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

Cai CQ, Zhang T, Breslin MB, Giraud M, Lan MS - Diabetes (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Research Institute for Children, Children’s Hospital, New Orleans, Louisiana, USA.

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.

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Related in: MedlinePlus

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.
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Figure 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.

Mentions: IDDM2 or INS is one of the multiple loci that encode susceptibility to type 1 diabetes, a prototype of human autoimmunity (10,22–24). INS-VNTR situated 5′-upstream of the INS promoter, is regarded as a causal variant at the locus after a close correlation was found between VNTR haplotypes and insulin thymic expression (7,8). However, comprehensive resequencing of the locus revealed two polymorphisms that are potentially functional and genetically indistinguishable from the VNTR (25–27). Therefore, direct evidence is required to support that the VNTR allele controls insulin transcript expression in the human thymus. To establish a functional role of the VNTR in the differential thymic insulin gene expression, six individual human genomic DNA samples were genotyped using the PstI+ polymorphism. The PstI+ (uncut by PstI) polymorphism is in strong linkage disequilibrium with the diabetes-predisposing class I allele and PstI− (cut by PstI) with diabetes-protective class III VNTR haplotypes (10). The insulin gene locus at 11p15.5 spanning the VNTR region was PCR amplified and digested with PstI to determine the haplotype. Class I homozygous individuals will display a single 290 bp band; class III homozygous individuals will display two bands 200 and 90 bp; and class I/III heterozygous individuals display three bands 290, 200, and 90 bp in size. Of the six DNA samples analyzed, samples TD and KE are heterozygous class I/III and the other four are homozygous class I (Fig. 1A). To determine the relative repeat number of each individual DNA sample, a NcoI/BglII double restriction endonuclease digest was performed. Each sample contains a 450, 230, and 100 bp band as shown in the schematic diagram in Fig. 1C. Using the approximate size of the class I VNTR band, the number of repeat units was estimated (Fig. 1D). Finally, sequence analysis revealed the exact number of repeat units contained in each of the unique human DNA samples and are shown according to the convention established by Owerbach and Gabbary (24) (Fig. 1E).


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

Cai CQ, Zhang T, Breslin MB, Giraud M, Lan MS - Diabetes (2010)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 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.
Mentions: IDDM2 or INS is one of the multiple loci that encode susceptibility to type 1 diabetes, a prototype of human autoimmunity (10,22–24). INS-VNTR situated 5′-upstream of the INS promoter, is regarded as a causal variant at the locus after a close correlation was found between VNTR haplotypes and insulin thymic expression (7,8). However, comprehensive resequencing of the locus revealed two polymorphisms that are potentially functional and genetically indistinguishable from the VNTR (25–27). Therefore, direct evidence is required to support that the VNTR allele controls insulin transcript expression in the human thymus. To establish a functional role of the VNTR in the differential thymic insulin gene expression, six individual human genomic DNA samples were genotyped using the PstI+ polymorphism. The PstI+ (uncut by PstI) polymorphism is in strong linkage disequilibrium with the diabetes-predisposing class I allele and PstI− (cut by PstI) with diabetes-protective class III VNTR haplotypes (10). The insulin gene locus at 11p15.5 spanning the VNTR region was PCR amplified and digested with PstI to determine the haplotype. Class I homozygous individuals will display a single 290 bp band; class III homozygous individuals will display two bands 200 and 90 bp; and class I/III heterozygous individuals display three bands 290, 200, and 90 bp in size. Of the six DNA samples analyzed, samples TD and KE are heterozygous class I/III and the other four are homozygous class I (Fig. 1A). To determine the relative repeat number of each individual DNA sample, a NcoI/BglII double restriction endonuclease digest was performed. Each sample contains a 450, 230, and 100 bp band as shown in the schematic diagram in Fig. 1C. Using the approximate size of the class I VNTR band, the number of repeat units was estimated (Fig. 1D). Finally, sequence analysis revealed the exact number of repeat units contained in each of the unique human DNA samples and are shown according to the convention established by Owerbach and Gabbary (24) (Fig. 1E).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Research Institute for Children, Children’s Hospital, New Orleans, Louisiana, USA.

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.

Show MeSH
Related in: MedlinePlus