Is there bidirectional control of mitochondrial DNA replication?
thesisposted on 03.03.2017, 00:53 by Lee, William
Mitochondrial DNA (mtDNA) plays an important role during development in eukaryotic cells. It encodes 13 subunits of electron transfer chain (ETC) and is extensively involved in the synthesis of ATP via the process of oxidative phosphorylation (OXPHOS). MtDNA copy number is strictly regulated during differentiation, which is closely linked to the expression of the catalytic subunit of mitochondrial DNA polymerase gamma (POLGA). It has been recently shown that the levels of DNA methylation in exon 2 of PolgA in non-transformed mouse cells are negatively correlated with mtDNA copy number in a tissue-specific manner. On the other hand, the polymorphisms within mtDNA establish the mtDNA haploytpes and it has been shown that mtDNA haplotypes exert effects on the expression of pluripotent genes in mouse embryonic stem cells harbouring different mtDNA. Increasing evidence shows that the regulation of mtDNA is critical for cancer cells. The question remains undetermined as to how the interactions between nuclear and mitochondrial genomes influence tumorigenesis in human. DNA methylation of exon 2 of POLGA is correlated with down-regulation of mtDNA copy number in proliferative stem cells and cancer cells. Differentiating human neural stem cells (hNSCs) and terminally differentiated tissues demonstrated that loss of DNA methylation corresponded to increased mtDNA copy number and expression of POLGA. Using demethylating agents, 5-azacytidine (5-azaC) and ascorbic acid (VitC), Glioblastoma multiforme (GBM) cells showed enhanced differentiation by demethylating at exon 2 of POLGA. However, a rebound effect was observed in the VitC-treated GBM cells, as it did not inhibit DNA methylation but instead only enhanced DNA demethylation. To address the influence of mtDNA on tumorigenesis, multiple myeloma (U266) cells, GBM and osteosarcoma (143B) cells were depleted of their mtDNA and were inoculated into immunocompromised mice to form tumours. 143B cells were further repopulated with mtDNA from different genotypes and all samples were analysed for genome-wide mRNA expression profiles using RNA-seq or microarray. The data demonstrated that extensive depletion of mtDNA reduced tumorigenicity in all 3 types of cancer models. However, HSR-GBM1 and 143B cells were able to rebound from depletion by either replenishing their mtDNA or acquisition of foreign mtDNA from the surrounding tumour microenvironment. The introduction of different mtDNA genotypes into 143B cells influenced the nuclear gene expression profiles in a mtDNA genotype-specific manner but also restored tumorigenicity even in those with wild-type NSC mtDNA. This suggests that the nuclear genome plays a major role in tumorigenic phenotypes but the mitochondrial genome also showed an impact on the nucleus. The xenomitochondrial 143B reconstituted cell lines further demonstrated changes in gene expression profiles at different stages of tumour progression, suggesting possible therapeutic targets. Overall, this thesis shows that the nuclear genome interacts closely and bi-directionally with the mitochondrial genome in human cells. Such interactions are influenced by numerous factors such as epigenetic status of the nuclear genome as well as the variants in mtDNA and its copy number. This results in maintenance of pluripotency and tumorigenicity in stem cells and cancer cells, respectively.