利用酵母菌雙雜交技術找尋在小鼠肝中與 LRH-1 有交互作用的蛋白質

Abstract

Liver receptor homolog-1 (LRH-1；NR5A2) 隸屬於NR5A 核受器家族，主要表現於肝臟、小腸、胰臟以及卵巢。LRH-1 對於發育、膽固醇的逆運輸、膽酸的恆定以及固醇類荷爾蒙的生成等扮演重要的角色。先前的研究指出，LRH-1 會和調控因子產生交互作用，進而調控許多下游基因的表現；然而，目前已知與 LRH-1 有交互作用的蛋白質並不多，因此我們希望藉由酵母菌雙雜交篩選，尋找與LRH-1 有交互作用的蛋白質，並且進一步探討它們調控 LRH-1 的功能。 將 LRH-1 建構至 LexA DNA binding domain (DBD) 下游，形成誘餌蛋白，針對建構於 GAL4 activation domain (AD) 的小鼠肝臟的 cDNA library 進行篩選。共得到58 種不同的蛋白質，從中挑選出7個蛋白質，分別為H3 histone, family 3B (H3f3b)、Methyl-CpG binding domain protein 1 (Mbd1)、Zinc finger protein 579 (Zfp579)、MYC-associated zinc finger protein (Maz)、Zinc finger and BTB domain containing 44 (Zbtb44)、Ring finger protein 128 (Rnf128) 和 Nuclear receptor subfamily 1 group H, member 4 (Nr1h4, FXR)。經由共同免疫沉澱法，證實這七個蛋 白質皆與 LRH-1 有交互作用。接著利用免疫螢光染色法，發現多數的蛋白質主要分佈於細胞核，與 LRH-1 的螢光表現重疊。後續經由功能性分析 (functional assay)，包括: 西方墨點法分析以及啟動子活性分析，觀察蛋白質是否會影響 LRH-1 蛋白質的表現量或是影響 LRH-1 調控啟動子的轉錄活性。

Liver receptor homolog-1 (LRH-1; NR5A2) is a member of the nuclear receptor NR5A subfamily. LRH-1 is predominantly found in liver, intestine, pancreas and ovary.In these tissues, LRH-1 plays a critical role in development, reverse cholesterol transport, bile-acid homeostasis and steroidogenesis. Previous studies have shown that LRH-1 can interact with coregulators and regulate the expression of many downstream genes. However, there are only a few proteins which were known as LRH-1-interacting proteins. Therefore, we carried out a yeast two-hybrid screen to identify proteins that will interact with LRH-1 and investigate their physiological functions with LRH-1. LRH-1 was fused to the LexA DNA binding domain (DBD) as a bait, and performed screening of the mouse liver cDNA library fused to a GAL4 activation domain (AD). Eventually, we identified 58 proteins and chose 7 of them for further investigation. These proteins are H3 histone, family 3B (H3f3b), Methyl-CpG binding domain protein 1 (Mbd1), Zinc finger protein 579 (Zfp579), MYC-associated zinc finger protein (Maz), Zinc finger and BTB domain containing 44 (Zbtb44), Ring finger protein 128 (Rnf128) and Nuclear receptor subfamily 1 group H, member 4 (Nr1h4, FXR). The co-immunoprecipitation assay verified that all of the proteins interact with LRH-1. Moreover, the immunofluorence assay results demonstrate that most of the proteins are colocalized with LRH-1 in the nucleus. We further performed the functional assay, such as: Western blotting and promoter activity assay, to identify whether these proteins will affect LRH-1 protein expression or modulate the promoter activity through LRH-1.