Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
ReviewMitochondrial transcription factor A regulates mitochondrial transcription initiation, DNA packaging, and genome copy number☆
Highlights
► We review TFAM–DNA interactions, including specificity, affinity, bending, and cooperativity. ► We review proposed mechanisms of TFAM regulation of mtDNA copy number. ► We propose a model in which TFAM dimerization, DNA looping, and cooperativity impact mtDNA packaging and promoter activity.
Section snippets
The importance of mtDNA copy number control
Mitochondrial DNA is a multicopy, circular genome that is absolutely required for respiratory function. Interference with mitochondrial genome sequence fidelity, transcription, or translation can cause ATP insufficiency and disease. There are a host of disorders in mitochondrial DNA (mtDNA) maintenance that can cause disease, although mitochondrial transcription factor A (TFAM) has yet to be identified as the cause. Nonetheless, a reduction in functional mtDNA abundance is now recognized as an
TFAM and mitochondrial transcription initiation
Several excellent reviews detail the identification and characterization of mtDNA sequence elements and other factors involved in mitochondrial transcription regulation [32], [33], [34], [35], [36]; here we present the promoter regions and their consequent coding sequences as they relate to TFAM activity (Fig. 1). MtDNA contains three promoters [36], [37], [38]; RNA synthesis from the light strand is controlled by the light strand promoter (LSP) [39], which generates transcripts encoding one
TFAM and mtDNA packaging
In addition to its role in transcription initiation, TFAM binds abundantly and non-specifically around the entire mitochondrial genome. In vivo, mtDNA is present as discrete, punctate protein–DNA structures termed nucleoids. TFAM is a major component of the nucleoid [47], [54], [55], where it fulfills an architectural [56] or scaffolding [57] role, presumably through its strong affinity for non-specific (NS) DNA sequences [45]. Furthermore, these NS–DNA interactions can condense DNA templates
The domains of TFAM
TFAM is a highly conserved, basic protein with a molecular weight of ~ 25 kDa (Fig. 2). Mature mouse and human TFAM (mTFAM and hTFAM, respectively) are 77% similar and 63% identical, and have a similar isoelectric point (pI) of ~ 10, a value attributed to the basic amino acids that comprise 23% of the protein. TFAM contains several well-defined domains, including an ~ 45 amino acid N-terminal mitochondrial targeting sequence (MTS), which is cleaved in a two-step reaction [59] upon import to the
TFAM affinity for DNA
Since the discovery of TFAM as a DNA binding protein, investigating the protein's affinity for DNA has been a priority for improving our understanding of its function. To characterize the DNA binding activity of TFAM quantitatively, we analyzed mouse TFAM–DNA affinity and specificity by Surface Plasmon Resonance (SPR), a technique that measures changes in mass associated with DNA immobilized in a flow cell. This method has two primary advantages over electrophoretic mobility shift assays
DNA bending by TFAM
HMGB-related proteins, including TFAM, bind to specific and nonspecific DNA sequences in a highly similar fashion [67] and frequently play an architectural role on chromatin [68]. During binding of the HMG box to DNA, polar amino acids interact with the phosphate backbone and hydrophobic side chains intercalate between adjacent bases. Intercalation separates the bases, expanding the minor groove and compressing the major groove on the opposite side of the helix. This compression imparts a local
TFAM cooperativity
Several lines of evidence support the idea that TFAM binds DNA with positive cooperativity, a characteristic of DNA binding in which the presence of one TFAM molecule bound encourages proximal binding of the next molecule, presumably by altering the local DNA structure. Cooperativity may facilitate the “spread” of TFAM molecules across large stretches of DNA, and could be mediated by the distortion of DNA described in the previous section. TFAM exhibits cooperative characteristics both by
TFAM multimerization
Understanding how TFAM acts on DNA requires a clear understanding of TFAM stoichiometry. Given that TFAM protein sequence includes a coiled-coil motif that could mediate homodimerization, several groups have attempted to determine the simplest DNA-binding unit of TFAM. The first experiments investigating a potential oligomeric form of TFAM were performed using enriched TFAM analyzed by glycerol gradient sedimentation analysis; the results suggested that the protein is monomeric [43]. In those
Nucleoid organization by TFAM
TFAM binds and kinks DNA, but does it function in nucleoid packaging? During our in vitro studies of TFAM-mediated DNA compaction [58], we found that minimal contour length is achieved at sub-saturating levels of TFAM via DNA looping. Electron microscopy of mtDNA from detergent-solubilized rat mitochondria has revealed rosette structures [75] that are strikingly similar to the AFM images generated in vitro. Furthermore, loading of additional TFAM molecules on DNA increases DNA rigidity,
How does TFAM regulate MTDNA copy number?
