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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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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 -Renilla luciferase reporter vector.
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Figure 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 -Renilla luciferase reporter vector.

Mentions: In the presence of AIRE, the insulin basal promoter alone (−365 bp) displays relatively low activity in the human TEC and rat primary TEC (Fig. 5A and B). The class I VNTR is two to threefold higher relative to the insulin basal promoter activity, whereas the class III VNTR increases seven to ninefold over the insulin basal promoter activity, suggesting that the AIRE transcriptional regulator could activate the INS-VNTR sequence in the context of the insulin basal promoter. To validate the interaction between the AIRE protein and the VNTR sequence, we performed a DNA-protein pull-down experiment using Adenovirus-AIRE transduced hTEC cell lysate and radiolabeled DNA probes (50,000 cpm) derived from the insulin basal promoter, class I-VNTR-INS-basal promoter, and class III-VNTR-INS-basal promoter sequences (Fig. 5C). Both class I and III DNA probes were pulled-down by the anti-AIRE antibody. A very weak signal, if any, was detected with the insulin basal promoter probe. This result suggests that AIRE protein can interact directly or indirectly through the recruitment of a transcriptional complex to bind the VNTR sequences.


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)

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 -Renilla luciferase reporter vector.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 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 -Renilla luciferase reporter vector.
Mentions: In the presence of AIRE, the insulin basal promoter alone (−365 bp) displays relatively low activity in the human TEC and rat primary TEC (Fig. 5A and B). The class I VNTR is two to threefold higher relative to the insulin basal promoter activity, whereas the class III VNTR increases seven to ninefold over the insulin basal promoter activity, suggesting that the AIRE transcriptional regulator could activate the INS-VNTR sequence in the context of the insulin basal promoter. To validate the interaction between the AIRE protein and the VNTR sequence, we performed a DNA-protein pull-down experiment using Adenovirus-AIRE transduced hTEC cell lysate and radiolabeled DNA probes (50,000 cpm) derived from the insulin basal promoter, class I-VNTR-INS-basal promoter, and class III-VNTR-INS-basal promoter sequences (Fig. 5C). Both class I and III DNA probes were pulled-down by the anti-AIRE antibody. A very weak signal, if any, was detected with the insulin basal promoter probe. This result suggests that AIRE protein can interact directly or indirectly through the recruitment of a transcriptional complex to bind the VNTR sequences.

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