Investigating the role of DNA (cytosine-5)-methyltransferase 1 in mitochondrial DNA methylation

Abstract

Mitochondrial DNA (mtDNA) that escapes from autophagy-mediated degradation after hemodynamic stress can cause inflammation in the heart by activating TLR9 signalling pathway, in which unmethylated CpG motifs bind to TLR9. DNA methyltransferases (DNMTs) such as DNMT1 and DNMT3A are involved in nuclear DNA methylation. It has also been reported that two additional potential transcription initiation codons exist at 5’ region to the original DNMT1 and that this 1st-to-3rd ATG sequence fused to GFP cDNA can localise in mitochondria, suggesting that DNMT1 might affect mtDNA methylation patterns. The aim of this study is to elucidate the role of DNMT1 in mtDNA methylation. To achieve this aim, three different isoforms of mouse DNMT1 sequences (“nuclear DNMT1”, “whole DNMT1” and “mtDNMT1”) as well as a mouse DNMT3A isoform were generated through molecular cloning. Mitoprot II software analysis predicted a high chance of mitochondrial localization (>90%) for both whole DNMT1 and mtDNMT1 isoforms. Western blotting analysis confirmed overexpression of all DNMT1 constructs, and immunocytochemistry experiments confirmed the predicted localisation of DNMT1 isoforms. DNMT3A localisation was detected at nuclear level, but not at mitochondrial level. Adenoviral vectors for nuclear DNMT1 and mtDNMT1 were generated and the methylated-DNA immunoprecipitation/qPCR methylation analysis of the D-Loop showed a 3-to-5 fold increase in total methylation levels in mtDNMT1 overexpressed mouse cardiac endothelial cells. However, global percentage of methylation is low. CoIP-MS analysis after mtDNMT1 overexpression failed to highlight any candidates for mtDNMT1 co-interaction. These data suggest that the 1st-to-3rd ATG sequence contains the mitochondrial localisation signal for DNMT1, and that DNMT1 can localise to mitochondria through it and methylate mtDNA.

Date of Award	2018
Original language	English
Awarding Institution	King's College London
Supervisor	Kinya Otsu (Supervisor) & Metin Avkiran (Supervisor)