title: Identification of NF1 as a silencer protein of the human adenine nucleotide translocase-2 gene.
abstract: The human adenine nucleotide translocase-2 (ANT2) promoter contains a silencer region that confers partial repression on the heterologous herpes simplex virus thymidine kinase (HSVtk) promoter [Barath, P., Albert-Fournier, B., Luciakova, K., Nelson, B.D. (1999) J. Biol. Chem.274, 3378-3384]. Two sequences in the silencer (Site-2 and Site-3) are protected in the DNase I assay in vitro, and one of these is a repeated GTCCTG element previously shown to act as the active repressor element. We have now purified the DNA binding protein, and identified it using MALDI-TOF MS as a 33-kDa member of the nuclear factor 1 (NF1) family of transcription factors. NF1 purified from rat liver and HeLa cell nuclei bind to both silencer Site-2 and Site-3, resulting in a DNase I footprint identical to that obtained with purified recombinant NF1. Furthermore, transient transfection experiments with reporter constructs containing mutated silencer Site-2 and/or Site-3 show that both sites contribute to repression of the HSVtk promoter. Finally, chromatin immunoprecipitation analysis reveals that NF1 is bound to both elements on the endogenous HeLa cell ANT2 promoter. Our data support the belief that NF1 acts as a repressor when bound to silencing Site-2 and Site-3 of the ANT2 gene.
authors.affiliation: Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: Barath
authors.forename: Daniela
authors.initials: D
authors.lastname: Poliakova
authors.forename: Katarina
authors.initials: K
authors.lastname: Luciakova
authors.forename: B Dean
authors.initials: BD
authors.lastname: Nelson
mesh_terms: D033742:Adenine Nucleotide Translocator 2 / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*
chemical_list: D033742:Adenine Nucleotide Translocator 2; D022762:CCAAT-Enhancer-Binding Proteins; D051839:NFI Transcription Factors; D012097:Repressor Proteins; D014157:Transcription Factors; D004247:DNA