Regulation of LRH-1 transcriptional activity

肝受器同族體-1 (liver receptor homolog-1; LRH-1) 屬於孤兒核受器NR5A的一員，會表現於類固醇生成的組織，具有調控類固醇生成基因表現的能力。本篇論文主要探討mouse LRH-1 (mLRH-1)轉錄活性的調控。 先前我們已證實mLRH-1可以調控CYP11A1啟動子的表現，且刪除N端後，mLRH-1的轉錄活性會提高2倍。為了解mLRH-1 C端的特性，我們建構了兩個刪除C端不同長度的mLRH-1。結果發現，刪除C端ligand binding domain的mLRH-11-240 ，其調控基因轉錄的活性幾乎完全喪失，並且對mLRH-1的轉錄活性有顯性抑制 (dominant negative) 的作用。 除LBD外，還刪除hinge region及部分DNA binding domain的mLRH 11-191，仍位於細胞核內，但不具顯性抑制的作用。 LRH-1會受small ubiquitin-related modifier-1 (SUMO-1) 轉譯後的修飾作用，我們藉由點突變mLRH-1上SUMO-1可能的結合位以了解SUMO-1修飾作用對mLRH-1轉錄活性的影響，結果顯示SUMO-1會與mLRH-1的lysine 289位置結合，並抑制其轉錄活性。而lysine 289分別與其他4個可能的SUMO-1結合位進行點突變的雙點突變與lysine 289的單點突變比較，則顯示雙點突變對mLRH-1調控基因轉錄的活性並沒有顯著差異性，證實了lysine 289是mLRH-1最主要的SUMO-1結合位置。 近來發現PIAS家族具有SUMO E3接合酶的活性，為了解PIAS家族是否參與SUMO-1對mLRH-1轉錄活性的調控，利用與mLRH-1共同轉染的實驗，發現PIAS家族的成員中PIASxα，PIASxβ和PIASy能有效地抑制mLRH-1轉錄活性，PIASy的抑制效果尤其明顯，而PIAS1及PIAS3則無顯著效果。藉由共同轉染的實驗，顯示lysine 289 及其他4個非SUMO-1結合位分別帶有點突變的mLRH-1仍會受到PIASy與PIASxα的抑制效果，表示PIASy或PIASxα可能不是透過mLRH-1 sumoylation抑制其轉錄活性。 此外，我們更進一步證明，PIASy與mLRH-1有直接的交互作用。 由本篇論文中我們發現： 1、mLRH-1其C端序列為其轉錄活性所需，且刪除mLRH-1 C端片段會增加mLRH-1的蛋白量。 2、SUMO-1對mLRH-1的修飾作用主要是藉由與mLRH-1的lysine 289結合，並造成其轉錄活性的下降。 3、具有SUMO E3接合酶活性的PIAS家族中，PIASxα，PIASxβ和PIASy會抑制mLRH-1轉錄活性，並且PIASy或PIASxα可能不是透過mLRH-1 sumoylation抑制其轉錄活性。 4、PIASy與mLRH-1有直接的交互作用。Liver receptor homolog-1 (LRH-1) is a member of nuclear receptor 5A subfamily. LRH-1 is expressed in steroidogenic tissues and has been shown to possess the ability to regulate the expression of steroidogenic genes. The aim of this study is to evaluate the regulation of mouse LRH-1 (mLRH-1) transcriptional activity. Our previous studies indicated that mLRH-1 stimulated the expression of human CYP11A1 promoter and N-terminal deletion of mLRH-1 enhanced the transcriptional activity of mLRH-1. In this study, two C-terminal deletions of mLRH-1 were constructed to examine the effect of C-terminus on mLRH-1. We found that mLRH-11-240 lacking of ligand binding domain almost lose transcriptional activity and had a dominant negative effect on mLRH-1 mediated transcription. The mLRH-11-191 with further deletion of hinge region and a part of DNA binding domain still located in the nucleus but had no dominant negative effect on mLRH-1-mediated transcription. LRH-1 can be conjugated with a post-translational modifier protein, small ubiquitin-related modifier-1 (SUMO-1). By mutating the potential SUMO-1 binding sites, we found that the lysine 289 of mLRH-1 is the major SUMO-1 conjugation site. In addition, the sumoylation at lysine 289 may inhibit mLRH-1 transcriptional activity. The members of PIAS family are recently known to be E3-like ligases that control the conjugation of SUMO-1 to target protein. Our cotransfection experiment showed that PIASxα, PIASxβ and PIASy can repress mLRH-1-induced transcription and PIASy had a most significant effect. And it’s suggested that PIASxαand PIASy-mediated repression of mLRH-1 is independent of mLRH-1 sumoylation. We further proved that mLRH-1 could interact with PIASy in vitro. In this study we found that: 1、The C-terminus of mLRH-1 is necessary for its transcriptional activity. Deletion of C-terminus increases the protein amount of mLRH-1. 2、SUMO-1 is mainly conjugated to lysine 289 of mLRH-1 that represses the transcriptional activity of mLRH-1. 3、SUMO E3 ligase PIASxα, PIASxβ and PIASy can repress mLRH-1-induced transcription. The repression of mLRH-1 transcriptional activity mediated by PIASxαand PIASy is independent of mLRH-1 sumoylation. 4、PIASy can directly interact with mLRH-1.目錄................................................Ⅰ 圖次...............................................Ⅳ 中文摘要 ...........................................Ⅴ 英文摘要 ............................................Ⅶ 第一章、 序論.............................................1 一、 核受器NR5A家族.......................................1 二、 LRH-1之作用.........................................2 1、膽酸合成代謝...........................................2 2、影響胚胎發育與分化...............................2 3、調控類固醇生成基因................................3 三、 轉譯後修飾蛋白SUMO-1............................4 1、SUMO-1的特性........................................4 2、SUMO-1的作用......................................5 四、 PIAS家族............................................6 1、PIAS家族的特性 ....................................................................... 6 2、PIAS於sumoylation作用的角色 ............................................. 