郭應誠臺灣大學：獸醫學研究所賴政分lai, Cheng-FenCheng-Fenlai2007-11-282018-07-092007-11-282018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/59975類固醇轉錄因子（Steroidogenic Factor1, SF-1）是孤核受體（orphan nuclear receptor）的一種，能調控許多類固醇生成酵素基因的表現，對腦下垂體、下視丘、腎上腺及性線的發育及分化非常重要。在小鼠與人的腎上腺皮質腫瘤細胞內（Y1 and H295 cells），使用組蛋白去乙醯基轉移酵素抑制劑 ，如Trichostatin A和Sodium butyrate，能增加Histone 4的乙醯化 (Acetylation)，卻減少SF-1與其下游膽固醇側鏈分切酵素（P450scc）的表現，這個抑制現象與劑量和作用時間成正比。報導基因表現分析的實驗中，TSA抑制了SF-1啟動子的活性；TSA能增加外源性SF-1蛋白質的表現，但減少內源性SF-1蛋白質的表現量。表示HDAC 抑制劑是透過SF-1啟動子來控制基因轉錄的表現。許多文獻指出，SF-1啟動子上面，具有一個重要的調控序列，E-box，與其結合的兩個主要蛋白質為上游刺激因子一及二 (Upstream Stimulatory Factor 1 and USF2)。膠體電泳位移分析 (EMSA) 結果表示，HDAC 抑制劑同時減少SF-1啟動子上調控單位（E box and CCAAT box）與其轉錄因子間的結合，也減少了USF1和USF2蛋白質的表現量。TSA抑制了P450scc及USF2的mRNA，但卻增加了USF1的mRNA；此結果與減少的USF1蛋白質相矛盾，故使用HDAC 抑制劑可能會影響特定蛋白質的穩定性並加速其降解。The orphan nuclear receptor steroidogenic factor 1 (SF-1, A4BP, or NR5A1) is a key transcription factor that regulates the expression of many steroidogenic enzymes. It has also been demonstrated to play an important role in the development and differentiation of hypothalamus, pituitary, adrenal glands, and gonads. Being such an important factor, the regulation of SF-1 expression is highly investigated. We used inhibitors of histone deacetylases which generally activate gene expression and used as anticancer drugs such as trichostatin A and sodium butyrate. Histone deacetylase inhibitors although increased the acetylation of Histone 4, reduced the protein content of SF1 and cytochrome P450 side-chain cleavage enzyme, a SF-1 controlled gene, in a dose and time-dependent manner in adrenocorticol cells, Y1 and H295 cells. In a reporter assay, TSA reduced the SF-1 promoter activity. In Y1 stable clone, which expresses SF-1-HA driven by CMV promoter, TSA increased exogenous SF-1-HA protein, however, reduced endogenous SF-1 protein. Both results indicate that SF-1 is transcriptionally down-regulated by these inhibitors. TSA reduced binding of E-box and CCAAT box binding factor to SF-1 promoter in Electorphoretic Mobility Shift Assay. The E-box binding proteins, USF1 (upstream stimulatory factor 1) and USF2 were down-regulated by TSA as well as SF-1. These results suggest that TSA-inhibited expression of SF-1 may provide insight to uncover the regulatory mechanism of SF-1 gene. Interesting, we found that TSA treatment increases mRNA of SCC and USF2 but reduces USF1 mRNA in Quantitative-Real-Time PCR. Increased USF1 mRNA and reduced USF1 protein content after TSA treatment provides a new association of protein stability and acetylation.Table of Contents Abbreviation 1 Abstract in Chinese 2 Abstract 3 Introduction 4 I、Steroid hormones 4 Classes & Functions 4 CYP11A1 (P450scc) 4 II、Steroidogenic Factor 1 5 Characterization 5 Structure 5 Regulation 6 III、Transcriptional regulation 7 Chromatin structure & Histone modification 7 Acetylation & Histone acetyltransferase 8 Dacetylation & Histone deacetylase 9 IV、HDAC inhibitors 9 Trichostatin A 10 Sodium Butyrate 10 V、HDAC inhibitors down regulate gene expression 10 VI、HDAC inhibitors inhibit SF-1 expression 10 In a dose- and time-dependent manner 11 Reduces SF-1-BS, E-box and CCAAT box binding 11 Reduces binding of USF1 and USF2 to E-box 12 Aim 13 Material and Methods 14 Cell culture and transfection 14 Plasmids construction 14 Western Blot analysis 15 Electrophoretic Mobility Shift Assay (EMSA) 15 Real-Time PCR 16 Administration of Trichostatin A and Sodium Butyrate 16 Results 18 I. HDAC inhibitors reduce SF-1 expression through SF-1 promoter region 18 II. TSA reduces SCC and USF2 mRNA but increases USF1 mRNA level 18 III. HDAC inhibitor decreases USF1 protein stability 19 IV. HDAC inhibitor effects in steroidogenesis in mice 19 Discussions 20 I. USF1 and USF2 have different response to TSA 20 The regulation of mRNA expression 20 The regulation of protein content 21 II. HDAC inhibitor effects in vivo 23 References 25 Table and Figures 331240401 bytesapplication/pdfen-US類固醇轉錄因子孤核受體腎上腺皮質腫瘤細胞組蛋白去乙醯基轉移酵素抑制劑乙醯化膽固醇側鏈分切酵素上游刺激因子一及二histone acetylatransferaseP450 side chain cleavage enzymethe cholesterol side-chain cleavage enzymesteroidogenic factor 1trichostatin A[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59975/1/ntu-94-R92629015-1.pdf