Functional role of a conformationally flexible homopurine/homopyrimidine domain of the androgen receptor gene promoter interacting with SP1 and a pyrimidine single strand DNA-binding protein
Functional role of a conformationally flexible homopurine/homopyrimidine domain of the androgen receptor gene promoter interacting with SP1 and a pyrimidine single strand DNA-binding protein
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DOI：
10.1210/me.11.1.3
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发表时间：
1997-01-01
影响因子：
--
通讯作者：
Roy, AK
中科院分区：
文献类型：
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作者：
Chen, S;Supakar, PC;Roy, AK
雄激素受体（AR）基因启动子不包含TATA或CAAT盒，但它包含一个长（类似于90 bp）的同型嘌呤/同型嘧啶（pur/pyr）延伸紧邻Sp1结合GC盒位点的上游。该pur/pyr延伸在大鼠、小鼠和人AR基因启动子中的相同近端位置处是保守的。该区域的突变导致启动子活性下降3倍，表明其具有重要的调控功能。用单链特异性S1核酸酶检查AR pur/pyr区域的构象状态表明，它能够形成涉及未配对单链的非B DNA结构。精细映射的S1-敏感位点显示了不对称的裂解模式指示的分子内三重螺旋H-型DNA构象。电泳迁移率变动分析表明，AR启动子的pur/pyr区域可以结合一种新的嘧啶单链特异性蛋白（ssPyrBF），也可以结合一种双链DNA结合蛋白。寡核苷酸交叉竞争和抗体超移位实验都确定了双链结合蛋白与Sp1等同。脱氧核糖核酸酶I（DNase I）足迹分析表明，多个Sp1结合的pur/pyr网站和较弱的Sp1相互作用，这一地区相比，相邻的GC盒，其中Sp1的功能，以招募TFIID复合物。这些结果表明，AR基因的pur/pyr结构域可以用来吸引额外的Sp1分子时，它存在于双链B-DNA构象。然而，ssPyrBF的结合和由此产生的非B DNA结构的稳定预期将阻止其与Sp1的相互作用。我们推测，在TATA-少AR基因启动子，多个弱Spl位点的pur/pyr区域相邻的GC盒可以提供一个现成的来源，这种转录因子的功能GC盒，从而促进组装的起始复合物。
The androgen receptor (AR) gene promoter does not contain the TATA or CAAT box, but it contains a long (similar to 90-bp) homopurine/homopyrimidine (pur/pyr) stretch immediately upstream of the Sp1-binding GC box site. This pur/pyr stretch is conserved at the same proximal position in the rat, mouse, and human AR gene promoters. Mutation of this region results in a 3-fold decline in promoter activity, indicating an important regulatory function. Examination of the conformational state of the AR pur/pyr region with the single-strand-specific S1 nuclease showed that it is capable of forming a non-B DNA structure involving unpaired single strands. Fine mapping of the S1-sensitive site revealed an unsymmetric cleavage pattern indicative of an intramolecular triple helical H-form DNA conformation. Electrophoretic mobility shift analyses showed that the pur/pyr region of the AR promoter can bind a novel pyrimidine single-strand-specific protein (ssPyrBF) and also a double-strand DNA-binding protein. Both oligonucleotide cross-competition and antibody supershift experiments established that the double-strand binding protein is equivalent to Sp1. Deoxyribonuclease I (DNase I) footprinting analysis showed multiple Sp1-binding to the pur/pyr site and a weaker Sp1 interaction to this region compared with the adjacently located GC box, where Sp1 functions to recruit the TFIID complex. These results suggest that the pur/pyr domain of the AR gene can serve to attract additional Sp1 molecules when it exists in the double-stranded B-DNA conformation. However, binding of ssPyrBF and the resultant stabilization of the non-B DNA structure is expected to prevent its interaction with Sp1. We speculate that in the TATA-less AR gene promoter, multiple weak Spl sites at the pur/pyr region adjacent to the GC box can provide a readily available source of this transcription factor to the functional GC box, thereby facilitating the assembly of the initiation complex.