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ABSTRACT: We earlier reported the formation of a unique nuclear NF1/Smad complex in serum-restricted fibroblasts that acts as an NF1-dependent repressor of the human adenine nucleotide translocase-2 gene (ANT2) [K. Luciakova, G. Kollarovic, P. Barath, B.D. Nelson, Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex, Biochem. J. 412 (2008) 123-130]. In the present study, we show that TGF-β, like serum-restriction: (a) induces the formation of NF1/Smad repressor complexes, (b) increases binding of the complexes to the repressor elements (Go elements) in the ANT2 promoter, and (c) inhibits ANT2 expression. Repression of ANT2 by TGF-β is eliminated by mutating the NF1 binding sites in the Go repressor elements. All of the above responses to TGF-β are prevented by inhibitors of TGF-β RI and MAPK p38. These inhibitors also prevent NF1/Smad4 repressor complex formation and repression of ANT2 expression in serum-restricted cells, suggesting that similar signaling pathways are initiated by TGF-β and serum-restriction. The present finding that NF1/Smad4 repressor complexes are formed through TGF-β signaling pathways suggests a new, but much broader, role for these complexes in the initiation or maintenance of the growth-inhibited state.
Biochemical and Biophysical Research Communications 08/2011; 411(3):648-53. · 2.48 Impact Factor
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ABSTRACT: NF1 (nuclear factor 1) binds to two upstream elements of the human ANT2 (adenine nucleotide translocator-2) promoter and actively represses expression of the gene in growth-arrested diploid skin fibroblasts [Luciakova, Barath, Poliakova, Persson and Nelson (2003) J. Biol. Chem. 278, 30624-30633]. ChIP (chromatin immunoprecipitation) and co-immunoprecipitation analyses of nuclear extracts from growth-arrested and growth-activated diploid cells demonstrate that NF1, when acting as a repressor, is part of a multimeric complex that also includes Smad and Sp-family proteins. This complex appears to be anchored to both the upstream NF1-repressor elements and the proximal promoter, Sp1-dependent activation elements in growth-arrested cells. In growth-activated cells, the repressor complex dissociates and NF1 leaves the promoter. As revealed by co-immunoprecipitation experiments, NF1-Smad4-Sp3 complexes are present in nuclear extracts only from growth-inhibited cells, suggesting that the growth-state-dependent formation of these complexes is not an ANT2 promoter-specific event. Consistent with the role of Smad proteins in the repression complex, TGF-beta (transforming growth factor-beta) can fully repress ANT2 transcription in normally growing fibroblasts. Finally, pull-down experiments of in vitro transcribed/translated NF1 isoforms by GST (glutathione transferase)-Smad and GST-Smad MH fusion proteins indicate direct physical interactions between members of the two families. These findings suggest a possible functional relationship between the NF1 and Smad proteins that has not been previously observed.
Biochemical Journal 06/2008; 412(1):123-30. · 4.90 Impact Factor
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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.
European Journal of Biochemistry 06/2004; 271(9):1781-8. · 3.58 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 09/2003; 278(33):30624-33. · 4.77 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 08/2003; 278(33):30624-30633. · 4.77 Impact Factor
