Transient overexpression of mitochondrial transcription factor A (TFAM) is sufficient to stimulate mitochondrial DNA transcription, but not sufficient to increase mtDNA copy number in cultured cells

Abstract

Mitochondrial transcription factor A (TFAM) stimulates transcription from mitochondrial DNA (mtDNA) promoters in vitro and in organello. To investigate whether changes of TFAM levels also modulate transcription and replication in situ, the protein was transiently overexpressed in cultured cells. Mitochondrial mRNAs were significantly elevated at early time points, when no expansion of the TFAM pool was yet observed, but were decreased when TFAM levels had doubled, resemb-ling in vitro results. HEK cells contain about 35 molecules of TFAM per mtDNA. High levels of TFAM were not associated with increases of full-length mtDNA, but nucleic acid species sensitive to RNAse H increased. Stimulation of transcription was more evident when TFAM was transiently overexpressed in cells pre-treated with ethidium bromide (EBr) having lowered mtDNA, TFAM and mitochondrial transcript levels. EBr rapidly inhibited mtDNA transcription, while decay of mtDNA was delayed and preferentially slowly migrating, relaxed mtDNA species were depleted. In conclusion, we show that transcription of mtDNA is submaximal in cultured cells and that a subtle increase of TFAM within the matrix is sufficient to stimulate mitochondrial transcription. Thus, this protein meets all criteria for being a key factor regulating mitochondrial transcription in vivo, but other factors are necessary for increasing mtDNA copy number, at least in cultured cells.

Figure 1

Transient overexpression of TFAM in HeLa cells. (A) TFAM levels analysed by western blotting at 5 h (lanes 1–4) and 15 h (lanes 5–8) after transfection with pKEX and pKEX-TFAM. Two representative lanes for each group are shown. Vectors used are indicated above the lanes. (B) Localization of the TFAM precursor and mature proteins, respectively, comparing total cell lysate with a mitochondrial fraction treated with proteinase K (μg/ml) under various conditions. (C) Expression of several TFAM isoforms after transfection with pKEX-TFAM (lanes 1 and 5), pKEX-ΔC-8-TFAM (lanes 2 and 6), pKEX-ΔC-15-TFAM (lanes 3 and 7), pKEX-ΔC-25-TFAM (lanes 4 and 8). Cells were harvested at 5 and 15 h as indicated. p, precursor; m, mature protein; PK, proteinase K; RT, room temperature.

Figure 2

Effect of transient overexpression of TFAM on mitochondrial transcript levels in HeLa cells. (A) Northern blot showing levels of CO I mRNA, RNA 6 and 12S rRNA (upper panel), ND1 mRNA, RNA 19, ATPase 6/8 mRNA and its precursor (lower panel) at 5 h and 15 h after transfection with pKEX (lanes 1–3 and 7–9), and pKEX-TFAM (lanes 4–6 and 10–12). Also shown are the signals obtained for cytosolic 28S rRNA used for normalization. Each lane contains RNA from pooled cells of three equally treated culture dishes. Vectors used are indicated above the lanes. (B) Levels of CO I mRNA and its precursor RNA 6, as well as 12S rRNA (arbitrary densitometric units/28S rRNA signal) at the indicated time points after transfection with pKEX-TFAM (black bars) or controls (pKEX, open bars). Numbers of RNA preparations analysed are given for each time point; data are mean ± SD. (C) Northern blot showing levels of ND1 mRNA, ATPase 6/8 mRNA and their respective precursors as well as 28S rRNA used for normalization after transfection with pKEX (lanes 1–3), pKEX-ΔC-8-TFAM (lanes 4–6) and pKEX-ΔC-15-TFAM (lanes 7–9). The cells were harvested after 5 h. Each lane contains RNA from pooled cells of three equally treated culture dishes. Asterisk indicates a statistically significant difference between cells transfected with pKEX-TFAM and controls (pKEX), P < 0.05.

