Dusing M R, Wiginton D A
Department of Pediatrics, University of Cincinnati College of Medicine, OH.
Nucleic Acids Res. 1994 Feb 25;22(4):669-77. doi: 10.1093/nar/22.4.669.
Tissue-specific expression of the human adenosine deaminase (ADA) gene is mediated by transcriptional activation over a thousand-fold range. Cis-regulatory regions responsible for high and basal levels of activation include an enhancer and the proximal promoter region. While analyses of the T-cell specific enhancer have been carried out, detailed studies of the the promoter region or promoter-enhancer interactions have not. Examination of the promoter region by homology searches revealed six putative Sp1 binding sites. DNase I footprinting showed that Sp1 is able to bind these sites. Deletion analysis indicated that the proximal Sp1 site is required for activation of a reporter gene to detectable levels and that the more distal Sp1 sites further activate the level of expression. Inclusion of an ADA enhancer-containing fragment in these deletion constructions demonstrated that Sp1 sites are also essential for enhancer function. Apparently Sp1 controls not only low level expression but is also an integral part of the mechanism by which the enhancer achieves high level ADA expression. Mutagenesis of a potential TBP binding site at base pairs -21 to -26 decreased activity only two-fold indicating that it is not essential for transcriptional activation or enhancement.
人类腺苷脱氨酶（ADA）基因的组织特异性表达是由转录激活介导的，其激活范围超过千倍。负责高水平和基础水平激活的顺式调控区域包括一个增强子和近端启动子区域。虽然已经对T细胞特异性增强子进行了分析，但尚未对启动子区域或启动子 - 增强子相互作用进行详细研究。通过同源性搜索对启动子区域进行检查，发现了六个推定的Sp1结合位点。DNase I足迹分析表明Sp1能够结合这些位点。缺失分析表明，近端Sp1位点是将报告基因激活到可检测水平所必需的，而更远端的Sp1位点进一步激活表达水平。在这些缺失构建体中包含一个含ADA增强子的片段表明，Sp1位点对于增强子功能也是必不可少的。显然，Sp1不仅控制低水平表达，而且也是增强子实现高水平ADA表达机制的一个组成部分。在碱基对-21至-26处对潜在的TBP结合位点进行诱变，活性仅降低两倍，表明它对于转录激活或增强不是必需的。
