Paul C, Simar-Blanchet A E, Ro H S, Le Cam A
INSERM U-376, hôpital Arnaud de Villeneuve, Montpellier, France.
Eur J Biochem. 1998 Jun 15;254(3):538-46. doi: 10.1046/j.1432-1327.1998.2540538.x.
The activity of the rat serine protease inhibitor 2.3 gene (spi 2.3) is controlled by several positive promoter elements [Simar-Blanchet, A.-E., Paul, C., Mercier, L. & Le Cam, A. (1996) Eur. J. Biochem. 236, 638-648] and a negative element located in the 3' untranslated gene region (3' UTR) [Le Cam, A. & Legraverend, C. (1996) Eur. J. Biochem. 231, 620-627]. In the present studies, we dissected the 348-bp spi 2.3 3' UTR silencer to precisely define repressor sites and look for specifically interacting proteins. Three short elements referred to as A (nucleotides 1751-1776 in the cDNA), B (nucleotides 1812-1827) and C (nucleotides 1958-1974) sites repressed transcription from the homologous spi 2.3 promoter as well as from a heterologous minimal promoter containing the spi GAGA box enhancer. All three sites harbor a (TTTC) motif whose mutation affected silencer activity that was also dependent on flanking sequences. Those sites share the (TTTC) motif and a CCAAT/enhancer-binding-protein(C/EBP)-binding site with a fatty-acid-binding-protein gene promoter element shown to interact specifically with a transcriptional repressor [He, G. P., Muise, A., Wu Li, A. & Ro, H.-S. (1995) Nature 378, 92-96]. This repressor is however unlikely to mediate spi 2.3 3' UTR silencer action since it was not detected in rat hepatocytes. In vitro footprinting of the spi 2.3 3' UTR silencer region revealed a strong interaction with liver nuclear proteins. Among the six identified footprints, three of them (F-II, FIII and F-IV) bound C/EBPs and mapped in regions harboring the repressor function. Binding of C/EBPs to all three spi 2.3 3' UTR repressor sites, although rather weak, was confirmed by electrophoretic mobility shift assays that otherwise failed to reveal specific interactions with other liver nuclear proteins in vitro. However, none of the most largely liver expressed C/EBP species (i.e. alpha, beta and delta) activated the spi 2.3 3' UTR silencer function in NIH 3T3 cells, suggesting that binding of those transcription factors did not mediate the transcriptional repression.
大鼠丝氨酸蛋白酶抑制剂2.3基因（spi 2.3）的活性受几个正向启动子元件[西马尔 - 布兰切特，A.-E.，保罗，C.，梅西埃，L.和勒坎，A.（1996年）《欧洲生物化学杂志》236卷，638 - 648页]以及位于3'非翻译基因区域（3'UTR）的一个负向元件[勒坎，A.和勒格拉韦伦德，C.（1996年）《欧洲生物化学杂志》231卷，620 - 627页]的调控。在本研究中，我们剖析了348 bp的spi 2.3 3'UTR沉默子，以精确确定阻遏位点并寻找特异性相互作用蛋白。三个短元件，分别称为A位点（cDNA中核苷酸1751 - 1776）、B位点（核苷酸1812 - 1827）和C位点（核苷酸1958 - 1974），可抑制同源spi 2.3启动子以及含有spi GAGA盒增强子的异源最小启动子的转录。所有这三个位点都含有一个（TTTC）基序，其突变会影响沉默子活性，而沉默子活性也依赖于侧翼序列。这些位点与一个脂肪酸结合蛋白基因启动子元件共享（TTTC）基序和一个CCAAT/增强子结合蛋白（C/EBP）结合位点，该启动子元件已显示可与一种转录阻遏物特异性相互作用[何，G.P.，缪斯，A.，吴丽，A.和罗，H.-S.（1995年）《自然》378卷，92 - 96页]。然而，这种阻遏物不太可能介导spi 2.3 3'UTR沉默子的作用，因为在大鼠肝细胞中未检测到它。对spi 2.3 3'UTR沉默子区域进行的体外足迹分析显示它与肝核蛋白有强烈相互作用。在六个已鉴定的足迹中，其中三个（F-II、F-III和F-IV）与C/EBP结合，并定位在具有阻遏功能的区域。通过电泳迁移率变动分析证实，C/EBP与所有三个spi 2.3 3'UTR阻遏位点的结合虽然相当弱，但在体外未能揭示与其他肝核蛋白的特异性相互作用。然而，在NIH 3T3细胞中，在肝脏中大量表达的C/EBP的任何一种类型（即α、β和δ）都未激活spi 2.3 3'UTR沉默子功能，这表明这些转录因子的结合并未介导转录抑制作用。
