Both polymorphic variable number of tandem repeats and autoimmune regulator modulate differential expression of insulin in human thymic epithelial cells

Abstract

Objective: Polymorphic INS-VNTR plays an important role in regulating insulin transcript expression in the human thymus that leads to either insulin autoimmunity or tolerance. The molecular mechanisms underlying the INS-VNTR haplotype-dependent insulin expression are still unclear. In this study, we determined the mechanistic components underlying the differential insulin gene expression in human thymic epithelial cells, which should have profound effects on the insulin autoimmune tolerance induction.

Research design and methods: A repetitive DNA region designated as a variable number of tandem repeats (VNTR) is located upstream of the human insulin gene and correlates with the incidence of type 1 diabetes. We generated six class I and two class III VNTR constructs linked to the human insulin basal promoter or SV40 heterologous promoter/enhancer and demonstrated that AIRE protein modulates the insulin promoter activities differentially through binding to the VNTR region.

Results: Here we show that in the presence of the autoimmune regulator (AIRE), the class III VNTR haplotype is responsible for an average of three-fold higher insulin expression than class I VNTR in thymic epithelial cells. In a protein-DNA pull-down experiment, AIRE protein is capable of binding to VNTR class I and III probes. Further, the transcriptional activation of the INS-VNTR by AIRE requires the insulin basal promoter. The VNTR sequence loses its activation activity when linked to a heterologous promoter and/or enhancer.

Conclusions: These findings demonstrate a type 1 diabetes predisposition encoded by the INS-VNTR locus and a critical function played by AIRE, which constitute a dual control mechanisms regulating quantitative expression of insulin in human thymic epithelial cells.

FIG. 1.

Six novel class I VNTR alleles and the restriction profile of the class I and III alleles of the constructs. A: Six human genomic DNA samples were genotyped using the +1,127 PstI polymorphism. PstI⁺ (uncut by PstI) and PstI⁻ (cut by PstI) variants are in strong linkage disequilibrium with diabetic class I and nondiabetic class III VNTR haplotypes, respectively (10). By designing a pair of primers (5′-TAAATGCAGAAGCGTGGCATTGTGGAAC-3′ and 5′-CTGCATGCTGGGCCTGGCCGG-3′), the PCR products amplified from genomic DNA were digested with PstI to generate a single band of 290 bp from class I homozygotes; two bands of 200 bp and 90 bp from class III homozygotes; and three bands of 290 bp, 200 bp, and 90 bp from I/III heterozygotes. TD and KE are heterozygous (I/III) and the other four are homozygous class I. PstI + and − are control DNA of class I and class III homozygotes, respectively. B: Restriction profile of the VNTR alleles contained in the reporter gene constructs. NcoI and BglII were used. C: Schematic explanation for the profile shown on the gel B or quantitated in table D. The band sizes are indicated by base pairs and marked by N (NcoI) and B (BglII); 230 + VNTR in C corresponds to VNTR in B or restriction profile in D. D: Estimation of the sizes of individual VNTR alleles. E: Sequence of six class I VNTR alleles in the format of repeat unit array and according to the convention set by previous reference (24). The number of repeat units is shown to the right of each sequence.

FIG. 2.

Pancreas-based insulin expression pattern associated with the IDDM2-VNTR locus. In comparison with the activity in thymus, the pancreatic insulin expression pattern, represented by the luciferase reporter activity, was confirmed in a HIT cell line (A and B). An average of 1.58-fold increase was observed in reporter activities driven by class I over class III VNTR haplotypes. The class I activity was expressed as an average of six constructs. The class III activity was expressed as an average of three experiments from one construct. The constructs assayed are the same set of reporter constructs as those used in the hTEC line or rat primary cells in Fig. 3. Both class I and III reporter constructs respond to the addition of AIRE similarly.

FIG. 3.

