Human mitochondrial DNA (mtDNA) is a 16.5-kb double-stranded circular molecule that encodes 13 essential protein components of the mitochondrial oxidative phosphorylation complexes. The maintenance of mtDNA integrity is essential for normal function of the respiratory chain that is responsible for aerobic ATP production. There are hundreds to thousands of copies of mtDNA in one cell. Because the level of mtDNA transcripts largely depends on the copy number of mtDNA, the regulation of its copy number is important for maintaining mitochondrial ATP production. However, the regulation of mtDNA copy number is still poorly understood.
Mitochondrial transcription factor A (TFAM) (11
), a transcription factor for mtDNA, enhances mtDNA transcription in a promoter-specific fashion in the presence of mitochondrial RNA polymerase and transcription factor B (TFB1 M or TFB2 M) (10
). TFAM is a member of the high-mobility group (HMG) proteins because it contains two HMG boxes. TFAM possesses DNA-binding properties regardless of sequence specificity, although it shows a higher affinity for the light- and heavy-strand promoters (LSP and HSP, respectively) (11
). In addition to these two HMG boxes, human TFAM has a linker region between the two HMG boxes and a carboxyl-terminal tail region (C-tail) composed of 27 and 25 residues, respectively (6
According to the strand-coupled model (4
), replication of the L-strand, i.e., lagging-strand replication, occurs simultaneously with that of the H-strand. On the other hand, in another mtDNA replication model, the strand displacement model (34
), replication of the nascent H-strand proceeds and displaces the parental H-strand until a replication origin of the L-strand, OL
, is exposed on a single strand. The process of mtDNA replication begins with the initiation of transcription at LSP. The transcript initiated from LSP forms an RNA-DNA hybrid at a replication origin for the H-strand, OH
. The RNA-DNA hybrid is processed to generate an RNA primer utilized by mitochondrial DNA polymerase γ (20
). Thus, in the latter model, the replication of mammalian mtDNA is proposed to be coupled with transcription, and therefore TFAM is thought to be essential for replication of mtDNA (34
). The role of transcription in the former model has not yet been clarified.
Abf2p, a TFAM homolog in Saccharomyces cerevisiae
, has two HMG boxes and a short linker region between them, but unlike TFAM, it does not have a C-tail (12
). Abf2p is abundant in mitochondria, with one Abf2p polypeptide present for every 15 bp of mtDNA (8
). Disruption of the ABF2 gene leads to a loss of mtDNA and a resultant loss of respiratory competence when cells are grown in the presence of glucose. Expression of human TFAM in the S. cerevisiae abf2
strain rescued the phenotype, implying a potential functional homology between human TFAM and Abf2p (30
). However, unlike mammalian TFAM, Abf2p is not required for the initiation of transcription in yeast mtDNA (8
). An in vitro transcription assay demonstrated that Abf2p or C-tail-deleted TFAM does not activate transcription, whereas a chimeric Abf2p containing the C-tail does (6
), suggesting that the C-tail of TFAM is necessary for transcriptional activation. This notion is also supported by a recent report that the C-tail of TFAM is necessary to bind mitochondrial transcription factor B (TFBM) and that this binding is required for transcription activation (24
). In agreement with this, the import of full-length TFAM into isolated mitochondria increases transcription, but import of TFAM lacking the C-tail does not (14
). Thus, the C-tail of TFAM is considered essential for the activation of transcription.
The mitochondrial nucleoids, protein-mtDNA complexes, have been studied extensively in the lower eukaryotes S. cerevisiae
) and Physarum polycephalum
). In S. cerevisiae
Abf2p is detected as a main component of the nucleoid and appears to function to maintain mtDNA and the nucleoid structure (17
). In P. polycephalum
, Glom, which also has two HMG boxes, shows a strong DNA-packaging activity (32
). Both of these HMG family proteins can be functionally replaced by an Escherichia coli
histone-like protein, HU (25
), implying that Abf2p and Glom package mtDNA.
There are several reports that mtDNA in higher eukaryotes is somewhat naked except for the D-loop region (2
), while the mtDNA of Xenopus laevis
is reported to be packaged into regular beaded structures (3
). These conflicting results on whether animal mtDNA takes on a higher nucleosome- or chromatin-like structure (mitochondrial nucleoid or mitochromatin/mitochondrial chromosome) are not fully resolved. Recent reports also suggest the existence of such a higher mtDNA structure in mammals (1
). Because the amount of human TFAM is sufficient to cover the entire region of mtDNA (36
) and because most TFAM molecules indeed bind to mtDNA (1
), TFAM has been proposed to be one of the main components of the human mtDNA higher structure.
Homozygous gene disruption of Tfam
is lethal in both mouse and chicken cells, at least in part due to mtDNA depletion and resultant loss of oxidative phosphorylation capacity (19
). In heterozygous cells, the expression of mouse and chicken TFAM was reduced by about 50% and the amount of mtDNA also decreased by about half (19
). These results suggest that TFAM is necessary to maintain mtDNA. There are two possibilities to explain mtDNA maintenance by TFAM. One is that, given that the replication of mtDNA is coupled to transcription (34
), TFAM affects the replication of mtDNA directly. The other is that TFAM binds and stabilizes mtDNA, as do other HMG family proteins (5
There have been no reports demonstrating overexpression of TFAM in mammalian cells. We established stable and inducible human cell lines overexpressing TFAM for the first time with a tetracycline-regulated gene expression system. In this study, with these TFAM-overexpressing cell lines and RNA interference (RNAi), we manipulated the amount of human TFAM in human HeLa cell lines and then analyzed mtDNA and mitochondrial transcription. We found that the amount of TFAM but not the transcription level is correlated to the amount of mtDNA.