The Study Of Mitochondrial DNA De Novo Methylation During Early Development Of Mouse Embryos

Mitochondria as the most abundant organelle, not only provide the energy, but also participate in the regulation of ion homeostasis, amino acid metabolism, fatty acid metabolism, and apoptosis, and so on. The orchestrated mitochondrial functions and dynamics is highly regulated by both the nuclear (nDNA) and mitochondrial genomes. In addition to the 37 mtDNA encoded genes, nDNA-encoded genes (approximately 1200 proteins) contribute significantly to mitochondrial function. During early embryonic development, mitochondrial functions and structures changed dramatically, which is very essential for survival and growth of early embryos. During this period, embryos underwent another important biological process named de novo methylation, which is critical for the establishment and maintenance of DNA methylation patterns in somatic cells. Interestingly, window of mitochondrial dynamics change best coincide with the window of de novo methylation of mtDNA.Using high-throughput analysis of methylome, methylated DNA immunoprecipitation-sequencing (MeDIP-seq), we found promoters of most of nDNA encoded mitochondria-related genes (MtGs), underwent de novo methylation, implying this epigenetic process is significantly implicated in the expression of MtGs during early development. Interestingly, we found the methylation levels of some regions of mtDNA appears to be increased during early development. Based on this novel observation, we hypothesized that mtDNA underwent de novo methylation during post-implantation period. Next, we confirmed that most selected regions of mtDNA underwent de novo methylation, which is independent on the futures of selected regions and CpG density. Furthermore, we explored the mechanism underlying mtDNA de novo methylation. Using ChIP-qPCR, we confirmed that DNMT3A and DNMT3B can bind to mtDNA during early development. In addition, we provided the evidences of mitochondrial-localized DNMT3A and DNMT3B using immunoelectron microscopy and Western blotting. Moreover, we overexpressed Dnmt3a exclusively in mitochondria of 293T cells using a commercial mitochondria-targeted sequence (MTS), which can lead to increase in mtDNA methylation level. Next, we screened out potential MTSs of Dnmt3a and Dnmt3b, and confirmed that the MTS is functionally active in targeting EGFP maker in mitochondria. This study, provide the novel insight in understanding the origin of mtDNA methylation that has been reported in different somatic cell types.Based on the essential role of mitochondrial functions and dynamics in early developemnt, we screened out nDNA and mtDNA encoded MtGs from transcriptome of in vivo (IVO) and in vitro fertilized (IVF) embryos at E3.5, E7.5 and E10.5. The filtered data was compared between IVO and IVF embryos. Functional analyses indicated that IVF-induced mitochondrial dysfunctions mainly included: (i) inhibited mitochondrial biogenesis and impaired maintenance of DNA methylation of mitochondria-related genes during the post-implantation stage; (ii) dysregulated glutathione/glutathione peroxidase (GSH/Gpx) system and increased mitochondria-mediated apoptosis; (iii) disturbed mitochondrial beta-oxidation, oxidative phosphorylation and amino acid metabolism; and (iv) disrupted mitochondrial transmembrane transport and membrane organization. Based on these observation, we supplemented melatonin, a mitochondria-enriched antioxidant that can improve mitochondrial functions, in vitro culture medium. This strategy can restore mitochondrial functions in IVF embryos and thereby rescuing the impaired development. This study, provided a promising strategy for improving IVF system by targeted restoring functions of organelles.