title: New PAH gene promoter KLF1 and 3'-region C/EBPalpha motifs influence transcription in vitro.
abstract: Phenylketonuria (PKU) is a metabolic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. Although the PAH genotype remains the main determinant of PKU phenotype severity, genotype-phenotype inconsistencies have been reported. In this study, we focused on unanalysed sequences in non-coding PAH gene regions to assess their possible influence on the PKU phenotype. We transiently transfected HepG2 cells with various chloramphenicol acetyl transferase (CAT) reporter constructs which included PAH gene non-coding regions. Selected non-coding regions were indicated by in silico prediction to contain transcription factor binding sites. Furthermore, electrophoretic mobility shift assay (EMSA) and supershift assays were performed to identify which transcriptional factors were engaged in the interaction. We found novel KLF1 motif in the PAH promoter, which decreases CAT activity by 50 % in comparison to basal transcription in vitro. The cytosine at the c.-170 promoter position creates an additional binding site for the protein complex involving KLF1 transcription factor. Moreover, we assessed for the first time the role of a multivariant variable number tandem repeat (VNTR) region located in the 3'-region of the PAH gene. We found that the VNTR3, VNTR7 and VNTR8 constructs had approximately 60 % of CAT activity. The regulation is mediated by the C/EBPalpha transcription factor, present in protein complex binding to VNTR3. Our study highlighted two novel promoter KLF1 and 3'-region C/EBPalpha motifs in the PAH gene which decrease transcription in vitro and, thus, could be considered as PAH expression modifiers. New transcription motifs in non-coding regions will contribute to better understanding of the PKU phenotype complexity and may become important for the optimisation of PKU treatment.
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010, Belgrade, Serbia.
authors.forename: Kristel
authors.initials: K
authors.lastname: Klaassen
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010, Belgrade, Serbia.
authors.forename: Biljana
authors.initials: B
authors.lastname: Stankovic
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010, Belgrade, Serbia.
authors.forename: Nikola
authors.initials: N
authors.lastname: Kotur
authors.affiliation: Mother and Child Health Care Institute of Serbia "Dr Vukan Cupic", School of Medicine, University of Belgrade, Radoja Dakića 6-8, 11070, Belgrade, Serbia.
authors.forename: Maja
authors.initials: M
authors.lastname: Djordjevic
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010, Belgrade, Serbia.
authors.forename: Branka
authors.initials: B
authors.lastname: Zukic
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010, Belgrade, Serbia.
authors.forename: Gordana
authors.initials: G
authors.lastname: Nikcevic
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010, Belgrade, Serbia.
authors.forename: Milena
authors.initials: M
authors.lastname: Ugrin
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010, Belgrade, Serbia.
authors.forename: Vesna
authors.initials: V
authors.lastname: Spasovski
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010, Belgrade, Serbia.
authors.forename: Sanja
authors.initials: S
authors.lastname: Srzentic
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010, Belgrade, Serbia.
authors.forename: Sonja
authors.initials: S
authors.lastname: Pavlovic
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010, Belgrade, Serbia. maja.stojiljkovic@imgge.bg.ac.rs.
authors.forename: Maja
authors.initials: M
authors.lastname: Stojiljkovic
mesh_terms: D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D005838:Genotype; D056945:Hep G2 Cells; D006801:Humans; D007438:Introns; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism*; D018598:Minisatellite Repeats; D010641:Phenotype; D010651:Phenylalanine Hydroxylase / Q000235:genetics*; D010661:Phenylketonurias / Q000235:genetics; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; C515848:CEBPA protein, human; D051741:Kruppel-Like Transcription Factors; C080774:erythroid Kruppel-like factor; D010651:Phenylalanine Hydroxylase
keywords: 3′ Non-coding region; Phenylalanine hydroxylase gene; Phenylketonuria; Promoter; Silencer; Transcription