The role of nuclear factor-1 in growth regulated expression of the human ANT2 gene.

Based on previous studies of human ANT2 (adenine nucleotide translocator-2) promoter, we describe novel interaction between NFI and Smad transcription factors. Immunoprecipitation analyses of nuclear extracts from growth-arrested diploid cells demonstrate that NFI is part of NF1–Smad4–Sp3 complex. In growth-activated cells, the repressor complex dissociates. Consistent with the role of Smad proteins, TGF-beta (transforming growth factor-beta) can repress transcription of ANT2 gene. Physical interaction of NFI isoforms with Smad proteins was confirmed by GST-pulldown assay. We developed purification strategy for recombinant NFI proteins. The fusion proteins can be expressed in detectable amounts, but were found mainly in the insoluble fractions. The final purification strategy represents a triple affinity chromatography consisting of Heparin-Sepharose with subsequent IMAC and glutathione affinity chromatography. Isolated proteins were functionally active and suitable for further proteomic studies.