title: Identification of a DNA methylation-dependent activator sequence in the pseudoxanthoma elasticum gene, ABCC6.
abstract: ABCC6 encodes MRP6, a member of the ABC protein family with an unknown physiological role. The human ABCC6 and its two pseudogenes share 99% identical DNA sequence. Loss-of-function mutations of ABCC6 are associated with the development of pseudoxanthoma elasticum (PXE), a recessive hereditary disorder affecting the elastic tissues. Various disease-causing mutations were found in the coding region; however, the mutation detection rate in the ABCC6 coding region of bona fide PXE patients is only approximately 80%. This suggests that polymorphisms or mutations in the regulatory regions may contribute to the development of the disease. Here, we report the first characterization of the ABCC6 gene promoter. Phylogenetic in silico analysis of the 5' regulatory regions revealed the presence of two evolutionarily conserved sequence elements embedded in CpG islands. The study of DNA methylation of ABCC6 and the pseudogenes identified a correlation between the methylation of the CpG island in the proximal promoter and the ABCC6 expression level in cell lines. Both activator and repressor sequences were uncovered in the proximal promoter by reporter gene assays. The most potent activator sequence was one of the conserved elements protected by DNA methylation on the endogenous gene in non-expressing cells. Finally, in vitro methylation of this sequence inhibits the transcriptional activity of the luciferase promoter constructs. Altogether these results identify a DNA methylation-dependent activator sequence in the ABCC6 promoter.
authors.affiliation: Institute of Enzymology, Hungarian Academy of Sciences, H-1113 Budapest, Hungary. aranyi@enzim.hu
authors.forename: Tamás
authors.initials: T
authors.lastname: Arányi
authors.forename: Marcin
authors.initials: M
authors.lastname: Ratajewski
authors.forename: Viola
authors.initials: V
authors.lastname: Bardóczy
authors.forename: Lukasz
authors.initials: L
authors.lastname: Pulaski
authors.forename: András
authors.initials: A
authors.lastname: Bors
authors.forename: Attila
authors.initials: A
authors.lastname: Tordai
authors.forename: András
authors.initials: A
authors.lastname: Váradi
mesh_terms: D002460:Cell Line; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018899:CpG Islands; D019175:DNA Methylation; D018076:DNA, Complementary / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005808:Genes, Recessive; D017930:Genes, Reporter; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D011561:Pseudoxanthoma Elasticum / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013447:Sulfites / Q000737:chemistry; D014158:Transcription, Genetic
chemical_list: D018076:DNA, Complementary; D008156:Luciferases; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012333:RNA, Messenger; D013447:Sulfites; C102029:ABCC6 protein, human