Transcriptional Regulation of the Nuclear Gene Encoding the α-Subunit of the Mammalian Mitochondrial F1F0 ATP Synthase Complex:  Role for the Orphan Nuclear Receptor, COUP-TFII/ARP-1

• Published in: Biochemistry (Easton) 2003-03, Vol.42 (9), p.2656-2663
• Main Authors: Jordan, Elzora M, Worley, Teri, Breen, Gail A. M
• Format: Article
• Language: English
• Subjects: Animals; Binding, Competitive; Cattle; Cloning, Molecular; COUP Transcription Factor II; COUP Transcription Factors; DNA-Binding Proteins - metabolism; DNA-Binding Proteins - physiology; Enzyme Repression - genetics; HeLa Cells; Humans; Mice; Mitochondrial Proton-Translocating ATPases - antagonists & inhibitors; Mitochondrial Proton-Translocating ATPases - biosynthesis; Mitochondrial Proton-Translocating ATPases - genetics; Mitochondrial Proton-Translocating ATPases - metabolism; Mutagenesis, Site-Directed; Promoter Regions, Genetic - genetics; Protein Binding - genetics; Protein Subunits - antagonists & inhibitors; Protein Subunits - biosynthesis; Protein Subunits - genetics; Protein Subunits - metabolism; Receptors, Cytoplasmic and Nuclear - metabolism; Receptors, Cytoplasmic and Nuclear - physiology; Receptors, Steroid; Repressor Proteins - physiology; Saccharomyces cerevisiae - genetics; Trans-Activators - metabolism; Transcription Factors - metabolism; Transcription Factors - pharmacology; Transcription Factors - physiology; Transcription, Genetic; Two-Hybrid System Techniques; Upstream Stimulatory Factors
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0268347

Our laboratory has been studying the transcriptional regulation of the nuclear gene (ATPA) that encodes the α-subunit of the mammalian mitochondrial F1F0 ATP synthase complex. We have previously determined that the regulatory factor, upstream stimulatory factor 2 (USF2), can stimulate transcription of the ATPA gene through the cis-acting regulatory element 1 in the upstream promoter of this gene. In this study, we used the yeast one-hybrid screening method to identify another factor, COUP-TFII/ARP-1, which also binds to the ATPA cis-acting regulatory element 1. Binding of the orphan nuclear receptor, COUP-TFII/ARP-1, to the ATPA regulatory element 1 was confirmed using electrophoretic mobility shift experiments, and COUP-TFII/ARP-1-containing complexes were detected in HeLa cell nuclear extracts. A mutational analysis indicated that the binding site for COUP-TFII/ARP-1 in the ATPA regulatory element 1 is an imperfect direct repeat of a nuclear receptor response element (A/GGGTCA) with a spacer of three nucleotides. Functional assays in HeLa cells showed that COUP-TFII/ARP-1 represses the ATPA promoter activity in a dose- and sequence-dependent manner. Furthermore, cotransfection assays demonstrated that COUP-TFII/ARP-1 inhibits the USF2-mediated activation of the wild-type ATPA gene promoter but not a mutant promoter that is defective in COUP-TFII/ARP-1-binding. Overexpression of USF2 reversed the COUP-TFII/ARP-1-mediated repression of the ATPA promoter. Mobility shift assays revealed that COUP-TFII/ARP-1 and USF2 compete for binding to the ATPA regulatory element 1. Thus, the ATPA gene is regulated by a multifunctional binding site through which the transcription factors, COUP-TFII/ARP-1 and USF2, bind and exert their antagonistic effects.