Nuclear respiratory factor 2 induces the expression of many but not all human proteins acting in mitochondrial DNA transcription and replication

Abstract

In mammals, NRF-2 (nuclear respiratory factor 2), also named GA-binding protein, is an Ets family transcription factor that controls many genes involved in cell cycle progression and protein synthesis as well as in mitochondrial biogenesis. In this paper, we analyzed the role of NRF-2 in the regulation of human genes involved in mitochondrial DNA transcription and replication. By a combination of bioinformatic and biochemical approaches, we found that the factor binds in vitro and in vivo to the proximal promoter region of the genes coding for the transcription termination factor mTERF, the RNA polymerase POLRMT, the B subunit of the DNA polymerase-gamma, the DNA helicase TWINKLE, and the single-stranded DNA-binding protein mtSSB. The role of NRF-2 in modulating the expression of those genes was further established by RNA interference and overexpression strategies. On the contrary, we found that NRF-2 does not control the genes for the subunit A of DNA polymerase-gamma and for the transcription repressor MTERF3; we suggest that these genes are under regulatory mechanisms that do not involve NRF proteins. Since NRFs are known to positively control the expression of transcription-activating proteins, the novelty emerging from our data is that proteins playing antithetical roles in mitochondrial DNA transcription, namely activators and repressors, are under different regulatory pathways. Finally, we developed a more stringent consensus with respect to the general consensus of NRF-2/GA-binding protein when searching for NRF-2 binding sites in the promoter of mitochondrial proteins.

FIGURE 1.

Location of predicted CAAT boxes and NRF-1, NRF-2, and Sp1 binding sites in the promoter proximal region of human genes coding for mtDNA transcription and replication proteins. Position numbers refer to the transcription start sites of each gene as deduced from mRNA sequence extracted by the Ensembl search engine. The −45 NRF-2 site in the POLRMT promoter was detected by visual inspection of the sequence.

FIGURE 2.

Conservation in different species of NRF-2 binding sites in the analyzed promoters. Shown are alignments of corresponding human (Hs), rat (Rn), and mouse (Mm) promoter sequences containing the predicted NRF-2 binding sites. Nucleotides conserved in all sequences are shaded in black; the numbers on both sides of the human sequences indicate nucleotide positions in reference to the transcription start site. Predicted NRF-2 site core sequences are indicated by brackets together with their respective positions.

FIGURE 3.

In vitro binding of NRF-2 to the predicted sites in the promoter of the selected genes. NRF-2 binding was evaluated by EMSA using a heparin-Sepharose-purified nuclear extract from HeLa cells and radiolabeled double-stranded oligonucleotide probes containing one or two NRF-2 putative binding sites. The unlabeled specific (RCOX4), nonspecific, and mutated (mut-61 and mut-45) competitor oligonucleotides (100-fold molar excess) and NRF-2 antisera are indicated above each lane. Probes are shown below the lanes. DNA-protein complexes are indicated by arrows on the left of each panel. Ab, antibody.

FIGURE 4.

ChIP assay for in vivo binding of NRF-2 to the analyzed promoters. A, nuclei obtained from formaldehyde-fixed HeLa cells were lysed, and chromatin was fragmented by sonication. Chromatin was immunoprecipitated either in the absence of antibodies (no Ab) or in the presence of p53 commercial antibodies (mock) or polyclonal NRF-2α antiserum. Immunoprecipitated DNAs were subjected to semiquantitative PCR using primers specific for each examined promoter. Template DNAs, immunoprecipitated in the different conditions, are indicated above each lane; input indicates PCRs containing as template 0.3% of the total amount of chromatin used in immunoprecipitation reactions. Analyzed promoters are indicated to the right. Reactions addressed to TFB2M and β-actin were used as positive and negative control, respectively. B, histogram showing the quantification of PCR products obtained on NRF-2α immunoprecipitated DNA, relative to input signal; the relative enrichment of β-actin DNA was fixed as 1. Data represent the average of independent quantifications (n = 6) from three ChIP experiments; values are expressed as a ratio, and results are means ± S.D. Statistical analysis was performed using paired two-tailed Student's t test (*, p < 0.05; **, p < 0.01).

FIGURE 5.

Effect of NRF-2 complex knockdown on the level of the mRNA from all analyzed genes. A, NRF-2α and -β subunits were knocked down in HeLa cells by means of RNAi. Cells, either untreated (control) or treated with lacZ (mock) or NRF-2α/NRF-2β siRNAs, were harvested 48 and 96 h after transfection; total cellular proteins were subjected to Western blotting analysis with polyclonal antibodies against NRF-2α, NRF-2β, and β-actin. B, total RNA was extracted from 96-h treated and control cells, and relative quantification of mRNAs was carried out by real-time reverse transcription-PCR. Bars indicate the relative content of transcripts, normalized to β-actin mRNA (endogenous control), in treated with respect to control cells, fixed as 1. The relative quantification was performed according to the Pfaffl equation (25). Values are expressed as a ratio, and results are means ± S.D. (n = 9). Statistical analysis was performed using paired two-tailed Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 6.

Effect of NRF-2 complex overexpression on the level of the mRNA from all analyzed genes. A, NRF-2α and -β subunit overexpression was obtained by co-transfecting HeLa cells with DNA constructs containing the cDNA of the two subunits under the control of the constitutive CMV promoter. Cells either untreated (control) or transfected with empty pcDNA3.1 vector or co-transfected with pcDNA/NRF-2α and pcDNA/NRF-2β₁ constructs (NRF-2 O.E.) were harvested 24 h after transfection; total cellular proteins were subjected to Western blotting analysis with polyclonal antibodies against NRF-2α, NRF-2β, and β-actin. B, total RNA was extracted from treated and control cells, and relative quantification of mRNAs was carried out by real-time reverse transcription-PCR as described in the legend to Fig. 5. Values are expressed as a ratio, and results are means ± S.D. (n = 5). Statistical analysis was performed using paired two-tailed Student's t test (*, p < 0.05; **, p < 0.01).

FIGURE 7.

Motif analysis of functional NRF-2 binding sites in mitochondrial gene promoters. The binding site alignment is shown as WebLogo output. The information content of the motifs is expressed in bits. The relative size of the letters is a measure of the relative representation of each nucleotide in a given position; letters are sorted in descending order depending on their frequencies.