ORIGINAL RESEARCH article
Front. Cell Dev. Biol.
Sec. Molecular and Cellular Pathology
Volume 13 - 2025 | doi: 10.3389/fcell.2025.1677402
This article is part of the Research TopicBeyond Energy Production: Exploring Mitochondrial Dynamics and DiseaseView all 7 articles
Cytosine methylase and hydroxymethylase activity in mammalian mitochondria
Provisionally accepted
- 1Virginia Commonwealth University, Richmond, United States
- 2Virginia Commonwealth University Massey Comprehensive Cancer Center, Richmond, United States
Select one of your emails
You have multiple emails registered with Frontiers:
Notify me on publication
Please enter your email address:
If you already have an account, please login
You don't have a Frontiers account ? You can register here
Mitochondria are integral components of eukaryotic cells, functioning as energy powerhouses and key mediators of diverse metabolic and signaling cascades. As endosymbiotic remnants, these unique organelles retain and express their own DNA. Mitochondrial DNA (mtDNA) is packaged into DNA-protein complexes called nucleoids, and is also subject to epigenetic modification. We identified a mitochondrial isoform of DNA methyltransferase 1 (mtDNMT1) that binds to mtDNA in critical control regions; however, its enzymatic activity remained unexplored. Here, we show that endogenously-tagged mtDNMT1 purified from mitochondria exhibits time-and concentration-dependent CpG-specific DNA methyltransferase activity, but it is not working alone: DNMT3b cooperates with mtDNMT1 to methylate mtDNA and regulate mitochondrial transcription. In addition, we detect ten-eleven translocase (TET)-like hydroxymethylase activity in mitochondria, demonstrating that mechanisms for both writing and erasing 5-methylcytosine marks are functional in this organelle. CRISPR/Cas9-mediated inactivation of mtDNMT1 and/or DNMT3b activity resulted in a stepwise decrease in mitochondrial methylation across the heavy and light strand promoters of mtDNA, with a significant reduction in transcription of several mtDNA-encoded OXPHOS genes. Interestingly, the effects of mtDNA methylation on mitochondrial transcription are diametrically opposed to the role of promoter methylation in the nucleus, suggesting a novel mode of gene regulation in mitochondria. Cells lacking mtDNMT1 and/or DNMT3b also exhibited a modest reduction in mtDNA content, suggesting that methylation impacts both mtDNA transcription and replication. These observations implicate mtDNA methylation in the fine-tuning of mitochondrial function and suggest a role for aberrant mitochondrial methylase activity in disease.
Keywords: Mitochondrial DNA (mtDNA), epigenetics, transcription, DNA Replication, DNAmethylation, DNA demethylation, DNA methyltransferase
Received: 31 Jul 2025; Accepted: 25 Sep 2025.
Copyright: © 2025 Shock, Thakkar, Robinson and Taylor. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Lisa S Shock, lisa.shock@vcuhealth.org
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.