Allele-specific transcription of the PAI-1 gene in human astrocytes.
The 4G allele of the PAI-1 -675(4G/5G) insertion/deletion promoter polymorphism has been associated with elevated plasma levels of PAI-1 and an increased risk of myocardial infarction. However, this allele has also been associated with a reduced risk of ischaemic stroke. In the brain, PAI-1 is mainly produced by astrocytes, and can reduce the neurotoxic effects exerted by tissue-type plasminogen activator during pathophysiologic conditions. The aim of the present study was to investigate whether the PAI-1 -675(4G/5G) polymorphism and the linked -844A/G polymorphism affect transcriptional activity of the PAI-1 gene in human astrocytes. Haplotype chromatin immunoprecipitation (haploChIP) was used in order to quantify allele-specific promoter activity in heterozygous cells. Protein-DNA interactions were investigated by electrophoretic mobility shift assay (EMSA). A clear allele-specific difference in PAI-1 gene expression was observed in astrocytes, where the haplotype containing the 4G and the -844A alleles was associated with higher transcriptional activity compared to the 5G and -844G-containing haplotype. EMSA revealed an allele-specific binding of nuclear proteins to the 4G/5G site as well as to the -844A/G site. Supershift experiments identified specific binding of the transcription factors Elf-1 and Elk-1 to the -844G allele. The relative impact of the different sites on allele-specific PAI-1 promoter activity remains to be determined. We demonstrate that common polymorphisms within the PAI-1 promoter affect transcriptional activity of the PAI-1 gene in human astrocytes, thus providing a possible molecular genetic mechanism behind the association between PAI-1 promoter variants and ischaemic stroke.
Hultman
Karin
K
Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, the Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
Tjarnlund-Wolf
Anna
A
Odeberg
Jacob
J
Eriksson
Per
P
Jern
Christina
C
D000483:Alleles; D001253:Astrocytes / Q000378:metabolism*; D001665:Binding Sites; D002545:Brain Ischemia / Q000235:genetics / Q000517:prevention & control; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D006239:Haplotypes; D006801:Humans; D054643:INDEL Mutation; D050836:Interferon Regulatory Factor-2 / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis / Q000235:genetics*; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism; D020521:Stroke / Q000235:genetics / Q000517:prevention & control; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D050807:ets-Domain Protein Elk-1 / Q000378:metabolism
C512897:ELF1 protein, human; C494112:ELK1 protein, human; C494229:IRF2 protein, human; D050836:Interferon Regulatory Factor-2; D009687:Nuclear Proteins; D017395:Plasminogen Activator Inhibitor 1; C051321:SERPINE1 protein, human; C495901:TP53 protein, human; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D050807:ets-Domain Protein Elk-1; D012319:RNA Polymerase II