The MODY1 gene HNF-4α regulates selected genes involved in insulin secretion
J. Clin. Invest. Rana K. Gupta, et al. 115:1006 doi:10.1172/JCI22365 [
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Figure 6HNF-4α directly activates the Kir6.2 gene. (
A) The consensus binding site for HNF-4α is 13 bp long and was derived from 71 known HNF-4α binding sequences from the literature (
55) using the program Weblogo (http://weblogo.berkeley.edu/). The size of the letters reflects the frequency at which the nucleotide appears at that position in the binding site. (
B) Putative HNF-4α binding site in the Kir6.2 promoter identified using NUBIScan, which uses a transcription factor–binding site–identification algorithm to identify nuclear receptor binding sites. Note that the site located at position –2,300 matches all determinant nucleotides in the HNF-4α consensus site shown in
A. (
C) EMSA demonstrates that HNF-4α binds to the identified binding site in the Kir6.2 gene as well as the HNF-4α consensus site. In supershift experiments using 2 different antibodies raised against HNF-4α, the identity of the bound protein is confirmed to be HNF-4α. (
D) Cotransfection of BHK cells with HNF-4α and pGL3-Kir6.2, expressing luciferase under the control of the 237-bp region of Kir6.2 containing the binding site, results in a dose-dependent increase in luciferase activity, indicating that this element serves as an HNF-4α–dependent enhancer. Mutation of this binding site abolishes the transcriptional activation. Statistical analysis was performed by ANOVA; n = 3 for each transfection condition.
