Structural determinants for transcriptional activation by cAMP-responsive DNA elements.
Structural determinants for transcriptional activation by cAMP-responsive DNA elements.
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发表时间：
1988-12
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通讯作者：
P. Deutsch;J. Hoeffler;J. Jameson;J. Lin;J. Habener
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作者：
P. Deutsch;J. Hoeffler;J. Jameson;J. Lin;J. Habener
CAMP反应增强子元件(CRE)由8个碱基对(BP)回文序列5‘TGACGTCA3’组成，存在于多种真核基因中。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，以及双重复Cre转录协同的要求。当作为带有限制酶连接点的寡核苷酸插入时，8-bp的Cre本身在赋予无增强型氯霉素乙酰转移酶报告质粒上的cAMP响应性方面与绒毛膜促性腺激素α(CGα)的单拷贝一样活跃，重复两次包含Cre的18-BP序列。Cre的第一(T到A)、第四(C到G)或第八(A到T)位置的点突变，当包含在CGα18BP序列中时，每个位置都抑制了90%以上的转录活性。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cAMP反应序列中，并且该突变序列在天然VIP序列相邻碱基的背景下，当插入到报告质粒中时，对cAMP反应最大。当合成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时，核心8-碱基Cre的活性大大降低，进一步强调了特定周围序列的限制性影响。含有8个碱基回文的活性寡核苷酸和不同但同样允许的上下文在转基因报告基因和凝胶迁移率移位分析中具有类似的性质。CGα基因中含有Cre的一对随机重复的18个碱基元件与成对的天然Cre或当一个天然Cre与一个突变Cre配对时协同刺激转录，这表明存在协同作用。串联插入两个以上18个碱基的序列，或在两个CRE之间插入额外的序列，会抑制转录。这些观察表明，与Cres相邻的碱基的环境对cAMP反应元件介导的转录活动产生了深远的影响。
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.