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. 2008 Feb 8;283(6):3120-3129.
doi: 10.1074/jbc.M707587200. Epub 2007 Dec 12.

Nuclear respiratory factor 1 regulates all ten nuclear-encoded subunits of cytochrome c oxidase in neurons

Shilpa S Dhar  1 , Sakkapol Ongwijitwat  1 , Margaret T T Wong-Riley  2
Affiliations

Nuclear respiratory factor 1 regulates all ten nuclear-encoded subunits of cytochrome c oxidase in neurons

Shilpa S Dhar et al. J Biol Chem. .

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.

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Figures

FIGURE 1
FIGURE 1. In vitro binding activity of NRF-1 to putative binding sites on all ten nuclear-encoded COX subunits as measured with EMSA and supershift assays
32P-labeled oligonucleotides (oligo), excess unlabeled oligonucleotides as competitors, excess unlabeled mutant NRF-1 as competitors, HeLa extract, and NRF-1 antibodies added to or absent from the reactions are indicated by a + or a − sign, respectively, above each lane. Arrowheads mark the specific NRF-1/probe shift complex and antibody-supershifted complex. Rat cytochrome c served as a positive control, showing shift band (A, lanes 1) and supershift band (A, lane 3) with labeled NRF-1 binding site. Excess unlabeled probe eliminated the shift band (A, lane 2). Labeled oligonucleotides with mutated NRF-1 site on COX 4i1 served as a negative control (A, lanes 14−16). Labeled oligonucleotides with putative NRF-1 binding sites on all ten mouse COX subunits showed specific shift and supershift bands that are eliminated by excess unlabeled competitors (A, lanes 5, 6, 8, 10, 11, and 13; B and C, all lanes). On the other hand, excess unlabeled oligonucleotides with mutated NRF-1 site were unsuccessful in competing for the binding (A, lanes 7 and 12). Labeled oligonucleotides for COX 4i1 and COX5a with NRF-1 antibodies alone did not yield any bands (A, lanes 4 and 9), ruling out nonspecific antibody-oligonucleotide interactions.
FIGURE 2
FIGURE 2. In vivo ChIP assays for NRF-1 interaction with COX nuclear-encoded subunit promoters
PCR reactions were performed on N2a cell chromatin precipitated with anti-NRF-1 antibodies (NRF-1 lanes) or anti-nerve growth factor receptor p75 antibodies (negative control, NGFR lanes). Control reactions were performed with 0.5% and 0.1% of input chromatin (input lanes). PCR products _targeting COX 4i1, 5a, 5b, 6a1, 6b, 6c, 7a2, 7b, 7c, and 8a promoters revealed that all ten COX promoters’ DNA co-immunoprecipitated with NRF-1. Reactions _targeting TFB2M promoter was used as a positive control, and β-actin was used as a negative control.
FIGURE 3
FIGURE 3. Mutational analysis of the promoter elements of COX genes
Relative luciferase activity of wild-type and site-directed mutations of NRF-1 binding sites on all ten nuclear-encoded COX promoters indicates significant reductions in luciferase activity in all mutants. (n = 6 for each construct). *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 4
FIGURE 4. RNA interference-mediated silencing of NRF-1 suppresses mRNAs in all 10 nuclear-encoded COX subunit genes and another six nuclear genes important in mitochondrial biogenesis
A, N2a cells transfected with NRF-1 shRNA expressed significantly less NRF-1 mRNA than those with empty vectors. B, Western blot reveals a down-regulation of NRF-1 protein in shRNA-transfected neurons. β-Actin served as a loading control. C, N2a cells were transfected with shRNA against NRF-1 (hatched bars) or with empty vectors (solid bars). NRF-2α served as a negative control. All ten COX subunit mRNAs show significant decreases in shRNA-treated samples as compared with those with empty vectors, whereas NRF-2α mRNA remained unchanged. n = 5−6 for each data point; *, p < 0.05; **, p < 0.01; ***, p < 0.001. D, mRNAs were quantified for six other known _target genes of NRF-1: TFAM, TFB1M, TFB2M, SURF1, VDAC1, and TOM20 in N2a cells. All six genes showed significant reductions in mRNA expression in NRF-1 shRNA-transfected cells (hatched bars) as compared with empty vector controls (solid bars). n = 5−6 for each data point; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 5
FIGURE 5. All ten nuclear-encoded COX subunit promoters from mouse, rat, and human contained conserved putative NRF-1 binding sites
Aligned partial sequences of COX promoters from human (H), mouse (M), and rat (R) genomes show conservation of typical and atypical NRF-1 binding sites. Conserved binding site sequences are in boldface. Solid boxes highlight NRF-1 sites that are highly conserved in all three or at least two species.
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