Smith M F, Eidlen D, Brewer M T, Eisenberg S P, Arend W P, Gutierrez-Hartmann A
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
J Immunol. 1992 Sep 15;149(6):2000-7.
To study the molecular mechanisms involved in transcriptional regulation of the human IL-1R antagonist (IL-1ra) we have isolated 1680-bp of 5'-flanking region DNA from the IL-1ra gene. This region of DNA was sequenced and cloned into the luciferase expression vector pA3Luc (pRA-1680.Luc) for use in gene transfer studies aimed at determining the cis-acting DNA elements required for IL-1ra expression. Sequence analysis of the IL-1ra promoter revealed a TATAA box at -26, with consensus sequences for possible NF-kB-, NFIL-1 beta A-, AP-1-, and CRE-binding sites located further upstream. When transfected into a variety of human and murine cell lines, the cloned IL-1ra promoter was preferentially active in those cell lines in which expression of the endogenous IL-1ra gene could be detected. The cloned promoter and the endogenous IL-1ra promoter utilized the same transcriptional start site. This promoter activity was LPS-inducible in the RAW 264.7 murine macrophage cell line. In the human monocytic cell line U937, IL-1ra promoter activity was inducible by LPS or PMA treatment, but the combination of LPS and PMA led to the greatest increase in promoter activity, identical to the pattern of expression of endogenous IL-1ra mRNA as detected by polymerase chain reaction analysis. A series of 5'-truncated promoter constructs having a common 3'-end at +27 were created to map potential cis-acting transcriptional elements important for full IL-1ra promoter activity. Removal of sequences between -294 and -148 led to a greater than 90% decrease in both unstimulated and LPS-induced promoter activity; further deletion to -85 led to an almost complete abrogation of promoter activity. These studies demonstrate that the cloned IL-1ra promoter behaves in a manner consistent with that of the endogenous gene. Two regions within the IL-1ra promoter are identified which are required for full promoter activity.
为了研究参与人类白细胞介素-1受体拮抗剂（IL-1ra）转录调控的分子机制，我们从IL-1ra基因中分离出了1680 bp的5'侧翼区DNA。对该DNA区域进行测序，并将其克隆到荧光素酶表达载体pA3Luc（pRA-1680.Luc）中，用于基因转移研究，旨在确定IL-1ra表达所需的顺式作用DNA元件。IL-1ra启动子的序列分析显示，在-26处有一个TATAA框，在更上游还存在可能的NF-κB、NFIL-1βA、AP-1和CRE结合位点的共有序列。当转染到多种人类和小鼠细胞系中时，克隆的IL-1ra启动子在那些能够检测到内源性IL-1ra基因表达的细胞系中具有优先活性。克隆的启动子和内源性IL-1ra启动子使用相同的转录起始位点。这种启动子活性在RAW 264.7小鼠巨噬细胞系中可被脂多糖（LPS）诱导。在人类单核细胞系U937中，IL-1ra启动子活性可被LPS或佛波酯（PMA）处理诱导，但LPS和PMA的组合导致启动子活性增加最大，这与通过聚合酶链反应分析检测到的内源性IL-1ra mRNA的表达模式相同。构建了一系列在+27处具有共同3'末端的5'截短启动子构建体，以定位对完整IL-1ra启动子活性重要的潜在顺式作用转录元件。去除-294至-148之间的序列导致未刺激和LPS诱导的启动子活性均下降超过90%；进一步缺失至-85导致启动子活性几乎完全丧失。这些研究表明，克隆的IL-1ra启动子的行为与内源性基因一致。确定了IL-1ra启动子内的两个区域，它们是完整启动子活性所必需的。
