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

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.

Crosstalk between Nuclear Factor I-C and Transforming Growth Factor-β1 Signaling Regulates Odontoblast Differentiation and Homeostasis

“…It was previously reported that Smad3, but not Smad2, binds to NFI-C through its MH1 domain [14]. Therefore, we looked for interactions between the Smads and NFI-C in immunoprecipitation assays.…”

“…The NFIC-binding site was found to overlap with the TGF-β response element in the promoter regions of extracellular matrix genes, including rat bone sialoprotein (BSP) [10], the plasminogen activator inhibitor gene [11], and ColIa1 [12]. In addition, experiments searching for DNA-binding domains of Smads and members of the NFI/CAAT box-binding family indicated that they belong to a new superfamily of genes [13], and that Smad3 and Smad4 interact strongly with NFI-C through their MH1 domains [14]. However, the exact mechanism of the interaction between NFI-C and TGF-β signaling remains unclear.…”

Differential CpG island methylation of murine adenine nucleotide translocase genes

“…Ant2 expression is ubiquitous but growth-dependent [8,21,22]. The GRBOX (glycolysis regulated box) motif, Sp1-binding elements, and NF1 binding site in the Ant2 gene promoter are proposed to regulate Ant2 expression in a cell cycle-dependent manner and also depending on cellular glycolysis status [4,23-25]. Ant4 expression is exclusively detected in male germ cells and is particularly high during meiosis [10].…”

Adenine nucleotide translocase family: four isoforms for apoptosis modulation in cancer

“…Two of the SP1 elements (conserved AB box) have been shown to activate ANT2 transcription, while the third element (C box organization) has been shown to inhibit gene transcription. In addition, Luciakova et al (2008) provided evidence for an upstream-binding site for NF1, an active repressor of the ANT2 gene. ANT2 transcription is up-regulated in growth-activated cells and down-regulated in cells exiting the cell cycle upon serum restriction or density-dependent growth inhibition.…”