膽固醇側鏈截切酵素基因之促進子的研究

Abstract

膽固醇側鏈切除酵素P450scc產生於類固醇生成組織中，負責催化類固醇生合成的第一步反應，將類固醇之側鏈切除成妊醇酮。本實驗室將P450scc基因的5′調節區截切成小片段，接在tkCAT載體中，以暫時轉染法將質體送入老鼠腎上腺細胞Y1中，分析P450scc基因的5′調節區的功能。在-1903／-1822的區域中，發現有一促進子的存在，其可以增加載體的基底轉錄活性達四至五倍，將此區域稱為AdE。我將AdE的促進子功能區定位到AdE1，為-1845／-1822的片段中，AdE1不只可以增加外源性的促進子（heterologous promoter）的活性，亦可增加其本身基因的促進子（native promoter）活性。利用體外（in vitro）的明膠上移法分析，知道AdE1可與Y1細胞核中許多的蛋白質結合，而由競爭試驗及抗體超上移分析的結果，可知與AdE1結合的蛋白質中有一類為類NF1的蛋白質，有一類為類Sp1的蛋白質。以突變的AdE1在Y1細胞中作功能測試，結果顯示AdE1上的Sp1蛋白質結合位對其促進子功能最為重要。我們另將P450scc基因上游2.5kb接在lacZ報導基因之前，以此DNA片段產生基因轉殖老鼠，我觀察到lacZ基因只表現在類固醇生成組織，如：腎上腺皮質、睪丸的管間細胞（Leydig cell）。在我的實驗中，利用了體外（in vitro）的細胞轉染分析，及體內（in vivo）的基因轉質老鼠分析，我證實了P450scc基因上游2.5kb的區域提供P450scc基因組織專一表現的特性。

The first and rate-limiting step in synthesis of steroids is the cleavage of the side chain from cholesterol to form pregnenolone. This step is catalyzed by P450scc (cholesterol side chain cleavage enzyme, CYP11A1). We have characterized some cis-regulatory elements of P450scc by transfection of deleted gene fragments linked to the CAT reporter gene. One of these is a positive regulatory element located within the region -1932 to -1822 of CYP11A1 gene, termed AdE, which can increase the expression of reporter gene 4 to 5 folds only in steroidogenic cells. I have narrowed down the enhancer element to a small fragment at -1822/-1845, termed AdE1. AdE1 can increase the basal transcroption activity in front of both heterologous promoter and native promoter. In vitro binding assay showed that AdE1 can form several complexes with Y1 nuclear proteins. I demonstrated that two major complexes are NF1-like proteins and one minor complex is SP1-like protein by the competition with NF1 and SP1 consensus sequences. The functional assay of mutant AdE1 shows that the SP1 site is the most important site for the enhancer function. We also generated transgenic mice with the 2.5 kb upstream region of hP450scc gene in front of LacZ reporter gene. By lacZ enzyme activity staining, I observed that the lacZ gene only expressed in steroidogenic tissue: adrenal cortex and testis Leydig cell. Both using in vitro cell transfection assay and in vivo transgenic mice assay, I demostrated that the 2.5kb upstream regulatory region provides the tissue-specificity of the CYP11A1 gene.