| 摘要: | 抗胰島素激素(resistin)是一種由脂肪組織所分泌的荷爾蒙，會阻抗胰島素的作用及脂肪細胞的分化，並會受到許多的轉錄因子所調節。為了充分地瞭解 FOXO3 轉錄因子在脂肪細胞內調節該激素基因的表現，本論文初步先找尋在抗胰島素激素驅動子上 FOXO3 的結合位。經由凝膠遷移滯後技術分析，結果發現 glutathione-S-transferase-FOXO3 融合蛋白會直接與抗胰島素激素驅動子裡分別位於 -3439 ~ -3377 bp 、 -3304 ~ -3255 bp 、 -3254 ~ -3205 bp 、 -2700 ~ -2615 bp 、 -2561 ~ -2480 bp 、 -2479 ~ -2397 bp 、 -2074 ~ -1985 bp 、 -1984 ~ -1895 bp 、 -1894 ~ -1805 bp 、 -1804 ~ -1715 bp 、 -1504 ~ -1405 bp 、 -1404 ~ -1285 bp 、 -1054 ~ -955 bp 、 -954 ~ -855 bp 以及 -838 ~ -759 bp等多個核苷酸區域相結合。在競爭實驗中也發現位移，加入未帶有放射性的競爭探針後 FOXO3 分別與以上所有的驅動子區域的結合都有減弱的情形，因此更加確認了FOXO3 與抗胰島素激素驅動子為專一性的結合。此外， FOXO1 也會直接與上面所列片斷序列中，例如 -3254 ~ -3205 bp 、 -1504 ~ -1405 bp 、 -1404 ~ -1285 bp 、 -1054 ~ -955 bp 以及 -954 ~ -855 bp 等五個驅動子區域相結合，但其它與 FOXO3 相結合的區域，並沒有發現有 FOXO1 的結合位。進一步利用染色質免疫沉澱分析法和 C3H10T1/2 細胞株，也發現在第四天及第六天分化中脂肪細胞， FOXO3 蛋白質會直接與位於抗胰島素激素驅動子上的核苷酸序列，例如 -3444 ~ -3315 bp 、 -3354 ~ -3155 bp 、 -2799 ~ -2615 bp 、 -2571 ~ -2380 bp 、 -2074 ~ -1895 bp 、 -1894 ~ -1705 bp 、 -1558 ~ -1385 bp 、 -1404 ~ -1285 bp 、 1104 ~ -925 bp 以及 -954 ~ -724 bp 等區域相結合，但在前脂肪細胞及已分化脂肪細胞內， FOXO3 並未與這些區域相結合，顯示 FOXO3 與抗胰島素激素驅動子之間的結合力會隨著細胞分化的程度而有變化。綜合以上的結果，本論文的結論是 FOXO3 轉錄因子會直接與多重區域之抗胰島素激素驅動子相結合，其結合區域有同於或異於 FOXO1 結合區域。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. |
