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HEK293 TFAM Knockout Methylation Study

ABSTRACT: Mitochondrial DNA copy number (mtDNA-CN) has been associated with a variety of aging-related diseases, including all-cause mortality. However, the mechanism by which mtDNA-CN influences disease is not currently understood. One such mechanism may be through regulation of nuclear gene expression via the modification of nuclear DNA (nDNA) methylation. To investigate this hypothesis, we assessed the relationship between mtDNA-CN and nDNA methylation in 2,507 African American (AA) and European Americans (EA) participants from the Atherosclerosis Risk in Communities (ARIC) study using the Infinium Human Methylation 450K Beadchip (485,764 CpGs). Thirty-four independent CpGs were associated with mtDNA-CN at genome-wide significance (P<5x10-8). To validate our findings we assayed an additional 2,528 participants from the Cardiovascular Health Study (CHS) (N=533) and Framingham Heart Study (FHS) (N=1995). Meta-analysis across all cohorts identified 6 mtDNA-CN associated CpGs to be validated ac ross cohorts at genome-wide significance (P<5x10-8). Additionally, over half of these CpGs were associated with phenotypes known to be associated with mtDNA-CN, including CHD, CVD, and mortality. Experimental modification of mtDNA-CN through knockout via CRISPR-Cas9 of TFAM, a regulator of mtDNA replication, demonstrated that modulation of mtDNA-CN directly drives changes in nDNA methylation and gene expression of specific CpGs and nearby transcripts. Strikingly, the ‘neuroactive ligand receptor interaction’ KEGG pathway was found to be highly overrepresented in the ARIC cohort (P= 5.24x10-12), as well as the TFAM knockout methylation (P=4.41x10-4) and expression (P=4.30x10-4) studies. These results demonstrate that changes in mtDNA-CN influence nDNA methylation at specific loci and result in differential gene expression of specific genes, including those acting in the ‘neuroactive ligand receptor interaction’ pathway that may impact human health and disease via altered cell signaling.

ORGANISM(S): Homo sapiens

PROVIDER: GSE133994 | GEO | 2019/07/09

REPOSITORIES: GEO

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Project description:HEK293 cells with CRISPR KO were analyzed using RNA-Seq. Abstract: Mitochondrial DNA copy number (mtDNA-CN) has been associated with a variety of aging-related diseases, including all-cause mortality. However, the mechanism by which mtDNA-CN influences disease is not currently understood. One such mechanism may be through regulation of nuclear gene expression via the modification of nuclear DNA (nDNA) methylation. To investigate this hypothesis, we assessed the relationship between mtDNA-CN and nDNA methylation in 2,507 African American (AA) and European Americans (EA) participants from the Atherosclerosis Risk in Communities (ARIC) study using the Infinium Human Methylation 450K Beadchip (485,764 CpGs). Thirty-four independent CpGs were associated with mtDNA-CN at genome-wide significance (P<5x10-8). To validate our findings we assayed an additional 2,528 participants from the Cardiovascular Health Study (CHS) (N=533) and Framingham Heart Study (FHS) (N=1995). Meta-analysis across all cohorts identified 6 mtDNA-CN associated CpGs to be validated across cohorts at genome-wide significance (P<5x10-8). Additionally, over half of these CpGs were associated with phenotypes known to be associated with mtDNA-CN, including CHD, CVD, and mortality. Experimental modification of mtDNA-CN through knockout via CRISPR-Cas9 of TFAM, a regulator of mtDNA replication, demonstrated that modulation of mtDNA-CN directly drives changes in nDNA methylation and gene expression of specific CpGs and nearby transcripts. Strikingly, the ‘neuroactive ligand receptor interaction’ KEGG pathway was found to be highly overrepresented in the ARIC cohort (P= 5.24x10-12), as well as the TFAM knockout methylation (P=4.41x10-4) and expression (P=4.30x10-4) studies. These results demonstrate that changes in mtDNA-CN influence nDNA methylation at specific loci and result in differential gene expression of specific genes, including those acting in the ‘neuroactive ligand receptor interaction’ pathway that may impact human health and disease via altered cell signaling.

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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HEK293 TFAM Knockout Methylation Study

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