Abstract
Cytochrome c oxidase (COX) is one of only four bigenomic proteins in mammalian cells, having ten subunits encoded in the nuclear genome and three in the mitochondrial DNA. The mechanism of its bigenomic control is not well understood. The ten nuclear subunits are on different chromosomes, and the possibility of their coordinate regulation by the same transcription factor(s) deserves serious consideration. The present study tested our hypothesis that nuclear respiratory factor 1 (NRF-1) serves such a role in subunit coordination. Following in silico analysis of murine nuclear-encoded COX subunit promoters, electrophoretic mobility shift and supershift assays indicated NRF-1 binding to all ten promoters. In vivo chromatin immunoprecipitation assays also showed NRF-1 binding to all ten promoters in murine neuroblastoma cells. Site-directed mutagenesis of putative NRF-1 binding sites confirmed the functionality of NRF-1 binding on all ten COX promoters. These sites are highly conserved among mice, rats, and humans. Silencing of NRF-1 with RNA interference reduced all ten COX subunit mRNAs and mRNAs of other genes involved in mitochondrial biogenesis. We conclude that NRF-1 plays a significant role in coordinating the transcriptional regulation of all ten nuclear-encoded COX subunits in neurons. Moreover, NRF-1 is known to activate mitochondrial transcription factors A and B, thereby indirectly regulating the expressions of the three mitochondrial-encoded COX subunits. Thus, NRF-1 and our previously described NRF-2 prove to be the two key bigenomic coordinators for transcriptional regulation of all cytochrome c oxidase subunits in neurons. Possible interactions between the NRFs will be investigated in the future.
Highlights
c oxidase (COX) is a complex of 13 different subunits, 3 of which (I, II, and III) are encoded in the mitochondrial DNA, and the remaining 10 are nuclear-encoded [7]
We found that the protein and mRNA levels of NRF-2 changed in response to changing neuronal activity and in concert with altered COX activity (16 –20)
90% reduction of nuclear respiratory factor 1 (NRF-1) mRNA as measured by real-time quan- nuclear-encoded COX subunit genes located on different chrotitative PCR relative to empty vector controls (Fig. 4A)
Summary
COX is a complex of 13 different subunits, 3 of which (I, II, and III) are encoded in the mitochondrial DNA, and the remaining 10 are nuclear-encoded [7]. Two redox-responsive transcription factors, nuclear respiratory factors 1 and 2, or NRF-1 and NRF-2, have been proposed to mediate such bigenomic coordination in non-neuronal cells [12,13]. Both NRF-1 and NRF-2 reportedly regulate the expression of a few nuclear-encoded COX subunit genes and indirectly regulate the three mitochondrial-encoded COX subunit genes by activating mitochondrial transcription factors A and B (TFAM, TFB1M, and TFB2M) [9, 10]. Whether NRF-1 plays an important role in regulating all ten nuclear-encoded COX subunit genes located on different chromosomes was entirely unknown
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