE transfected HEK-293 cell lysate with anti-AIRE or actin antibody shows AIRE protein expression in transfected cells. B, a biotin-labeled INS-VNTR probe and an unrelated probe were generated by PCR amplification. An anti-biotin dot blot (DB) of INS-VNTR and unrelated probes are shown underneath. C, anti-AIRE antibody Western blot analysis of biotin-INS-VNTR pulled-down complexes. The WT or mutant AIRE-transfected HEK-293 cell lysate was mixed with biotin-INS-class I-VNTR probe or biotin-unrelated probe separately at 4 °C overnight. The DNA-protein complex was pulled down by streptavidin beads, washed, and separated by SDS-PAGE. An anti-AIRE Western blot analysis revealed WT or mutant AIRE binding ability to the biotin-INS-class I-VNTR probe. D, anti-AIRE antibody Western blot analysis of unrelated biotin-probe pulldown.

FIGURE 4.

Dominant negative AIRE mutants do not affect WT AIRE binding. A, heterozygous WT and R257X, C311fs, L397fs, or R433fs were co-transfected into HEK293 cells and showed as Western blot analysis. B, the WT and mutant AIRE lysates were subjected to biotin-labeled INS-VNTR or control probe pulldown assay.

FIGURE 5.

LXXLL-3 motif is essential for INS-VNTR binding, and PHD1, LXXLL-3, PHD2, and LXXLL-4 motifs are critical for transcriptional activation. A, chimeric AIRE R257-LXXLL-3 (R257-L3) composed of amino acids 1–257 and 397–433 and chimeric R257-LXXLL-3-PHD2 (R257-L3-P2) composed of amino acids 1–257 and 397–545. B, Western blot analysis of R257X mutant and the chimeric AIREs (R257X-L3 and R257-L3-P2) to confirm the expression of AIRE proteins (left). Biotin-labeled INS-class I-VNTR probe pulldown assay was performed to show whether the chimeric AIRE can restore binding activity (right). C, the WT, mutant, and chimeric AIREs are subjected to an assay for transcriptional activity of INS-class III-VNTR reporter in hTEC. Renilla was used as an internal control. Chimeric AIRE transcriptional activities were calculated relative to the WT AIRE (as 100%). The significance of mutant and chimeric AIRE versus WT AIRE has a p value of <0.001 (***).

FIGURE 6.

Cellular localization and nuclear pattern study of mutated AIRE in hTEC. The WT or mutated AIRE proteins were expressed in hTEC by transient transfection for 2 days, fixed, treated with anti-AIRE antibody, and stained with Alexa Fluor 488 secondary antibody. The nucleus of the cell is counterstained with DAPI.