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Modulation of Mitochondrial DNA Copy Number in a Model of Glioblastoma

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Modulation of Mitochondrial DNA Copy Number in a Model of Glioblastoma Sun and St John Epigenetics & Chromatin (2018) 11:53 https://doi.org/10.1186/s13072-018-0223-z Epigenetics & Chromatin RESEARCH Open Access Modulation of mitochondrial DNA copy number in a model of glioblastoma induces changes to DNA methylation and gene expression of the nuclear genome in tumours Xin Sun1,2 and Justin C. St John1,2* Abstract Background: There are multiple copies of mitochondrial DNA (mtDNA) present in each cell type, and they are strictly regulated in a cell-specifc manner by a group of nuclear-encoded mtDNA-specifc replication factors. This strict regu- lation of mtDNA copy number is mediated by cell-specifc DNA methylation of these replication factors. Glioblastoma multiforme, HSR-GBM1, cells are hyper-methylated and maintain low mtDNA copy number to support their tumori- genic status. We have previously shown that when HSR-GBM1 cells with 50% of their original mtDNA content were inoculated into mice, tumours grew more aggressively than non-depleted cells. However, when the cells possessed only 3% and 0.2% of their original mtDNA content, tumour formation was less frequent and the initiation of tumori- genesis was signifcantly delayed. Importantly, the process of tumorigenesis was dependent on mtDNA copy number being restored to pre-depletion levels. Results: By performing whole genome MeDIP-Seq and RNA-Seq on tumours generated from cells possessing 100%, 50%, 0.3% and 0.2% of their original mtDNA content, we determined that restoration of mtDNA copy number caused signifcant changes to both the nuclear methylome and its transcriptome for each tumour type. The afected genes were specifcally associated with gene networks and pathways involving behaviour, nervous system development, cell diferentiation and regulation of transcription and cellular processes. The mtDNA-specifc replication factors were also modulated. Conclusions: Our results highlight the bidirectional control of the nuclear and mitochondrial genomes through modulation of DNA methylation to control mtDNA copy number, which, in turn, modulates nuclear gene expression during tumorigenesis. Keywords: Mitochondrial DNA, DNA methylation, Gene expression, Tumorigenesis, POLG Background of the electron transfer chain that conducts oxidative Mammalian cells have multiple copies of the mitochon- phosphorylation (OXPHOS) and is the cell’s major gen- drial genome (mtDNA) within each mitochondrion. erator of ATP. It also encodes 22 transfer RNAs and 2 Te human mitochondrial genome is a circular, double- ribosomal RNAs [1]. stranded genome that is 16.6 kb in size. It is essential for mtDNA replication is driven by nuclear-encoded the production of cellular ATP, as it encodes 13 subunits mtDNA-specifc replication factors that translocate to the mitochondrion. Te primary factor is the mtDNA- specifc polymerase, polymerase gamma [2, 3], which is *Correspondence: [email protected] a heterotrimer enzyme composed of a catalytic subunit, 1 Mitochondrial Genetics Group, Hudson Institute of Medical Research, 27‑31 Wright Street, Clayton, VIC 3168, Australia subunit A (POLG), and two supporting subunits, subu- Full list of author information is available at the end of the article nits B (POLG2) [4]. Te process of mtDNA replication © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sun and St John Epigenetics & Chromatin (2018) 11:53 Page 2 of 18 is also supported by the mitochondrial helicase twin- to establish a new DNA methylation profle directed by kle (TWNK), the single-stranded DNA binding pro- the DNA methyltransferase family (DNMTs), which tein (SSBP1) and the mitochondrial topoisomerase primes naïve cells for cellular diferentiation [27, 28]. (TOP1MT) [5–7]. Tere are several other key factors To this extent, cell-specifc gene expression profles are including mitochondrial transcription factors A (TFAM), established during diferentiation in synchrony with B1 (TFB1M) and B2 (TFB2M), which generate the pre- changes to DNA methylation patterns. Likewise, the cursor transcript used to initiate mtDNA replication DNA methylation status at exon 2 of POLG is a determi- [8, 9]. Furthermore, some upstream regulators of these nant of when this gene is expressed and, in turn, regu- mtDNA replication factors are highly involved in mito- lates mtDNA copy number in a cell-specifc manner [15, chondrial biogenesis and function. Tese include nuclear 16]. Tis is supported by experiments using