Banks E B, Crish J F, Welter J F, Eckert R L
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 2109 Adelbert Road, Cleveland, OH 44106-4970, USA.
Biochem J. 1998 Apr 1;331 ( Pt 1)(Pt 1):61-8. doi: 10.1042/bj3310061.
Human involucrin (hINV) is an important precursor of the keratinocyte cornified envelope that is specifically expressed in the suprabasal layers of stratifying epithelia. Previous truncation and mutagenesis experiments have shown that an activator protein 1 (Ap1) site, AP1-5, located 2100bp upstream of the transcription start site, is required for optimal promoter activity. These previous studies suggest that AP1-5 is part of a distal regulatory region spanning nucleotides -2473 to -2088. In the present report, we study the distal regulatory region (DRR), which surrounds AP1-5. Our studies show that this region contains weak and strong activator elements spanning nucleotides -2473/-2216 and -2140/-2088, respectively. The strong activator element contains AP1-5 and an adjacent specificity protein 1 (Sp1) site. The AP1-5 site is absolutely required for DRR activity, as its mutation reduces transcription to basal levels. Mutagenesis studies of the AP1-5 and Sp1 sites in the presence or absence of the weak activator element indicate that the Sp1 site and the weak activator element synergistically activate the AP1-5 site-dependent transcription. The cooperation between the Sp1 and AP1-5 sites is also observed in the context of the full-length promoter. Gel mobility shift and supershift studies show that Sp1, but not Sp2, Sp3 or Sp4 binds to the Sp1 site. When the Sp1 site is mutated or the distance between the AP1-5 and Sp1 site is increased, the binding of AP1 factors to AP1-5 is markedly reduced. Surprisingly, gel shift studies suggest that activation does not require the formation of a stable AP1/Sp1/DNA ternary complex. These studies suggest that the AP1-5 site is absolutely required for transcriptional activation, that the weak activator element and Sp1 sites serve to enhance this activation, and that the Sp1 site is required for optimal AP1 factor binding at the AP1-5 site.
人内披蛋白（hINV）是角质形成细胞角化包膜的重要前体，在分层上皮的基底层以上各层中特异性表达。先前的截短和诱变实验表明，转录起始位点上游2100bp处的激活蛋白1（Ap1）位点AP1-5是最佳启动子活性所必需的。这些先前的研究表明，AP1-5是跨越核苷酸-2473至-2088的远端调控区域的一部分。在本报告中，我们研究了围绕AP1-5的远端调控区域（DRR）。我们的研究表明，该区域分别包含跨越核苷酸-2473 / -2216和-2140 / -2088的弱激活元件和强激活元件。强激活元件包含AP1-5和相邻的特异性蛋白1（Sp1）位点。AP1-5位点是DRR活性绝对必需的，因为其突变会将转录降低到基础水平。在存在或不存在弱激活元件的情况下对AP1-5和Sp1位点进行诱变研究表明，Sp1位点和弱激活元件协同激活AP1-5位点依赖性转录。在全长启动子的背景下也观察到了Sp1和AP1-5位点之间的协同作用。凝胶迁移率变动和超迁移研究表明，Sp1而非Sp2、Sp3或Sp4与Sp1位点结合。当Sp1位点发生突变或AP1-5与Sp1位点之间的距离增加时，AP1因子与AP1-5的结合会明显减少。令人惊讶的是，凝胶迁移研究表明激活并不需要形成稳定的AP1 / Sp1 / DNA三元复合物。这些研究表明，AP1-5位点是转录激活绝对必需的，弱激活元件和Sp1位点用于增强这种激活，并且Sp1位点是AP1因子在AP1-5位点进行最佳结合所必需的。
