人凝血酶原基因增强子中HNF1-α、HNF3-β（FOXA2）、HNF4-α、Sp1和Sp3转录因子结合位点的功能特性

Functional characterization of transcription factor binding sites for HNF1-alpha, HNF3-beta (FOXA2), HNF4-alpha, Sp1 and Sp3 in the human prothrombin gene enhancer.

作者信息

Ceelie H, Spaargaren-Van Riel C C, De Jong M, Bertina R M, Vos H L

机构信息

Department of Haematology, Leiden University Medical Center, Leiden, the Netherlands.

出版信息

J Thromb Haemost. 2003 Aug;1(8):1688-98. doi: 10.1046/j.1538-7836.2003.00393.x.

DOI:10.1046/j.1538-7836.2003.00393.x

PMID:12911579

Abstract

BACKGROUND

Prothrombin is a key component in blood coagulation. Overexpression of prothrombin leads to an increased risk of venous thrombosis. Therefore, the study of the transcriptional regulation of the prothrombin gene may help to identify mechanisms of overexpression.

OBJECTIVES

The aim of our study was to localize the regions within the prothrombin enhancer responsible for its activity, to identify the proteins binding to these regions, and to establish their functional importance.

METHODS

We constructed a set of prothrombin promoter 5' deletion constructs containing the firefly luciferase reporter gene, which were transiently transfected in HepG2, HuH7 and HeLa cells. Putative transcription factor (TF) binding sites were evaluated by electrophoretic mobility shift assays. The functional importance of each TF binding site was evaluated by site directed mutagenesis and transient transfection of the mutant constructs.

RESULTS

We confirmed the major contribution of the enhancer region to the transcriptional activity of the prothrombin promoter. Analysis of this region revealed putative binding sites for hepatocyte nuclear factor HNF4, HNF3-beta and specificity protein(Sp)1. We identified six different TFs binding to three evolutionary conserved sites in the enhancer: HNF4-alpha (site 1), HNF1-alpha, HNF3-beta and an as yet unidentified TF (site 2) and the ubiquitously expressed TFs Sp1 and Sp3 (site 3). Mutagenesis studies showed that loss of binding of HNF3-beta resulted in a considerable decrease of enhancer activity, whereas loss of HNF4-alpha or Sp1/Sp3 resulted in milder reductions.

CONCLUSIONS

The prothrombin enhancer plays a major role in regulation of prothrombin expression. Six different TFs are able to bind to this region. At least three of these TFs, HNF4-alpha, HNF3-beta and Sp1/Sp3, are important in regulation of prothrombin expression.

摘要

背景

凝血酶原是血液凝固中的关键成分。凝血酶原的过表达会导致静脉血栓形成风险增加。因此，研究凝血酶原基因的转录调控可能有助于确定过表达的机制。

目的

我们研究的目的是定位凝血酶原增强子中负责其活性的区域，鉴定与这些区域结合的蛋白质，并确定它们的功能重要性。

方法

我们构建了一组含有萤火虫荧光素酶报告基因的凝血酶原启动子5'缺失构建体，将其瞬时转染到HepG2、HuH7和HeLa细胞中。通过电泳迁移率变动分析评估假定的转录因子（TF）结合位点。通过定点诱变和突变构建体的瞬时转染评估每个TF结合位点的功能重要性。

结果

我们证实了增强子区域对凝血酶原启动子转录活性的主要贡献。对该区域的分析揭示了肝细胞核因子HNF4、HNF3-β和特异性蛋白（Sp）1的假定结合位点。我们鉴定出六种不同的TF与增强子中的三个进化保守位点结合：HNF4-α（位点1）、HNF1-α、HNF3-β和一种尚未鉴定的TF（位点2）以及普遍表达的TF Sp1和Sp3（位点3）。诱变研究表明，HNF3-β结合的丧失导致增强子活性显著降低，而HNF4-α或Sp1/Sp3的丧失导致的降低较轻微。