6 3、PIASy作為輔調控子 ................................................................ 7 五、 研究動機 .................................................................................... 9 第二章、 材料與方法 ................................................................................. 10 一、 質體建構 ..................................................................................... 10 二、 PCR定點突變 ........................................................................... 13 三、 細胞培養 ..................................................................................... 13 四、 暫時性轉染法 (Transient transfection) .................................... 14 五、 Luciferase 活性分析 .................................................................. 15 六、 免疫螢光染色法 ......................................................................... 15 七、 SDS-PAGE .................................................................................. 16 八、 Coomassie blue R-250 染色 ...................................................... 16 九、 西方墨染法 ................................................................................. 16 十、 GST重組蛋白於大腸桿菌內表現的誘導及分離 ................... 17 十一、 胞外轉錄、轉譯作用 ............................................................. 18 十二、 GST pull-down assay .............................................................. 18 第三章、 結果 ............................................................................................. 20 一、 mLRH-1 C端片段的特性 ......................................................... 20 1、蛋白質表現 ................................................................................ 20 2、轉錄活性 ..................................................................................... 20 二、 SUMO-1的轉譯後修飾作用對mLRH-1調控CYP11A1 之影響 ......................................................................................... 21 三、 PIAS對mLRH-1調控CYP11A1之影響 .................................... 22 四、 PIASy與mLRH-1之交互作用 .................................................... 22 1、PIASy抑制mLRH-1的轉錄活性與mLRH-1 sumoylation 無關 ........................................................................................... 23 2、PIASy與mLRH-1有直接的交互作用 ....................................... 23 (1) PIASy在E.coli的表現 ..................................................... 24 (2) 蛋白質純化 ........................................................................ 25 (3) GST pull-down .................................................................. 25 五、 LRH-1在乳癌細胞內的表現 ................................................... 26 第四章、 討論 ............................................................................................ 27 一、mLRH-1 C端片段影響其蛋白質穩定性及轉錄活性 ............... 27 二、SUMO-1的轉譯後修飾作用對mLRH-1調控CYP11A1 之影響 ........................................................................................... 28 三、PIAS對mLRH-1調控CYP11A1之影響 ..................................... 29 四、PIASy與mLRH-1之交互作用 ....................................................... 30 五、LRH-1在乳癌細胞內的表現 ..................................................... 33 參考文獻 ....................................................................................................... 34 圖 ................................................................................................................... 41 圖次 圖一、 mLRH-1及刪除C端片段的蛋白質表現 ……………………… 41 圖二、 mLRH-1及C端片段刪除在細胞內的分布 .............................. 42 圖三、 mLRH11-240調控人類CYP11A1啟動子的作用.......................... 43 圖四、 mLRH11-240對mLRH-1調控人類CYP11A1啟動子的影響 ..... 44 圖五、 mLRH11-191對mLRH-1調控人類CYP11A1啟動子的影響 ..... 45 圖六、 SUMO-1結合位單點突變對mLRH-1調控人類CYP11A1 啟動子的影響 ………....………………………………………... 46 圖七、 SUMO-1結合位雙重突變對mLRH-1調控人類CYP11A1 啟動子的影響 ............................................................................... 47 圖八、 PIAS家族對mLRH-1轉錄活性的影響 ..................................... 48 圖九、 PIASy抑制mLRH-1的轉錄活性 ………………………........... 49 圖十、 PIASy抑制mLRH-1的轉錄活性與mLRH-1 sumoylation 無關............ .................................................................................... 50 圖十一、PIASxα抑制mLRH-1 的轉錄活性與mLRH-1 sumoylation 無關 ............................................................................................... 51 圖十二、GST及GST-PIASy1-159蛋白於大腸桿菌之表現 ........................ 52 圖十三、GST及GST- PIASy1-159蛋白之純化 ......................................... 53 圖十四、PIASy1-159與mLRH-1蛋白交互作用之測試 ............................. 54 圖十五、LRH-1於乳癌細胞之表現 .......................................................... 5