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.
Highlights
Ricerca di Ateneo, and Telethon-Italy (Grant GGP06233). □S The on-line version of this article contains supplemental Tables S1 and S2 and Figs
This favors a POLRMT recycling mechanism that accounts for the high rate of rDNA transcription and for the stimulatory effect exerted by mTERF on transcription initiation [2]. mtDNA expression tuning requires activation factors and the transcription repressor MTERF3 [3], which, together with mTERF, belongs to the MTERF protein family [4]; it exerts its function by binding the promoter region of mtDNA. mtDNA synthesis is carried out by the DNA polymerase-␥ (Pol-␥) [5]
With the only exceptions being the transcription initiation factors TFAM and TFBM, for which the control by both NRF-1 and NRF-2 factors and by PGC-1 family co-activators was well established [17, 18], still limited is the information concerning the regulation of the expression of the mitochondrial transcription and replication proteins
Summary
For each probe and competitor, the complementary sequences are shown. Oligonucleotide positions are numbered with respect to the transcription initiation site. The transcriptional activation is not restricted to oxidative phosphorylation polypeptides because it was shown to control the expression of a variety of proteins, including enzymes [12], TOM complex receptors [13], complex IV assembly factors [14, 15], and peroxiredoxin V [16]. Regarding NRF-2 control on mtDNA transcription and replication proteins, the only available information indicates that it activates the expression of the transcription initiation factors TFAM [17] and TFB2M [18]. Under NRF-2 control is TFB1M (mitochondrial transcription factor B1), the TFB2M paralogue, that was recently shown to be mainly involved in mitochondrial protein synthesis [18, 19]. By a combination of in vitro and in vivo experiments, we demonstrate that NRF-2 positively regulates the expression of POLRMT, mTERF, Pol-␥B, TWINKLE, and mtSSB; on the contrary, no control by NRF-2 was observed for MTERF3 and Pol-␥A genes
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