Hepatoma-derived growth factor binds DNA through the N-terminal PWWP domain.
Hepatoma-derived growth factor binds DNA through the N-terminal PWWP domain.
复制标题
肝癌衍生的生长因子通过N末端pwwp结构域结合DNA。
DOI：
10.1186/1471-2199-8-101
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发表时间：
2007-10-31
影响因子：
--
通讯作者：
Everett, Allen D.
中科院分区：
文献类型：
--
作者：
Yang, Jun;Everett, Allen D.
肝癌衍生生长因子（HDGF）是一种核蛋白，具有促有丝分裂活性所需的核靶向作用。近年来，我们发现HDGF是一种转录抑制因子，但HDGF是否与DNA结合，结合DNA的特异性以及需要什么样的蛋白结构域尚不清楚。本研究的目的是鉴定HDGF是否为DNA结合蛋白，定位HDGF的功能性DNA结合结构域和DNA结合元件。使用染色质免疫沉淀（ChIP）的人类DNA，我们分离出10个DNA序列共享一个保守的~200 bp的元素。同源性分析鉴定了结合序列作为SMYD 1基因（HDGF靶基因）启动子内的基序。电泳迁移率变动分析（EMSA）证实了HDGF与该保守序列的结合。结果表明，SMYD 1基因启动子区有一段80 bp的保守序列与GST-HDGF紧密结合。使用缺失作图策略从5'和3'端将HDGF的结合核心序列缩小至37 bp。此外，ChIP和DNase I足迹分析显示HDGF特异性结合该80 bp的DNA片段。HDGF的功能性过表达抑制由含有80 bp DNA元件的SV-40启动子控制的报告基因。使用GST-HDGF的连续截短，我们将HDGF的DNA结合结构域映射到N-末端PWWP结构域。HDGF是一种DNA结合蛋白，特异性结合DNA，并且优选最小37 bp长的DNA片段。HDGF的N-末端PWWP结构域是DNA结合所必需的。HDGF通过与靶基因启动子中的保守DNA元件结合而发挥其转录抑制作用。
Hepatoma Derived Growth Factor (HDGF) is a nuclear protein with nuclear targeting required for mitogenic activity. Recently we demonstrated that HDGF is a transcriptional repressor, but whether HDGF binds DNA, the specificity of DNA binding and what protein domain is required are still unknown. In this study, we aimed to identify if HDGF is a DNA binding protein, map the functional DNA binding domain and DNA binding element for HDGF. Using chromatin immunoprecipitation (ChIP) of human DNA, we isolated 10 DNA sequences sharing a conserved ~200 bp element. Homology analysis identified the binding sequences as a motif within the promoter of the SMYD1 gene, a HDGF target gene. Electrophoretic Mobility Shift Assays (EMSA) confirmed the binding of HDGF to this conserved sequence. As a result, an 80 bp conserved sequence located in the SMYD1 promoter bound GST-HDGF tightly. The binding core sequence for HDGF was narrowed down to 37 bp using a deletion mapping strategy from both the 5' and 3' ends. Moreover, ChIP and DNase I footprinting analysis revealed that HDGF binds this 80 bp DNA fragment specifically. Functionally overexpression of HDGF represses a reporter gene which is controlled by an SV-40 promoter containing the 80 bp DNA element. Using serial truncations of GST-HDGF, we mapped the DNA binding domain of HDGF to the N-terminal PWWP domain. HDGF is a DNA binding protein, binds DNA specifically, and prefers a minimum of 37 bp long DNA fragment. The N-terminal PWWP domain of HDGF is required for DNA binding. HDGF exerts its transcription repressive effect through binding to a conserved DNA element in the promoter of target genes.
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