A search for the FOXO3-binding site of mouse resistin promoter

• Main Authors: Chao-Chun Ma, 馬兆駿
• Other Authors: Yung-Hsi Kao
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/01180032756062151032

碩士 === 國立中央大學 === 生命科學研究所 === 100 === Resistin is known as an adipose tissue-specific secretory hormone that can cause insulin resistance and inhibit adipocyte differentiation. It can be regulated by many transcriptional factors. To fully understand the regulation of resistin gene expression by FOXO3 transcription factor in adipocytes, we initially searched the FOXO3-binding site in the resistin gene promoter in vitro and in vivo. An in vitro analysis of electrophoretic mobility shift assay (EMSA) showed that glutathione-S-transferase-FOXO3 (GST-FOXO3) fusion protein could directly bind several nucleotide regions of the resistin promoter, including -3439~3377, -3304~-3255, -3254~-3205, -2700~-2615, -2561~-2480, -2479~-2397, -2074~-1985, -1984~-1895, -1894~-1805, -1804~-1715, -1504~-1405, -1404~-1285, -1054~-955, -954~-855, and -838~-759 bp. The binding specificity was confirmed by an EMSA competition experiment since the added competitive probe blocked the binding of GST-FOXO3 to each individual resistin promoter region. In contrast, GST-FOXO1 fusion protein could directly bind the nucleotide regions of resistin promoter at -3254~-3205, -1504~-1405, -1404~-1285, -1054~-955, and -954~-855 bp, but not at other FOXO3-binding regions. Chromatin Immunoprecipitation (ChIP) assay indicated that the fourth day and the sixth day of differentiating C3H10T1/2 adipocytes, but not preadipocytes or differentiated adipocytes, exhibited the strong binding of the endogenous FOXO3 protein to the following nucleotide regions of resistin promoter: -3444~-3315, -3354~-3155, -2799~-2615, -2571~-2380, -2074~-1895, -1894~-1705, -1558~-1385, -1404~-1285, -1104~-925, and -954~-724 bp. Consistent with the in vitro EMSA findings, these ChIP data suggest that the binding of FOXO3 to resistin promoter varies with the developmental status of fat cells. We also conclude that multiple FOXO3-binding sites are located on mouse resistin promoter, and some of these nucleotide positions are the FOXO1-binding site as well.