Identification and characterization of cis-acting elements residing in the walleye dermal sarcoma virus promoter.
Identification and characterization of cis-acting elements residing in the walleye dermal sarcoma virus promoter.
复制标题
存在于白斑眼真皮肉瘤病毒启动子中的顺式作用元件的鉴定和表征。
DOI：
10.1128/jvi.78.14.7590-7601.2004
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Quackenbush,SandraL
中科院分区：
文献类型：
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作者：
Hronek,BrettW;Meagher,Ashley;Rovnak,Joel;Quackenbush,SandraL
瓦莱耶皮肤肉瘤病毒（WDSV）是一种复杂的逆转录病毒，发现与肿瘤的出现和消退的季节性基础上。在发展中的肿瘤和退化的肿瘤之间，病毒表达量存在定量和定性差异。为了了解宿主细胞因子在WDSV表达中的作用，采用了DNA酶I足迹分析、电泳迁移率变动分析（EMSA）和报告基因分析。DNA酶I的足迹分析的WDSV长末端重复的U3区域与核提取物制备的walleye细胞系显示保护的Oct 1，AP 1，Whn，和两个E4 BP 4网站。另外，三个不含推定的转录因子结合位点的区域被保护。EMSA证实了受保护位点的特异性结合，并揭示了另外三个位点，NF 1，AP 3和LVa，在DNA酶I足迹分析中不受保护。在荧光素酶报告质粒的背景下，对各个位点的定点诱变揭示了NF 1、Oct 1、AP 1、E4BP4#2、AP 3和LVa位点有助于由WDSV U3区域驱动的转录激活。Novel#2的突变导致荧光素酶活性的增加，表明Novel#2位点可以起到结合转录的负调节因子的作用。抗-Jun和抗-Fos抗血清特异性抑制蛋白质-DNA复合物的形成，表明在walleye细胞核提取物中存在c-Jun和c-Fos，并且它们参与与AP 1位点的结合。有趣的是，在U3区发现的变性15-bp重复序列在DNA酶I足迹分析中受到海象细胞系核提取物和退化肿瘤核提取物的差异保护。利用15 bp重复探针的EMSA显示，有与W12细胞和发展中的肿瘤细胞核提取物的结合的相似性，并与观察到的退化肿瘤细胞核提取物的结合不同。
Walleye dermal sarcoma virus (WDSV) is a complex retrovirus found associated with tumors that appear and regress on a seasonal basis. There are quantitative and qualitative differences in the amount of virus expression between developing and regressing tumors. To understand the role of host cell factors in WDSV expression, DNase I footprint analysis, electrophoretic mobility shift assays (EMSA), and reporter gene assays were employed. DNase I footprint analysis of the U3 region of the WDSV long terminal repeat with nuclear extract prepared from a walleye cell line revealed protection of an Oct1, AP1, Whn, and two E4BP4 sites. Additionally, three regions that contained no putative transcription factor binding sites were protected. EMSA confirmed the specific binding of the protected sites and revealed three additional sites, NF1, AP3, and LVa, not protected in DNase I footprint analysis. Site-directed mutagenesis of the individual sites, in the context of a luciferase reporter plasmid, revealed that the NF1, Oct1, AP1, E4BP4#2, AP3, and LVa sites contributed to transcription activation driven by the WDSV U3 region. Mutation of Novel#2 resulted in an increase in luciferase activity, suggesting the Novel#2 site may function to bind a negative regulator of transcription. Anti-Jun and anti-Fos antiserum specifically inhibited protein-DNA complex formation, indicating the presence of c-Jun and c-Fos in the walleye cell nuclear extracts and their participation in binding to the AP1 site. Interestingly, degenerative 15-bp repeats found in the U3 region are differentially protected in DNase I footprint analysis by the walleye cell line nuclear extract and regressing-tumor nuclear extract. EMSA utilizing the 15-bp repeat probe revealed that there are similarities of binding with W12 cell and developing-tumor nuclear extracts and that the binding differs from that observed with regressing-tumor nuclear extract.