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Whole mitochondrial genome bisulfite sequencing (WmGBS) analysis for the mouse brain, liver, and embryonic stem cells

ABSTRACT: Whilst 5-methylcytosine (5mC) is a major epigenetic mark in the nuclear DNA in mammals, whether or not mitochondrial DNA (mtDNA) receives 5mC modification remains controversial. Herein, we exhaustively analysed mouse mtDNA using three methods that are based upon different principles for detecting 5mC. Next-generation bisulfite sequencing did not give any significant signatures of methylation in mtDNAs of liver, brain and embryonic stem cells (ESCs). Also, treatment with methylated cytosine-sensitive endonuclease McrBC resulted in no substantial decrease of mtDNA band intensities in Southern hybridisation. Furthermore, mass spectrometric nucleoside analyses of highly purified liver mtDNA preparations did not detect 5-methyldeoxycytidine at the levels found in the nuclear DNA but at a range of only 0.3–0.5% of deoxycytidine. Taken together, we propose that 5mC is not present at any specific region(s) of mtDNA and that levels of the methylated cytosine are fairly low, provided the modification occurs. It is thus unlikely that 5mC plays a universal role in mtDNA gene expression or mitochondrial metabolism.

ORGANISM(S): Mus musculus

PROVIDER: GSE104104 | GEO | 2018/04/11

REPOSITORIES: GEO

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Project description:Mitochondria generate signals of adaptation that regulate nuclear genes expression via retrograde signaling. But this phenomenon is complexified when qualitatively different mitochondria and mitochondrial DNA (mtDNA) coexist within cells. Although this cellular state of heteroplasmy leads to divergent phenotypes clinically, its consequences on cellular function and the cellular transcriptome are unknown. To interrogate this phenomenon, we generated somatic cell cybrids harboring increasing levels of a common mtDNA mutation (tRNALeu(UUR) 3243A>G) and mapped the resulting cellular phenotypes and transcriptional profiles across the complete range of heteroplasmy. Small increases in mutant mtDNAs caused relatively modest defect in mitochondrial oxidative capacity, but resulted in sharp transitions in mitochondrial ultrastructure and in the nuclear and mitochondrial transcriptomes, with the critical functional threshold corresponding to the induction of epigenetic regulatory systems. Principal component analysis underscores how each heteroplasmy level occupies a different "transcriptional space", with low levels heteroplasmy (20-30%) producing a dose-response linear progression in one direction, and mutationload of 50, 60 and 90% producing changes in the opposite direction. Hence, subtle changes in mitochondrial energetics can act through the epigenome to generate the phenotypes of the common “complex” diseases. Cells were generated by transferring the wildtype (3243A) and mutant (3243G) mtDNAs from a heteroplasmic 3243A>G patient’s lymphoblastoid cell line into 143B(TK-) mtDNA-deficient (ρo) cells and selected for transmitochondrial cybrids. Subsequent mtDNA depletion, reamplification, and cloning (Wiseman and Attardi, 1978) resulted in a series of stable cybrids harboring approximately 0, 20, 30, 50, 60, 90, and 100% 3243G mutant mtDNAs. Total RNA extracted from each cell line was then extracted, depleted of rRNA, and measured in sequenced in triplicates.

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Whole mitochondrial genome bisulfite sequencing (WmGBS) analysis for the mouse brain, liver, and embryonic stem cells

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