title: Effect of Regulatory Element DNA Methylation on Tissue-Type Plasminogen Activator Gene Expression.
abstract: Expression of the tissue-type plasminogen activator gene (t-PA; gene name PLAT) is regulated, in part, by epigenetic mechanisms. We investigated the relationship between PLAT methylation and PLAT expression in five primary human cell types and six transformed cell lines. CpG methylation was analyzed in the proximal PLAT gene promoter and near the multihormone responsive enhancer (MHRE) -7.3 kilobase pairs upstream of the PLAT transcriptional start site (TSS, -7.3 kb). In Bowes melanoma cells, the PLAT promoter and the MHRE were fully unmethylated and t-PA secretion was extremely high. In other cell types the region from -647 to -366 was fully methylated, whereas an unmethylated stretch of DNA from -121 to +94 was required but not sufficient for detectable t-PA mRNA and t-PA secretion. DNA methylation near the MHRE was not correlated with t-PA secretion. Specific methylation of the PLAT promoter region -151 to +151, inserted into a firefly luciferase reporter gene, abolished reporter gene activity. The region -121 to + 94 contains two well-described regulatory elements, a PMA-responsive element (CRE) near -106 and a GC-rich region containing an Sp1 binding site near +59. Methylation of double-stranded DNA oligonucleotides containing the CRE or the GC-rich region had little or no effect on transcription factor binding. Methylated CpGs may attract co-repressor complexes that contain histone deacetylases (HDAC). However, reporter gene activity of methylated plasmids was not restored by the HDAC inhibitor trichostatin. In conclusion, efficient PLAT gene expression requires a short stretch of unmethylated CpG sites in the proximal promoter.
authors.affiliation: Division of Angiology and Hemostasis, University Medical Center, University of Geneva, Geneva, Switzerland.
authors.forename: Sylvie
authors.identifier: http://orcid.org/0000-0001-5443-0063
authors.initials: S
authors.lastname: Dunoyer-Geindre
authors.affiliation: Division of Angiology and Hemostasis, University Medical Center, University of Geneva, Geneva, Switzerland.
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Rivier-Cordey
authors.affiliation: Division of Angiology and Hemostasis, University Medical Center, University of Geneva, Geneva, Switzerland.
authors.forename: Carlos
authors.initials: C
authors.lastname: Caetano
authors.affiliation: Department of Genetic Medicine and Development, University Medical Center, University of Geneva, Geneva, Switzerland.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Fish
authors.affiliation: Division of Angiology and Hemostasis, University Medical Center, University of Geneva, Geneva, Switzerland.
authors.forename: Egbert K O
authors.initials: EK
authors.lastname: Kruithof
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D019175:DNA Methylation / Q000235:genetics*; D044127:Epigenesis, Genetic / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000235:genetics*; D024363:Transcription Initiation Site / Q000502:physiology; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D010959:Tissue Plasminogen Activator