Mitochondrial DNA haplotypes define gene expression patterns in pluripotent and differentiating embryonic stem cells.

Mitochondrial DNA haplotypes are associated with various phenotypes, such as altered susceptibility to disease, environmental adaptations, and aging. Accumulating evidence suggests that mitochondrial DNA is essential for cell differentiation and the cell phenotype. However, the effects of different mitochondrial DNA haplotypes on differentiation and development remain to be determined. Using embryonic stem cell lines possessing the same Mus musculus chromosomes but harboring one of Mus musculus, Mus spretus, or Mus terricolor mitochondrial DNA haplotypes, we have determined the effects of different mitochondrial DNA haplotypes on chromosomal gene expression, differentiation, and mitochondrial metabolism. In undifferentiated and differentiating embryonic stem cells, we observed mitochondrial DNA haplotype-specific expression of genes involved in pluripotency, differentiation, mitochondrial energy metabolism, and DNA methylation. These mitochondrial DNA haplotypes also influenced the potential of embryonic stem cells to produce spontaneously beating cardiomyocytes. The differences in gene expression patterns and cardiomyocyte production were independent of ATP content, oxygen consumption, and respiratory capacity, which until now have been considered to be the primary roles of mitochondrial DNA. Differentiation of embryonic stem cells harboring the different mitochondrial DNA haplotypes in a 3D environment significantly increased chromosomal gene expression for all haplotypes during differentiation. However, haplotype-specific differences in gene expression patterns were maintained in this environment. Taken together, these results provide significant insight into the phenotypic consequences of mitochondrial DNA haplotypes and demonstrate their influence on differentiation and development. We propose that mitochondrial DNA haplotypes play a pivotal role in the process of differentiation and mediate the fate of the cell.

Mitochondrial DNA copy number is regulated in a tissue specific manner by DNA methylation of the nuclear-encoded DNA polymerase gamma A.

DNA methylation is an essential mechanism controlling gene expression during differentiation and development. We investigated the epigenetic regulation of the nuclear-encoded, mitochondrial DNA (mtDNA) polymerase γ catalytic subunit (PolgA) by examining the methylation status of a CpG island within exon 2 of PolgA. Bisulphite sequencing identified low methylation levels (<10%) within exon 2 of mouse oocytes, blastocysts and embryonic stem cells (ESCs), while somatic tissues contained significantly higher levels (>40%). In contrast, induced pluripotent stem (iPS) cells and somatic nuclear transfer ESCs were hypermethylated (>20%), indicating abnormal epigenetic reprogramming. Real time PCR analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) immunoprecipitated DNA suggests active DNA methylation and demethylation within exon 2 of PolgA. Moreover, neural differentiation of ESCs promoted de novo methylation and demethylation at the exon 2 locus. Regression analysis demonstrates that cell-specific PolgA expression levels were negatively correlated with DNA methylation within exon 2 and mtDNA copy number. Finally, using chromatin immunoprecipitation (ChIP) against RNA polymerase II (RNApII) phosphorylated on serine 2, we show increased DNA methylation levels are associated with reduced RNApII transcriptional elongation. This is the first study linking nuclear DNA epigenetic regulation with mtDNA regulation during differentiation and cell specialization.