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.
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.
Luciakova
Katarina
K
Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 83391 Bratislava, Slovak Republic. Katarina.Luciakova@savba.sk
Kollarovic
Gabriel
G
Barath
Peter
P
Nelson
B Dean
BD
D033742:Adenine Nucleotide Translocator 2 / Q000235:genetics*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D006801:Humans; D008954:Models, Biological; D005810:Multigene Family / Q000502:physiology; D046912:Multiprotein Complexes / Q000378:metabolism / Q000502:physiology; D025542:Neurofibromin 1 / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D012097:Repressor Proteins / Q000378:metabolism / Q000502:physiology; D051785:Smad Proteins / Q000378:metabolism / Q000502:physiology*; D051703:Sp Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic
D033742:Adenine Nucleotide Translocator 2; D016895:Culture Media, Serum-Free; D046912:Multiprotein Complexes; D025542:Neurofibromin 1; D012097:Repressor Proteins; D051785:Smad Proteins; D051703:Sp Transcription Factors