Liu Y, Chen H, Chiu J F
Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405.
Mol Cell Endocrinol. 1994 Jul;103(1-2):149-56. doi: 10.1016/0303-7207(94)90082-5.
Retinoic acid (RA) is known to have potent effects on development and differentiation. alpha-Fetoprotein (AFP), an oncodevelopmental protein, is transcriptionally activated by RA in several cell lines, but little is known about the mechanism of RA regulation of AFP gene expression. In the present study, we have identified a RA response element (RARE) in the 5'-flanking region of the AFP gene. Using deletion mapping, the RARE was located between -6337 to -6266 of the rat AFP 5'-flanking region, which confers RA responsiveness in a heterologous promoter. Further sequence analysis of this cis-acting element demonstrated a RARE direct repeat sequence of AGGTCA and RARE-like motifs at -6327 and -6319, respectively. This far upstream RARE (AFP-RARE1) can specifically bind to both RAR and RXR proteins in gel mobility shift assays. In co-transfections with RAR alpha, beta, gamma and RXR alpha expression vectors, a reporter gene construct consisting of the AFP-RARE1 sequence ligated upstream of the chloramphenicol acetyltransferase (CAT) gene showed strong RA responsiveness to RAR alpha and RXR alpha with 15- and 25-fold increases in CAT activity, respectively. Furthermore, responsiveness of AFP-RARE1 to RA was independent of orientation. These studies present a novel target for RA action by identifying a RARE in the AFP gene.
维甲酸（RA）已知对发育和分化具有强大作用。甲胎蛋白（AFP）是一种肿瘤发育蛋白，在多种细胞系中可被RA转录激活，但关于RA调节AFP基因表达的机制知之甚少。在本研究中，我们在AFP基因的5'侧翼区域鉴定出一个RA反应元件（RARE）。通过缺失定位，该RARE位于大鼠AFP 5'侧翼区域的-6337至-6266之间，其在异源启动子中赋予RA反应性。对该顺式作用元件的进一步序列分析显示，在-6327和-6319处分别有一个AGGTCA的RARE直接重复序列和类RARE基序。在凝胶迁移率变动分析中，这个位于上游远处的RARE（AFP-RARE1）能特异性结合RAR和RXR蛋白。在与RARα、β、γ和RXRα表达载体的共转染中，由连接在氯霉素乙酰转移酶（CAT）基因上游的AFP-RARE1序列组成的报告基因构建体对RARα和RXRα表现出强烈的RA反应性，CAT活性分别增加了15倍和25倍。此外，AFP-RARE1对RA的反应性与方向无关。这些研究通过在AFP基因中鉴定出一个RARE，提出了一个新的RA作用靶点。