TFAM–DNA interactions have been well studied in vitro, but significant controversy remains regarding how TFAM regulates mtDNA copy number in vivo. Two mechanisms have been proposed for TFAM-mediated increase in mtDNA content: one in which a higher frequency of TFAM binding at LSP increases transcription-mediated priming of replication, and another in which genome-wide binding by TFAM stabilizes steady-state levels of mtDNA, perhaps by reducing the rate of DNA turnover. We believe these models
Transcription-mediated copy number control
Because genome replication requires priming by a short RNA molecule originating from the LSP, increased transcription activation caused by TFAM overexpression (TFAM OE) would lead to increased mtDNA content. Import of TFAM into isolated mitochondria [82] and overexpression in cells following transient transfection [83] both increase mitochondrial transcription. Furthermore, in vitro titration experiments demonstrate that TFAM stimulates transcription from LSP strongly and at lower
Genome stability-mediated copy number control
This model is based on three major observations: TFAM abundance is sufficient to coat mtDNA; TFAM is associated with the entire genome and not just the promoter region; and elevation of copy number during TFAM overexpression does not require a transcriptionally active form. In human cell lines in which endogenous TFAM is reduced by RNAi knockdown, overexpression of TFAM-∆C results in severely diminished transcription activation activity, but is sufficient to increase mtDNA copy number [56].
Potential models of genome compaction and promoter selection by TFAM dimerization and cooperative binding
Several models have been put forward to explain how TFAM controls genome compaction and promoter activity [56], [81], [95], [96], [97], [98]. These models generally do not take into account TFAM binding as a dimer, DNA looping, or cooperativity as factors. Fig. 3 provides a series of illustrations of TFAM dimer-mediated genome compaction with increasing TFAM loading by a non-specific, cooperative mechanism as TFAM abundance increases (represented by the blue triangle). Because the abundance of
Regulation of TFAM–DNA binding
Sufficient evidence suggests that TFAM is a multifunctional protein whose activities would need to be carefully regulated to balance compaction and transcription or replication. To date, little is known about how TFAM activity may be regulated. While it might be tempting to associate TFAM activity with its mRNA levels, TFAM transcripts do not necessarily reflect TFAM protein levels. One clear example of this, and a potential mechanism for regulating TFAM activity, comes from experiments
Concluding remarks
The coordination of mtDNA transcription, replication and turnover is essential for maintaining oxidative capacity and cellular homeostasis. TFAM has been implicated in controlling genome compaction, activation of transcription, promoter selection, initiation of genome replication, and the regulation of mtDNA copy number. Here we have presented a description of TFAM's DNA binding characteristics, and by emphasizing TFAM dimerization and looping, present new ideas about the regulation of
Acknowledgements
This article was supported by a pilot award (BAK) from the DRC at the University of Pennsylvania from a grant sponsored by NIH DK 19525, from an award from the United Mitochondrial Disease Foundation (11-111) and CTC was supported in part from NIH grant T32GM008216.
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This article is part of a Special Issue entitled: Mitochondrial Gene Expression.