Finotti Alessia, Treves Susan, Zorzato Francesco, Gambari Roberto, Feriotto Giordana
Department of Biochemistry and Molecular Biology, Molecular Biology Section, University of Ferrara, Via Fossato di Mortara 74, 44100 Ferrara, Italy.
BMC Mol Biol. 2008 Dec 16;9:110. doi: 10.1186/1471-2199-9-110.
Alternative splicing of the locus A beta H-J-J generates functionally distinct proteins: the enzyme aspartyl (asparaginyl) beta-hydroxylase (AAH), truncated homologs of AAH with a role in calcium homeostasis humbug and junctate and a structural protein of the sarcoplasmic reticulum membranes junctin. AAH and humbug are over expressed in a broad range of malignant neoplasms. We have previously reported that this locus contains two promoters, P1 and P2. While AAH and humbug are expressed in most tissues under the regulation of the P1 promoter, AAH, junctin and junctate are predominantly expressed in excitable tissues under the control of the P2 promoter. We previously demonstrated that Sp transcription factors positively regulate the P1 promoter.
In the present study, we extended the functional characterization of the P1 promoter of the A beta H-J-J locus. We demonstrated by quantitative Real-time RT-PCR that mRNAs from the P1 promoter are actively transcribed in all the human cell lines analysed. To investigate the transcription mechanism we transiently transfected HeLa cells with sequentially deleted reporter constructs containing different regions of the -661/+81 P1 nucleotide sequence. Our results showed that (i) this promoter fragment is a powerful activator of the reporter gene in HeLa cell line, (ii) the region spanning 512 bp upstream of the transcription start site exhibits maximal level of transcriptional activity, (iii) progressive deletions from -512 gradually reduce reporter expression. The region responsible for maximal transcription contains an E-box site; we characterized the molecular interactions between USF1/2 with this E-box element by electrophoretic mobility shift assay and supershift analysis. In addition, our USF1 and USF2 chromatin immunoprecipitation results demonstrate that these transcription factors bind the P1 promoter in vivo. A functional role of USF1/USF2 in upregulating P1-directed transcription was demonstrated by analysis of the effects of (i) in vitro mutagenesis of the P1/E-box binding site, (ii) RNA interference targeting USF1 transcripts.
Our results suggest that USF factors positively regulate the core of P1 promoter, and, together with our previously data, we can conclude that both Sp and USF DNA interaction and transcription activity are involved in the P1 promoter dependent expression of AAH and humbug.
β-H-J-J基因座的可变剪接产生功能不同的蛋白质：天冬氨酰（天冬酰胺酰）β-羟化酶（AAH）、在钙稳态中起作用的AAH截短同源物humbug和连接蛋白以及肌浆网膜连接蛋白的一种结构蛋白。AAH和humbug在多种恶性肿瘤中过度表达。我们之前报道过该基因座包含两个启动子，P1和P2。虽然AAH和humbug在P1启动子的调控下在大多数组织中表达，但AAH、连接蛋白和连接点蛋白主要在P2启动子的控制下在可兴奋组织中表达。我们之前证明Sp转录因子正向调控P1启动子。
在本研究中，我们扩展了β-H-J-J基因座P1启动子的功能特性。我们通过定量实时逆转录聚合酶链反应证明，来自P1启动子的mRNA在所有分析的人类细胞系中均有活跃转录。为了研究转录机制，我们用含有-661/+81 P1核苷酸序列不同区域的连续缺失报告基因构建体瞬时转染HeLa细胞。我们的结果表明：（i）该启动子片段是HeLa细胞系中报告基因的强大激活剂；（ii）转录起始位点上游512 bp的区域表现出最大水平的转录活性；（iii）从-512开始的逐步缺失逐渐降低报告基因的表达。负责最大转录的区域包含一个E-box位点；我们通过电泳迁移率变动分析和超迁移分析表征了USF1/2与该E-box元件之间的分子相互作用。此外，我们的USF1和USF2染色质免疫沉淀结果表明，这些转录因子在体内与P1启动子结合。通过分析（i）P1/E-box结合位点的体外诱变、（ii）靶向USF1转录本的RNA干扰的作用，证明了USF1/USF2在上调P1指导的转录中的功能作用。
我们的结果表明，USF因子正向调控P1启动子的核心，并且，结合我们之前的数据，我们可以得出结论，Sp和USF的DNA相互作用及转录活性均参与AAH和humbug的P1启动子依赖性表达。
