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. Type 1 diabetes (T1D)2 is defined by absolute loss of insulin secretion due to the autoimmune destruction of insulin-producing ␤ cells in the pancreas. There are a few target autoantigens identified in ␤ cell autoimmune destruction with insulin and pro-insulin auto-antibodies detected in 23 and 34% of T1D patients, respectively (1, 2). The autoimmune polyendocrinopathy type 1 syndrome (APS-1 also known as APECED) is an autosomal recessive, monogenic form of human autoimmunity that is characterized by autoimmune destruction of multiple endocrine organs and defective cell-mediated immunity (3, 4).In 1997 an autoimmune regulator (AIRE) gene, which underlies APS-1, was identified on chromosome 21q22.3 using a positional cloning strategy (5, 6). The specific domains that the AIRE protein contains include (i) the N terminus of a homogenously staining region (HSR) domain and/or caspase recruitment domain (CARD), which is believed to be involved in dimerization and/or caspase recruitment (7, 8), (ii) a nuclear localization signal, (iii) a SAND (Sp100, AIRE-1, NucP41/75, DEAF-1) domain, which is thought to be involved in DNA binding (9), and (iv), two plant homeodomain (PHD) type zinc fingers (10, 11) and four LXXLL motifs (12). The AIRE protein has been shown to activate hundreds of tissue-restricted anti...

Association of common type 1 and type 2 diabetes gene variants with latent autoimmune diabetes in adults: A meta‐analysis

“…The INS gene plays a vital role in the process of insulin secretion and metabolism . Evidence suggests that polymorphism in the INS VNTR locus increases the risk of T1D or LADA by affecting the expression of INS in the thymus, thereby resulting in insulin autoimmunity . Therefore, we performed a meta‐analysis of the INS rs689 polymorphism using nine studies to determine its association with LADA.…”

“…35,44,45 The period of insulin independence in LADA patients could be due to the slower destruction of insulin-producing 46,47 Evidence suggests that polymorphism in the INS VNTR locus increases the risk of T1D or LADA by affecting the expression of INS in the thymus, thereby resulting in insulin autoimmunity. 15,48 Therefore, we performed a meta-analysis of the INS rs689 polymorphism using nine studies to determine its association with LADA. The analysis revealed a significant inverse association between INS and LADA in all genetic models.…”

Antioxidant, Anti-Inflammatory, and Immunomodulatory Properties of Tea—The Positive Impact of Tea Consumption on Patients with Autoimmune Diabetes

“…15% of the genetic predisposition for developing type 1 diabetes is attributed to the insulin promotor (insulinlinked variable number of tandem repeats INS-VNTR, chromosome 1p5,511p15), the cytotoxic T-Lymphocyte Antigen-4 receptor (CTLA-4, chromosome 2q33), protein tyrosine phosphatase N22 (PTPN2), and the immune signaling regulator (PTPN22, chromosome 1p13) as well as other genes [28,29]. INS-VNTR polymorphism is responsible for the expression of the insulin gene not only in the pancreas, but also thymus [29]. Mutation or polymorphism of the CTLA-4 gene leads to an uncontrolled proliferation response, which can be the cause of autoimmune diseases including type 1 diabetes [30].…”

Novel Findings into AIRE Genetics and Functioning: Clinical Implications

“…Moreover, Sparks et al identified additional dominant-negative AIRE mutations 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 ( 51 ).…”