陳宏文臺灣大學：生化科學研究所莊筱菁Chuang, Hsiao-ChingHsiao-ChingChuang2007-11-262018-07-062007-11-262018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/52723　　人類胎盤特殊轉錄因子hGCMa，屬於GCM基因家族，在胎盤的發育上扮演重要的角色；hGCMa促進胎盤中細胞滋養層(cytotrophoblast)細胞融合形成融合滋養層(syncytiotrophoblast)，為胎兒的發育所必須。 　　在實驗室先前研究中發現，在PKA的刺激之下，組蛋白乙醯轉移酶(histone acetyl-transferase，HAT)中的CBP會與hGCMa的結合並促進CBP對hGCMa進行乙醯化。而當hGCMa乙醯化後其蛋白的穩定度與轉錄活性皆上升，必定會誘發下游的訊息傳遞的進行，因而推測應有一個負調控機制存在，對hGCMa進行去乙醯化以降低hGCMa的活性。而在此次研究中發現，加入組蛋白去乙醯酶(histone deacetylase，HDAC)的抑制劑trichostatin A (TSA)後，hGCMa的乙醯化狀態增加且轉錄活性也更為上升。接著更進一步確定了HDACα, HDACγ, HDACκ和HDACλ可與hGCMa相結合而促進hGCMa進行去乙醯化。因為HDACγ呈現了較強的去乙醯化的效果與結合能力，透過進一步的短暫表現(transient expression)的實驗發現了HDACγ可抑制hGCMa的轉錄活性。 　　上述結果顯示乙醯化的hGCMa可做為數個去乙醯酶(deacetylase)分子的受質(substrate)。本篇報告同時呈現了一個新的機制－－hGCMa的活性透過組蛋白乙醯轉移酶及去乙醯酶兩大類分子而達到後轉譯(posttranslational)層次上的調控。Human GCMa is a zinc-containing transcription factor that plays a critical role for placenta development. hGCMa regulates the fusion of cytotrophoblasts into a syncytiotrophoblast layer, which is indispensable for the proper development of fetus. In our previous studies, we have demonstrated that PKA stimulates the association of hGCMa with CBP and increases hGCMa acetylation by CBP. Because hGCMa acetylation enhances its stability and transcriptional activity, there should be a negative regulation system, such as a deacetylation mechanism, to reverse hGCMa activity. In the current study, we observed that trichostatin A (TSA), an inhibitor of multiple histone deacetylases (HDACs), enhances the acetylation state of hGCMa and hGCMa-mediated transcriptional activity. We further found that hGCMa can be deacetylated through the interaction with HDACα, HDACγ, HDACκ and HDACλ Because HDACγ displayed the strongest deacetylation effect and binding efficiency, we also demonstrated that HDACγinhibits hGCMa-mediated transcriptional activity in transient expression experiments. These results suggest that acetylated hGCMa is a substrate for several deacetylases and reveal a new mechanism for the posttranslational regulation of hGCMa activity by histone acetyl-transferase and deacetylases.目　錄 I 圖表目錄 III 縮寫表 IV 中文摘要 VI 英文摘要　Abstract VII 第一章　緒論 1.1　　胎盤 1 1.2　　gcm轉錄因子基因家族 4 1.3　　轉錄因子相關的後轉譯修飾 8 1.4　　組蛋白去乙醯酶 (Histone deacetylase) 11 1.5　　研究動機及目標 12 第二章　材料及方法 2.1　　重組質體的構築 14 2.2　　細胞培養 15 2.3　　短暫轉染 (Transient transfection) 15 2.4　　細胞蛋白萃取及處理 16 2.5　　西方墨點法 (Western blotting) 17 2.6　　共同免疫沉澱法 (Co-immunoprecipatation) 17 2.7　　蛋白表現 18 2.8　　GST沈澱試驗 (GST pull-down assay) 19 2.9　　螢光報告基因Luciferase活性檢測 (Luciferase assay) 19 2.10 　免疫染色 (Immunostaining) 20 第三章　實驗結果 3.1　　TSA促進了hGCMa的乙醯化與轉錄活性。 22 3.2　　HDAC分子與hGCMa的交互作用及去乙醯化作用。 24 3.3　　HDACγ與hGCMa在活體內外的交互作用。 27 3.4　　定位HDACγ與hGCMa的結合區域。 29 3.5　　HDACγ抑制hGCMa的轉錄活性。 30 第四章　討論與總結 32 第五章　圖表 37 第六章　參考文獻 45en-US人類胎盤特殊轉錄因子組蛋白去乙醯酶human placeata-specific transcription factor hGCMahistone deacetylase (HDAC)other