Functional analyses of the mutation nt-128 TG in the hepatocyte nuclear factor-1α promoter region in Chinese diabetes pedigrees
Article first published online: 7 OCT 2012
© 2012 The Authors. Diabetic Medicine © 2012 Diabetes UK
Volume 29, Issue 11, pages 1456–1464, November 2012
How to Cite
Fang, Q., Chen, S., Wang, Y., Jiang, S., Zhang, R., Hu, C., Wang, C., Liu, F., Xiang, K. and Jia, W. (2012), Functional analyses of the mutation nt-128 TG in the hepatocyte nuclear factor-1α promoter region in Chinese diabetes pedigrees. Diabetic Medicine, 29: 1456–1464. doi: 10.1111/j.1464-5491.2012.03626.x
- Issue published online: 7 OCT 2012
- Article first published online: 7 OCT 2012
- Accepted manuscript online: 13 MAR 2012 06:25AM EST
- Accepted 6 March 2012
Aims Hepatocyte nuclear factor-1α (HNF-1α) regulates the expression of genes encoding proteins involved in glucose metabolism and insulin secretion. Mutations in the HNF-1α gene cause maturity-onset diabetes of the young Type 3. However, the mechanism leading to this disease has not been completely ascertained. Previously, we found a novel mutation in the regulatory element of the human HNF-1α gene in two Chinese diabetes pedigrees. The nucleotide at position -128 T was substituted by G (nt-128 TG). In this study, we analysed the functional defect of nt-128 TG in HNF-1α transcription activity.
Methods Luciferase reporter gene assays were carried out to examine the functional characteristics of this mutant. Electrophoretic mobility shift assays and chromatin immunoprecipitation were performed to confirm the binding of nuclear proteins to oligonucleotides.
Results The variant construct (nt-128 TG) had a 1.65-fold increase in promoter activity compared with that of the wild-type construct in HepG2 cells and a 1.33-fold increase in MIN6 cells, respectively. The variant resided at a FOXA/HNF-3 binding site identified by a series of competitive electrophoretic mobility shift assays and antibody supershift analyses. The assays showed a differential binding affinity in the wild-type and the nt-128 TG mutant fragments by FOXA/HNF-3. Chromatin immunoprecipitation indicated that FOXA/HNF-3 bound to this region in vivo. One nucleotide substitution in the FOXA/HNF-3 site in the human HNF-1α regulatory element caused an increase of HNF-1α transcriptional activity.
Conclusions Our data suggested that this substitution in the promoter region affects DNA–protein interaction and HNF-1α gene transcription. The mutant may contribute to the development of diabetes in these two nt-128 TG pedigrees of Chinese.