title: The effects of nuclear reprogramming on mitochondrial DNA replication.
abstract: Undifferentiated mouse embryonic stem cells (ESCs) possess low numbers of mitochondrial DNA (mtDNA), which encodes key subunits associated with the generation of ATP through oxidative phosphorylation (OXPHOS). As ESCs differentiate, mtDNA copy number is regulated by the nuclear-encoded mtDNA replication factors, which initiate a major replication event on Day 6 of differentiation. Here, we examined mtDNA replication events in somatic cells reprogrammed to pluripotency, namely somatic cell-ES (SC-ES), somatic cell nuclear transfer ES (NT-ES) and induced pluripotent stem (iPS) cells, all at low-passage. MtDNA copy number in undifferentiated iPS cells was similar to ESCs whilst SC-ES and NT-ES cells had significantly increased levels, which correlated positively and negatively with Nanog and Sox2 expression, respectively. During pluripotency and differentiation, the expression of the mtDNA-specific replication factors, PolgA and Peo1, were differentially expressed in iPS and SC-ES cells when compared to ESCs. Throughout differentiation, reprogrammed somatic cells were unable to accumulate mtDNA copy number, characteristic of ESCs, especially on Day 6. In addition, iPS and SC-ES cells were also unable to regulate ATP content in a manner similar to differentiating ESCs prior to Day 14. The treatment of reprogrammed somatic cells with an inhibitor of de novo DNA methylation, 5-Azacytidine, prior to differentiation enabled iPS cells, but not SC-ES and NT-ES cells, to accumulate mtDNA copies per cell in a manner similar to ESCs. These data demonstrate that the reprogramming process disrupts the regulation of mtDNA replication during pluripotency but this can be re-established through the use of epigenetic modifiers.
authors: Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria, 3168, Australia.
authors: Richard D W
authors: RD
authors: Kelly
authors: Huseyin
authors: H
authors: Sumer
authors: Matthew
authors: M
authors: McKenzie
authors: Joao
authors: J
authors: Facucho-Oliveira
authors: Ian A
authors: IA
authors: Trounce
authors: Paul J
authors: PJ
authors: Verma
authors: Justin C
authors: JC
authors: St John
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D056915:DNA Copy Number Variations; D004265:DNA Helicases / Q000096:biosynthesis / Q000378:metabolism; D000074002:DNA Polymerase gamma; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000235:genetics; D024101:Mitochondrial Proteins / Q000096:biosynthesis / Q000378:metabolism; D000071317:Nanog Homeobox Protein; D053652:Nuclear Transfer Techniques; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D012235:Rhodamines / Q000494:pharmacology; D055748:SOXB1 Transcription Factors / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D004272:DNA, Mitochondrial; D004791:Enzyme Inhibitors; D018398:Homeodomain Proteins; D024101:Mitochondrial Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D012235:Rhodamines; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; C026188:rhodamine 6G; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C498803:Polg protein, mouse; C513299:Twnk protein, mouse; D004265:DNA Helicases; D001374:Azacitidine