Heckert L L, Schultz K, Nilson J H
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Biol Chem. 1995 Nov 3;270(44):26497-504. doi: 10.1074/jbc.270.44.26497.
To identify elements of the human alpha subunit gene necessary for cell-specific expression, we generated an array of block mutations spanning approximately 400 base pairs (bp) of promoter proximal region and examined them using transient transfection analysis in pituitary (alpha T3) and placental (BeWo) cell lines. Comparison of promoter activity in the two cell types revealed both common and unique elements required for transcription in pituitary and placenta. Two strong elements, the cyclic AMP response element (CRE) and the upstream regulatory element (URE), regulate expression of the alpha subunit gene in BeWo cells. In contrast, promoter activity in alpha T3 cells requires an array of weaker elements. These include the CREs, the URE, as well as two previously described elements, pituitary glycoprotein hormone basal element (PGBE) and gonadotrope-specific element (GSE), and two new elements we designated as the alpha basal elements 1 and 2 (alpha BE1 and alpha BE2). These new elements reside between -316 and -302 bp (alpha BE1) and -296 and -285 bp (alpha BE2) of the human alpha subunit promoter and bind distinct proteins designated alpha BP1 and alpha BP2, respectively. Southwestern blot analysis revealed that alpha BE1 specifically binds 54- and 56-kDa proteins. Additional studies disclosed several potential interactions between proteins that bind the CRE and proteins that occupy PGBE, alpha BE1, and alpha BE2, suggesting that gonadotrope-specific expression occurs through a unique composite regulatory element that includes components of the placenta-specific enhancer.
为了确定细胞特异性表达所需的人α亚基基因元件，我们构建了一系列跨越启动子近端区域约400个碱基对（bp）的阻断突变体，并在垂体（αT3）和胎盘（BeWo）细胞系中通过瞬时转染分析对其进行检测。两种细胞类型中启动子活性的比较揭示了垂体和胎盘中转录所需的共同元件和独特元件。两个强元件，即环磷酸腺苷反应元件（CRE）和上游调节元件（URE），调节BeWo细胞中α亚基基因的表达。相比之下，αT3细胞中的启动子活性需要一系列较弱的元件。这些元件包括CREs、URE，以及两个先前描述的元件，垂体糖蛋白激素基础元件（PGBE）和促性腺激素细胞特异性元件（GSE），还有我们命名为α基础元件1和2（αBE1和αBE2）的两个新元件。这些新元件位于人α亚基启动子的-316至-302 bp（αBE1）和-296至-285 bp（αBE2）之间，分别结合名为αBP1和αBP2的不同蛋白质。蛋白质印迹分析显示αBE1特异性结合54 kDa和56 kDa的蛋白质。进一步的研究揭示了结合CRE的蛋白质与占据PGBE、αBE1和αBE2的蛋白质之间的几种潜在相互作用，这表明促性腺激素细胞特异性表达是通过一个独特的复合调节元件实现的，该元件包括胎盘特异性增强子的成分。
