アクティブボード・２０１０年１０月
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研究発表を行った学会；
・第１４回国際内分泌学会議サテライトシンポジウム
　 ～Nuclear Receptor and its Frontier～.
　２０１０年　３月３１日（京都）

タイトル；Analysis of a novel HNF4alpha target gene Anks4b in pancreatic beta-cells.
　
発表者；　八田　光世　氏
　　　（熊本大学　大学院生命科学研究部　病態生化学分野）

Abstract；
Hepatocyte nuclear factor (HNF)4alpha, a transcription factor
belonging to the nuclear receptor superfamily, is expressed in
pancreatic islets as well as in the liver, kidney, and intestine. Mutations in HNF4alpha gene cause a form of maturity-onset diabetes of the young (MODY1) that is characterized by impairment of glucose-stimulated insulin secretion by pancreatic beta-cells. Previous studies revealed that beta-cell-specific HNF4alpha knock-out (betaHNF4alphaKO) mice exhibited impairment of glucose-stimulated insulin secretion, which is a characteristic of MODY1.
However, the target genes regulated by HNF4alpha in the beta-cell are still elusive. In this study, we identified Anks4b as a novel HNF4alpha target gene by meaning of the microarray expression
analysis of isolated islets from the betaHNF4alphaKO mice. Real-
time PCR analysis revealed that HNF4alpha knockdown by RNAi down-
regulates Anks4b mRNA levels in beta-cell derived Min6 cells.
Additionally, the tissue distribution of Anks4b mRNA was correlated with expression of HNF4alpha mRNA. We next investigated whether Anks4b is a direct transcriptional target of HNF4alpha in vivo.
Computational analysis identified a putative HNF4alpha binding site in the proximal promoter region of mouse Anks4b gene. Chromatin immunoprecipitation (ChIP) assay detected the binding of HNF4alpha to Anks4b promoter in vivo. We next performed Luciferase assay to determine if HNF4alpha activates Anks4b promoter activity. HNF4alpha increased promoter activity in dose-dependent manner, and this transcriptional activation was abolished by using the HNF4alpha binding site mutated reporter construct and a dominant negative form HNF4alpha. Anks4b was originally identified as a Harmonin interacting protein, however its functional role has been unknown. Anks4b possesses three ankyrin repeats in the N-terminal, and a sterile alpha motif (SAM) domain in the C-terminal. Localization analysis by using GFP-fusion Anks4b revealed that Anks4b localizes in the cytoplasm, and the SAM domain is essential to its localization pattern. We finally investigated Anks4b mRNA expression of isolated islets from model mice of obese and diabetic conditions. Anks4b mRNA levels were increased in isolated islets from mice under obese condition, but decreased under diabetic condition. The results suggest that Anks4b expression correlates with expansion of beta-cell mass and increased insulin secretion.