Yang Jun, Everett Allen D
Department of Pediatrics, Division of Pediatric Cardiology, Johns Hopkins University, Baltimore, MD 21205, USA.
BMC Mol Biol. 2007 Oct 31;8:101. doi: 10.1186/1471-2199-8-101.
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.
肝癌衍生生长因子（HDGF）是一种核蛋白，其核靶向作用是有丝分裂活性所必需的。最近我们证明HDGF是一种转录抑制因子，但HDGF是否结合DNA、DNA结合的特异性以及需要何种蛋白质结构域仍不清楚。在本研究中，我们旨在确定HDGF是否为DNA结合蛋白，绘制HDGF的功能性DNA结合结构域和DNA结合元件图谱。
通过对人类DNA进行染色质免疫沉淀（ChIP），我们分离出10个共享约200 bp保守元件的DNA序列。同源性分析确定这些结合序列为HDGF靶基因SMYD1基因启动子内的一个基序。电泳迁移率变动分析（EMSA）证实HDGF与该保守序列结合。结果，位于SMYD1启动子中的一个80 bp保守序列与GST-HDGF紧密结合。使用从5'和3'末端的缺失定位策略，将HDGF的结合核心序列缩小至37 bp。此外，ChIP和DNase I足迹分析表明HDGF特异性结合这个80 bp的DNA片段。在功能上，HDGF的过表达抑制了一个由含有80 bp DNA元件的SV-40启动子控制的报告基因。通过对GST-HDGF进行连续截短，我们将HDGF的DNA结合结构域定位到N端PWWP结构域。
HDGF是一种DNA结合蛋白，能特异性结合DNA，且偏好至少37 bp长的DNA片段。HDGF的N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元件结合发挥其转录抑制作用。
