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標題: | hCGβ受到轉錄因子GCM1及AP-2c的調控機制 Regulation of human chorionic gonadotropin β gene expression by GCM1 and AP-2c |
作者: | Pei-Yun Chuang 莊珮雲 |
指導教授: | 陳宏文(Hung-Wen Chen) |
關鍵字: | human chorionic gonadotropin β (hCGβ),glial cells missing 1 (GCM1),activating protein 2 (AP-2), |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 胎盤是主要提供母體與胎兒之間進行物質交換的場所,在胎盤樹狀絨毛的部分,絨毛膜內層的細胞性滋養層細胞 (cytotrophoblast, CTB) 可以經由細胞融合 (cell fusion) 分化成多細胞核的融合滋養層細胞 (syncytiotrophoblast, STB),且融合滋養層細胞會製造與分泌生長因子以及荷爾蒙,調控母體與胎兒之間的物質交換。從過去實驗已知,胎盤專一性表現的轉錄因子glial cells missing 1 (GCM1),可以藉由活化syncytin-1、syncytin-2來促進胎盤細胞融合。根據實驗室所做的染色質免疫沉澱結合晶片分析技術 (chromatin immunoprecipitation-on-chip (ChIP-chip) analysis) 找到GCM1轉錄因子相關的下游標的基因,發現GCM1轉錄因子會與hCGβ基因前方的啟動子結合。我們想了解GCM1轉錄因子調控hCGβ的機制,另外,GCM1與hCGβ皆會受到cAMP/PKA訊號傳遞的調控,是否會透過cAMP路徑活化GCM1進而去影響hCGβ的表現。而過去文獻指出轉錄因子activating protein 2 (AP-2) 家族會調控hCGβ的表現,在hCGβ啟動子上含有兩個AP-2的結合位置在-311到-275與-250到-200的區域,可以增加hCGβ的基因表現,本篇針對GCM1與AP-2轉錄因子之間的調控進行討論,幫助我們了解hCGβ受到GCM1與AP-2轉錄因子的調控機制。
首先,在BeWo建立穩定表現GCM1 shRNA的細胞株,由Q-PCR與西方墨點法的實驗結果發現GCM1會直接影響到由forskolin所刺激hCGβ基因的轉錄與蛋白質的表現。利用不含有內生性AP-2的人類肝癌細胞HepG2,進行冷光報導基因活性檢測實驗,發現在forskolin刺激下當GCM1與CBP coactivator同時存在時,GCM1可以增加hCGβ的啟動子活性。在hCGβ啟動子-1674~+103的區域中包含四個可能的GCM1結合區域 (GCM1-binding site, GBS),為了確定四個GCM1結合區域是否會與GCM1有交互作用,將結合區域個別進行突變,發現GBS2為最重要的GCM1調控hCGβ啟動子活性的區域。進一步利用電泳遷移確定GCM1與AP-2c會直接結合在GBS2的位置上,另外,在BeWo建立穩定表現AP-2c shRNA的細胞株,結果發現GCM1與hCGβ的蛋白質表現明顯下降,且證明AP-2c會去調控GCM1的啟動子活性,由以上的結果知道在胎盤細胞中hCGβ的轉錄機制受到GCM1與AP-2c的調控。 Human cytotrophoblasts in the chorionic villi may undergo cell-cell fusion to form syncytiotrophoblasts to mediate gas and nutrient transport between mother and fetus. The placental transcription factor glial cells missing 1 (GCM1) has been shown to promote placental cell-cell fusion through transcriptional activation of membrane fusogenic proteins, syncytin-1 and -2. We recently performed chromatin immunoprecipitation-on-chip (ChIP-chip) analysis and identified the gene for human chorionic gonadotropin β (hCGβ) as a GCM1 target gene. A major function of hCGβ is to maintain the production of progesterone hormone and other growth factors in the corpus luteum until the 7th week of gestation. The activating protein 2 (AP-2) family has been shown to play an important role in hCGβ expression. The mechanism of regulation of hCGβ gene expression by GCM1and AP-2 is not clear. In this study, we first demonstrated that the hCGβ mRNA and protein levels are significantly decreased in the GCM1-konckdown BeWo cells by Q-PCR and Western blotting. Luciferase reporter assays were performed to study the regulation of hCGβ reporter activity by GCM1 in HepG2 cells, which are human hepatoma-derived cells without endogenous AP-2 activity. Both GCM1 and CBP coactivator are required for the stimulation of hCGβ reporter activity by forskolin in HepG2 cells. The reporter construct harbors the hCGβ promoter region, nt -1674~+103 relative to the transcriptional start site, containing four potential GCM1-binding sites (GBSs). By site-directed mutagenesis, we identified GBS2 as a critical element for GCM1 and CBP to upregulate hCGβ promoter activity in response to forskolin. We further demonstrated that both GCM1 and AP-2c can bind GBS2 by EMSA. Interestingly, the GCM1 protein level was decreased by AP-2c knockdown in BeWo cells. Correspondingly, AP-2c upregulates GCM1 promoter activity in luciferase reporter assays. Our study reveals an underlying mechanism for regulation of hCGβ expression by GCM1 and AP-2c. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16723 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科學研究所 |
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