Growth-dependent repression of human adenine nucleotide translocase-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex

Nuclear factor 1 (NF1) binds to two upstream elements of the human adenine nucleotide translocator-2 (ANT-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). Chromatin immunoprecipitation and co-immunoprecipitation analysis 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-immnoprecipitation 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-β can fully repress ANT2 transcription in normally growing fibroblasts. Finally, pull-down experiments of in vitro transcribed/translated NF1 isoforms by GST-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.