Figure 9

NRF-1 is a component of a protein complex that binds to the GABPβ promoter. (a) The NRF-1 consensus sequence [22], and the sequences of Gb-270 and mutant oligos are shown. Mutations are in lowercase and bold. (b) Gb-270 and the mutant oligos were used as probes in an EMSA with MCF-7 nuclear extracts. Binding complexes (S), non-specific binding complexes (NS) and free probe (F) are indicated. Bands corresponding to NRF-1, and predicted NRF-1 + X and NRF-1 + X +Y complexes (see part e) are also indicated. (c) Mutant oligos mT4 and mT5 were used as probes in an EMSA with MCF-7 nuclear extracts. An NRF-1 antibody was added to the binding reactions as indicated (+), or PBS was added as a negative control (-). Binding complexes (S) were supershifted (SS) in the presence of the NRF-1 antibody. (d) The transcriptional activities of the GABPβ promoter constructs -268 (contains the NRF-1 binding site) and -251 as well as promoter constructs with the mutations specified in part (a) were assessed in MCF-7 cells via dual luciferase assay in the presence of siRNA against GAPDH (siGAPDH, negative control) and NRF-1 (siNRF-1). Promoter activity expressed as relative light units. (e) Model of the NRF-1 complex that activates GABPβ gene expression. Banding patterns observed by EMSA suggest that NRF-1 binds to the GABPβ promoter in complex with at least two other proteins (refer to part b). We propose that NRF-1 binds as a homodimer to the GABPβ promoter from nucleotide -266 to -255 (underlined). Protein X binds NRF-1 and makes limited contact with the promoter upstream of the NRF-1 binding site stabilizing its interactions with NRF-1. A third protein (Y) binds to the NRF-1 + X complex. Mutations to the promoter (mT4, mT5 and mA6) disrupt formation of the complex (refer to part b), but oligonucleotides with mT4 and mT5 mutations retain NRF-1 binding capability (refer to part c).