Repression of the human adenine nucleotide translocase-2 gene in growth-arrested human diploid cells: the role of nuclear factor-1.
Adenine nucleotide translocase-2 (ANT2) catalyzes the exchange of ATP for ADP across the mitochondrial membrane, thus playing an important role in maintaining the cytosolic phosphorylation potential required for cell growth. Expression of ANT2 is activated by growth stimulation of quiescent cells and is down-regulated when cells become growth-arrested. In this study, we address the mechanism of growth arrest repression. Using a combination of transfection, in vivo dimethyl sulfate mapping, and in vitro DNase I mapping experiments, we identified two protein-binding elements (Go-1 and Go-2) that are responsible for growth arrest of ANT2 expression in human diploid fibroblasts. Proteins that bound the Go elements were purified and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as members of the NF1 family of transcription factors. Chromatin immunoprecipitation analysis showed that NF1 was bound to both Go-1 and Go-2 in quiescent human diploid cells in vivo, but not in the same cells stimulated to growth by serum. NF1 binding correlated with the disappearance of ANT2 transcripts in quiescent cells. Furthermore, overexpression of NF1-A, -C, and -X in NIH3T3 cells repressed expression of an ANT2-driven reporter gene construct. Two additional putative repressor elements in the ANT2 promoter, an Sp1 element juxtaposed to the transcription start site and a silencer centered at nucleotide -332, did not appear to contribute to growth arrest repression. Thus, enhanced binding of NF1 is a key step in the growth arrest repression of ANT2 transcription. To our knowledge, this is the first report showing a role for NF1 in growth arrest.
Luciakova
Katarina
K
Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden. Katarina.Luciakova@savba.sk
Barath
Peter
P
Poliakova
Daniela
D
Persson
Annika
A
Nelson
B Dean
BD
D016475:3T3 Cells; D033742:Adenine Nucleotide Translocator 2 / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D004268:DNA-Binding Proteins; D004171:Diploidy; D015536:Down-Regulation / Q000502:physiology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic / Q000502:physiology; D016192:Resting Phase, Cell Cycle / Q000502:physiology*; D012867:Skin / Q000166:cytology; D013489:Suppression, Genetic; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000502:physiology; D051840:Y-Box-Binding Protein 1
D033742:Adenine Nucleotide Translocator 2; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human