Figure 2

Effect of transient overexpression of TFAM on mitochondrial transcript levels in HeLa cells. (A) Northern blot showing levels of CO I mRNA, RNA 6 and 12S rRNA (upper panel), ND1 mRNA, RNA 19, ATPase 6/8 mRNA and its precursor (lower panel) at 5 h and 15 h after transfection with pKEX (lanes 1–3 and 7–9), and pKEX-TFAM (lanes 4–6 and 10–12). Also shown are the signals obtained for cytosolic 28S rRNA used for normalization. Each lane contains RNA from pooled cells of three equally treated culture dishes. Vectors used are indicated above the lanes. (B) Levels of CO I mRNA and its precursor RNA 6, as well as 12S rRNA (arbitrary densitometric units/28S rRNA signal) at the indicated time points after transfection with pKEX-TFAM (black bars) or controls (pKEX, open bars). Numbers of RNA preparations analysed are given for each time point; data are mean ± SD. (C) Northern blot showing levels of ND1 mRNA, ATPase 6/8 mRNA and their respective precursors as well as 28S rRNA used for normalization after transfection with pKEX (lanes 1–3), pKEX-ΔC-8-TFAM (lanes 4–6) and pKEX-ΔC-15-TFAM (lanes 7–9). The cells were harvested after 5 h. Each lane contains RNA from pooled cells of three equally treated culture dishes. Asterisk indicates a statistically significant difference between cells transfected with pKEX-TFAM and controls (pKEX), P < 0.05.

Figure 3

Effect of transient overexpression of TFAM on mtDNA copy number in HEK cells. (A) Southern blot showing linearized mtDNA at 20 h after transfection as well as new nucleic acid species hybridizing to a D-Loop probe (left panel), which are sensitive to RNAse H (right panel). Also shown is the signal for 18S rDNA used for normalization of mtDNA. M, double-stranded DNA size marker. (B) Western blot showing levels of TFAM and ΔC-15-TFAM at the indicated time points after transfection (four samples per group). The same blot was incubated with a β-tubulin antibody for normalization. (C) Southern blot showing levels of linearized mtDNA at the indicated time points after transfection with empty vector pKEX, pKEX-ΔC-15-TFAM and pKEX-TFAM, respectively (two samples per group). The blot was re-hybridized with an 18S rDNA probe for normalization.

Figure 4

Determination of the stoichiometry of TFAM to mtDNA in HEK cells. (A) Indicated amounts of HEK DNA as well as plasmid containing the ND1 gene were run in the same (left) or separate lanes (two right lanes), the Southern blot was hybridized to an ND1 probe and data were analysed densitometrically. (B) Indicated amounts of HEK protein as well as recombinant full-length TFAM protein were run in the same lanes (lower panel) or separate lanes (upper panel), the western blot was incubated with a TFAM antiserum and data were analysed densitometrically.

Figure 5

Effect of EBr treatment and removal in HEK cells. (A) Effect of EBr treatment for 4 days and drug removal (w/o) for 3, 6 and 24 h on levels of mtDNA (upper panel) and TFAM (lower panel). Also shown are the signals for 18S rDNA and β-tubulin used for normalization, respectively. (B) Kinetics of changes of TFAM and mitochondrial ND1 mRNA upon EBr treatment for up to 24 h.

Figure 6

Transient overexpression of TFAM in HEK cells with lowered mtDNA and TFAM content. (A) Levels of TFAM protein after transfection of cells pre-treated with EBr for 4 days. Control sample of untreated cells shows normal level of TFAM (lane 1); untransfected cells harvested after 18 h (lane 2) and cells which were transfected with pKEX-TFAM for 5 h (lane 3), 12 h (lane 4) and 18 h (lane 5) are shown. p, precursor; m, mature protein. (B) Northern blot showing levels of ND1 mRNA, RNA 19, ATPase 6/8 mRNA, pre-ATPase 6/8 and CO I mRNA as well as the signal of the 28S rRNA used for normalization. Each lane contains RNA from pooled cells of three equally treated culture dishes. Control sample of untreated cells shows normal wild-type level of various RNA species (lane 1), and lanes 2–5 show RNA levels in cells pre-treated with EBr for 4 days. Untransfected cells harvested after 18 h (lane 2) and cells that were transfected with pKEX-TFAM for 5 h (lane 3), 12 h (lane 4) and 18 h (lane 5) are shown. These samples were derived from the same plates as the samples shown on the western blot in Figure 5A.

Figure 7

Transient overexpression of TFAM and its C-terminal truncated isoform by nucleoporation in HEK cells with lowered mtDNA content. (A) Western blot showing levels of TFAM and precursor proteins in untreated control cells, after 4 days of EBr treatment and 6, 18 and 30 h after nucleoporation of EBr treated cells with various plasmids or in untransfected cells. Also shown is the level of β-tubulin to demonstrate equal loading. (B) Northern blot showing levels of ND1 mRNA in the samples described in (A). (C) Southern blots showing levels of linearized mtDNA (upper panel) or conformations of mtDNA (lower panel) in the samples described in A and B.