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High levels of TFAM repress in vivo transcription of mitochondrial DNA

ABSTRACT: Mammalian mitochondrial DNA (mtDNA) is coated with mitochondrial transcription factor A (TFAM) and compacted into nucleoids. TFAM is not only the main component of mitochondrial nucleoids but its levels can also control mtDNA copy number. Here we show that the TFAM-to-mtDNA ratio is critical for maintaining normal mtDNA expression in different tissues of the mouse. BAC transgenic mice with a 1.5-fold increase in TFAM protein levels maintain a normal TFAM-to-mtDNA ratio in different tissues and as a consequence mitochondrial gene expression, nucleoid distribution and whole animal metabolism are all unaltered. In contrast, mice expressing TFAM from the CAG promoter in the ROSA26 locus have 4.5-fold increase of TFAM protein levels in heart and skeletal muscle and develop pathology leading to early postnatal lethality. The TFAM-to-mtDNA ratio varies widely between tissues in these mice and is very high in skeletal muscle where it causes strong repression of mtDNA expression and deficient oxidative phosphorylation (OXPHOS) despite normal mtDNA levels. In heart, mtDNA copy number is increased leading to a near normal TFAM-to-mtDNA ratio and maintained OXPHOS capacity. In the liver, mtDNA expression is maintained despite increased TFAM levels and normal mtDNA levels. Here, tissue-specific induction of the LONP1 protease and mitochondrial RNA polymerase (POLRMT) expression counteracts the silencing effect of high TFAM levels. We conclude that the TFAM-to-mtDNA ratio has an important role in maintaining mtDNA expression in vivo. TFAM acts as a general repressor of mtDNA expression and this effect can be counterbalance by tissue-specific expression of regulatory factors.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Heart, Skeletal Muscle, Liver

SUBMITTER: Ilian Atanassov

LAB HEAD: Nils-Göran Larsson

PROVIDER: PXD023050 | Pride | 2021-08-18

REPOSITORIES: Pride

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Project description:The goal of this analysis was to utilize microarray profiling to identify basal alterations in gene expression in response to TFAM depletion and mtDNA stress. Mitochondrial DNA (mtDNA) is normally present at thousands of copies per cell and is packaged into several hundred higher-order structures termed nucleoids. The abundant mtDNA-binding protein, TFAM (transcription factor A,mitochondrial), regulates nucleoid architecture, abundance and segregation. Complete mtDNA depletion profoundly impairs oxidative phosphorylation, triggering calcium-dependent stress signalling and adaptive metabolic responses. However, the cellular responses to mtDNA instability, a physiologically relevant stress observed in many human diseases and ageing, remain poorly defined. Here we show that moderate mtDNA stress elicited by TFAM deficiency engages cytosolic antiviral signalling to enhance the expression of a subset of interferon-stimulated genes. Mechanistically, we find that aberrant mtDNA packaging promotes escape of mtDNA into the cytosol, where it engages the DNA sensor cGAS (also known as MB21D1) and promotes STING (also known as TMEM173)–IRF3-dependent signalling to elevate interferon-stimulated gene expression, potentiate type I interferon responses and confer broad viral resistance. Furthermore, we demonstrate that herpesviruses induce mtDNA stress, which enhances antiviral signalling and type I interferon responses during infection. Our results further demonstrate that mitochondria are central participants in innate immunity, identify mtDNA stress as a cell-intrinsic trigger of antiviral signaling and suggest that cellular monitoring of mtDNA homeostasis cooperates with canonical virus sensing mechanisms to fully engage antiviral innate immunity. Murine embryonic fibroblasts were isolated from wild-type or Tfam+/- E13.5 littermate embryos. RNA from passage-matched wild-type and Tfam+/- MEF lines was extracted in duplicate and hybridized onto Affymetrix microarrays. Four arrays were performed in total with two technical replicates per genotype.

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High levels of TFAM repress in vivo transcription of mitochondrial DNA

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