Deutsch P J, Hoeffler J P, Jameson J L, Lin J C, Habener J F
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Boston.
J Biol Chem. 1988 Dec 5;263(34):18466-72.
A transcriptional cAMP-responsive enhancer element (CRE) consisting of the 8-base pair (bp) palindrome, 5' TGACGTCA 3', is found in several eukaryotic genes. We analyzed the effects on gene transcription of point mutations within the CRE, the influence of the bases surrounding the CRE, and the requirements for transcriptional synergism of tandemly repeated CREs. When inserted as an oligonucleotide with restriction enzyme linker sites, the 8-bp CRE itself is as active in conferring cAMP responsivity on an enhancerless chloramphenicol acetyltransferase reporter plasmid as is a single copy of the choriogonadotropin alpha (CG alpha), twice repeated 18-bp sequence containing the CRE. Point mutations in the first (T to A), fourth (C to G), or eighth (A to T) positions of the CRE, when contained within the CG alpha 18-bp sequence, each inhibited transcriptional activity greater than 90%. However, the identical eighth position A to T mutation occurs in the cAMP-responsive sequence of the vasoactive intestinal peptide (VIP) gene, and that mutant sequence in the context of the adjacent bases of the native VIP sequence is maximally cAMP responsive when inserted in the reporter plasmid. The substantially reduced activity of the core 8-bp CRE when synthesized as a cassette including the adjacent bases of the rat glucagon or bovine parathyroid hormone gene further emphasizes the restrictive influence of particular surrounding sequences. Active oligonucleotides containing the 8-bp palindrome and different but equally permissive contexts have comparable properties in transfected reporter genes and gel mobility-shift assays. The pair of tandemly repeated 18-bp elements containing the CRE in the CG alpha gene synergistically stimulate transcription either with paired native CREs or when one native CRE is paired with one mutant CRE, suggesting the presence of cooperative interactions. Tandem insertion of more than two 18-bp sequences, or insertion of additional sequences between the two CREs, inhibits transcription. These observations indicate that the contexts of the bases adjacent to CREs exert profound influences on the transcriptional activities mediated by the cAMP-responsive elements.
一种由8个碱基对（bp）的回文序列5'TGACGTCA 3'组成的转录性环磷酸腺苷反应增强子元件（CRE），存在于多个真核基因中。我们分析了CRE内点突变对基因转录的影响、CRE周围碱基的影响以及串联重复CRE的转录协同作用的要求。当作为带有限制性内切酶连接位点的寡核苷酸插入时，8 bp的CRE本身在赋予无增强子的氯霉素乙酰转移酶报告质粒环磷酸腺苷反应性方面，与含有CRE的促性腺激素α（CGα）的单拷贝、重复两次的18 bp序列一样活跃。当包含在CGα 18 bp序列中时，CRE的第一（T到A）、第四（C到G）或第八（A到T）位置的点突变，每个都抑制转录活性超过90%。然而，相同的第八位A到T突变发生在血管活性肠肽（VIP）基因的环磷酸腺苷反应序列中，并且当插入报告质粒时，该突变序列在天然VIP序列相邻碱基的背景下具有最大的环磷酸腺苷反应性。当作为包含大鼠胰高血糖素或牛甲状旁腺激素基因相邻碱基的盒式结构合成时，核心8 bp CRE的活性大幅降低，这进一步强调了特定周围序列的限制作用。含有8 bp回文序列且具有不同但同样宽松背景的活性寡核苷酸，在转染的报告基因和凝胶迁移率变动分析中具有可比的性质。CGα基因中包含CRE的一对串联重复18 bp元件，与配对的天然CRE一起，或当一个天然CRE与一个突变CRE配对时，协同刺激转录，表明存在协同相互作用。串联插入两个以上的18 bp序列，或在两个CRE之间插入额外的序列，会抑制转录。这些观察结果表明，与CRE相邻的碱基背景对环磷酸腺苷反应元件介导的转录活性有深远影响。
