Transcriptional regulation of the nuclear gene encoding the alpha-subunit of the mammalian mitochondrial F1F0 ATP synthase complex: role for the orphan nuclear receptor, COUP-TFII/ARP-1.
Our laboratory has been studying the transcriptional regulation of the nuclear gene (ATPA) that encodes the alpha-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.
Jordan
Elzora M
EM
Department of Molecular and Cell Biology, The University of Texas at Dallas, P. O. Box 830688, Richardson, Texas 75083-0688, USA.
Worley
Teri
T
Breen
Gail A M
GA
D000818:Animals; D001667:Binding, Competitive; D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D002417:Cattle; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004794:Enzyme Repression / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D025261:Mitochondrial Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism / Q000502:physiology*; D011987:Receptors, Steroid; D012097:Repressor Proteins / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D051720:Upstream Stimulatory Factors
D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D004268:DNA-Binding Proteins; D025261:Mitochondrial Proton-Translocating ATPases; D021122:Protein Subunits; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497587:Nr2f2 protein, mouse; C497184:USF2 protein, human; C497186:Usf2 protein, mouse; C095569:NR2F2 protein, human