IDDM2-VNTR locus transduces a quantitative insulin expression signal in the presence of AIRE. A: Diagrammatic representation of the TH-INS-IGF2 interval on chromosome 11p15.5. INS or IDDM2 locus is bordered by TH (tyrosine hydroxylase) and IGF2 (insulin-like growth factor 2) loci. The three polymorphisms are designated by their positions with respect to the first base of the INS start codon that is represented by a solid triangle and marked as +1. The allelic variations at the VNTR begin at −603, positioned immediately before the basal promoter. The average length of a class I VNTR is ∼0.5 kb among the alleles we cloned and the class III used is ∼2.0 kb. A gray arrow depicts the basal insulin promoter region (365 bp) and open boxes denote the three insulin exons. The black bold lines represent the regions cloned into the luciferase reporter constructs including the VNTR, its 5′-flanking 1.1 kb region, the basal insulin promoter, and excluding the other two polymorphisms. The longer version bearing the 5′-flanking 1.1 kb in addition to the VNTR region and basal promoter construct, as well as a shorter construct without the 1.1 kb 5′-flanking region, were analyzed. B: A luciferase reporter gene is driven by class I or class III VNTR with insulin basal promoter (50 ng) and transfected in a hTEC line. Six class I and one class III haplotypes were tested. The relative activities both without (β-gal control) and with exogenous AIRE (50 ng) are shown. The class I activity was expressed as an average of six constructs. The class III activity was expressed as an average of three experiments from one construct. C: Detailed activities of each individual VNTR allele in the presence of AIRE were tabulated. D and E: The same set of constructs was assayed using rat primary thymic epithelial cells following the same strategy as in B and C. F and G: To rule out the possibility that the upstream 5′-flanking genomic region (∼1.1 kb) might contribute to the differential insulin expression in thymus, another set of constructs was made by removing this region (A) and assaying in the hTEC line. The average difference in thymic expression is consistently a threefold increase of class III over I VNTR haplotypes. H: Western blot analysis to verify AIRE expression in the transfected cell line. As a loading control, α-actin was used.

FIG. 4.

VNTR responded sensitively and differentially to AIRE in a human thymic epithelial cell line. A: The VNTR responded to AIRE in a hTEC line. A class I VNTR (PQ) and a class III VNTR reporter construct (50 ng) were cotransfected with AIRE expression vector or control vector β-gal DNA into a hTEC line. The amount of AIRE expression vector added to each sample of a 24-well plate format is indicated. B: Western blot analysis was performed to confirm that the increasing expression of AIRE protein corresponded to the amounts of added expression vector. Equal loading of each sample was confirmed by incubation with an anti-α-actin antibody.

FIG. 5.

AIRE regulates the insulin promoter activity mediated through the INS-VNTR element. A and B: Insulin basal promoter, VNTR-class I-INS-basal and VNTR-class III-INS-basal vectors demonstrated differential promoter activities in both hTEC and rat primary TEC in response to AIRE. C: DNA probes derived from insulin basal promoter (0.4 kb), VNTR-class I-INS-basal (2.0 kb), and VNTR-class III-INS-basal (3.5 kb) were radiolabeled with T4 polynucleotide kinase and γ-³²P-ATP as shown in the input lanes. An Ad-AIRE transduced hTEC total lysate (100 μg) was mixed with 50,000 cpm of each radiolabeled DNA probe for 6 h at room temperature and subsequently added 1 μg of goat anti-AIRE antibody for an overnight incubation. The immune complex was precipitated by salmon sperm DNA-treated protein G-agarose beads, washed, and eluted from beads for separation. Normal goat IgG was used as a negative control. The immunoprecipitated AIRE complexes were verified using an anti-AIRE antibody. D: VNTR linked to a heterologous promoter failed to respond to AIRE. The insulin basal promoter was replaced by a SV40 promoter in a reporter construct. Transfection of the construct lacking the insulin basal promoter with the AIRE expression vector in human TEC was measured for luciferase reporter activity. E: Addition of a SV40 enhancer to the construct greatly enhanced SV40 promoter activity, but the class I or III VNTR had no effect on the promoter/enhancer combination. Transfection efficiency was normalized with the null-Renilla luciferase reporter vector.