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


OBJECTIVE
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.
Ceelie
H
H
Department of Haematology, Leiden University Medical Center, Leiden, the Netherlands. hceelie@lumc.nl
Spaargaren-Van Riel
C C
CC
De Jong
M
M
Bertina
R M
RM
Vos
H L
HL
D000818:Animals; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019992:Databases as Topic; D004742:Enhancer Elements, Genetic; D017353:Gene Deletion; D006367:HeLa Cells; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D051542:Hepatocyte Nuclear Factor 3-beta; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry*; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011516:Prothrombin / Q000235:genetics*; D012306:Risk; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000737:chemistry*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000737:chemistry*; D014158:Transcription, Genetic; D014162:Transfection; D020246:Venous Thrombosis / Q000378:metabolism
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C496502:FOXA2 protein, human; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051542:Hepatocyte Nuclear Factor 3-beta; D051539:Hepatocyte Nuclear Factor 1-beta; D051705:Sp3 Transcription Factor; D011516:Prothrombin; D008156:Luciferases