DNA dem- respiratory factors 1 and 2 (NRF1/2), peroxisome prolif- ethylation agents, such as 5-azacytidine [29] and vitamin erator-activated receptor γ (PPARG) and its co-activator C [30], where modulation of DNA methylation at exon 2 PGC1α (PPARGC1A), the Sirtuin family of genes (SIRT1- of POLG increased mtDNA copy number in HSR-GBM1 3), and the oestrogen-related receptors (ESRRA/B/G) cells derived from a glioblastoma multiforme (GBM) [10–14]. tumour [16, 18, 31]. mtDNA replication is strictly regulated during devel- Te HSR-GBM1 cell line is a high-grade malignant opment and diferentiation, which enables mature cells GBM cell line that is characterized as being similar to to acquire the requisite numbers of mtDNA copy to stem-like neural precursors and is extensively DNA support their specifc functions [15–18]. Initially, this is methylated, which contributes to its tumorigenic gene achieved by pluripotent (naïve) cells having established profle [32, 33]. However, its hyper-methylated profle the mtDNA set point (reviewed in [19]). Tese naïve is not established by the overexpression of the isocitrate cells each possess around 200 copies of mtDNA, which dehydrogenases (IDH1/2) that harbour onco-muta- promotes glycolysis as the favoured form of energy pro- tions, as the alleles for these genes are wild type [34, duction, and cellular proliferation [20]. Tese copies of 35]. Under normal circumstances, IDH enzymes act on mtDNA serve as the initial template for mtDNA replica- the citric acid cycle to generate α-ketoglutarate, which tion, allowing cells to acquire the appropriate numbers of is a co-factor of the TET enzymes that modulate DNA mtDNA copy as they diferentiate into mature cell types demethylation patterns [36–40]. However, overexpres- [16–18, 20, 21]. sion of and mutations to the IDH genes in GBM result Te inability to regulate mtDNA copy number afects in a metabolic switch that produces 2-hydroxyglutarate cellular function and impedes developmental potential. and restricts DNA demethylation induced by the TET For example, oocytes at the metaphase II stage that pos- enzymes [36–40]. Consequently, HSR-GBM1 cells enable sess too few copies of mtDNA frequently fail to fertilize the analysis of modifcations to DNA methylation profles [22–25]. Likewise, certain types of cancer cells exhibit to be undertaken whereby the DNA methylation status low mtDNA copy number and are unable to successfully of the cells is not infuenced by mutations to key regu- complete diferentiation [16, 18]. Indeed, cancer cells lators of DNA demethylation and thus allows the efects mainly rely on aerobic glycolysis for energy production, of mtDNA copy number to be studied independently of which allows for higher rates of cellular proliferation and these infuences. prevents diferentiation from taking place [16]. However, Interestingly, mtDNA depletion of HSR-GBM1 cells when they are induced to diferentiate, they appear to be to varying amounts of mtDNA copy number afected trapped in a ‘pseudo-diferentiated’ state as they had not tumour progression and frequency when these cells were previously maintained the mtDNA set point and, as a inoculated into mice [18]. Progression and frequency result, the nuclear and mitochondrial genomes do not act were greatest in cells depleted to 50% of their original in synchrony [19, 26]. Interestingly, when cancer cells are content, but tumour formation was less frequent and partially depleted of their mtDNA content, they undergo took signifcantly longer when cells possessed only 3% dediferentiation and are then able to replicate mtDNA and 0.2% of their original mtDNA content [18]. Notably, as they undergo diferentiation, suggesting that they mtDNA copy was restored to similar levels during in vivo have re-established the mtDNA set point and synchrony tumorigenesis accompanied by DNA demethylation at between the nuclear and mitochondrial genomes [18]. exon 2 of POLG [17]. During the early stages of development, the nuclear In order to determine whether global DNA methyla- genome undergoes DNA demethylation mediated by tion profles were modulated following the restoration the ten-eleven translocation methylcytosine dioxyge- and maintenance of mtDNA copy number in end point nases (TET enzymes) to erase parental DNA methyla- tumours, we investigated the DNA methylation profles tion profles. De novo DNA methylation then takes place of GBM tumours derived from HSR-GBM1 cells that Sun and St John Epigenetics & Chromatin (2018) 11:53 Page 3 of 18 possessed varying amounts of mtDNA copy number 1.5 100 and exhibited diferent frequencies
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