Programmable aging function-related mitochondrial DNA 5-methylcytosine (m5C) modification with a TALE-directed...

Area of Science:

• Epigenetics
• Mitochondrial Biology
• Molecular Genetics

Background:

• Mitochondrial DNA (mtDNA) harbors genes crucial for cellular respiration and is implicated in aging and disease.
• Epigenetic modifications, like DNA methylation, can regulate gene expression without changing the DNA sequence.
• Targeting mtDNA for epigenetic modifications presents a therapeutic avenue for mitochondrial dysfunction.

Purpose of the Study:

• To develop and characterize a novel mitochondrial epigenetic editor (MEE) for site-specific mtDNA methylation.
• To assess the efficacy and specificity of MEE in cellular and in vivo models.
• To explore the functional consequences of MEE-mediated mtDNA methylation in aging-associated contexts.

Main Methods:

• Construction of MEE by fusing mitochondrion-targeted TALE modules with Dnmt3A and Dnmt3L methyltransferases.
• Application of MEE in human cells to induce site-specific 5mC methylation at targeted mtDNA loci.
• In vivo administration of MEE in mice to assess mtDNA methylation changes in brain tissue and downstream effects on gene expression and biomarkers.

Main Results:

• MEE achieved efficient and site-specific 5mC methylation in cellular mtDNA with low off-target activity.
• Methylation at the C12191 (H) site exceeded 53%, correlating with a ~95% reduction in MT-ND5 mRNA levels.
• In vivo, MEE increased methylation at the aging-associated C11168 (H) site by 11.76%, reducing MT-ND4 expression and altering plasma t-Tau and NFL levels in mice.

Conclusions:

• MEE serves as a powerful tool for precise epigenetic engineering of mitochondrial DNA.
• MEE enables the experimental investigation of specific 5mC modifications and their functional roles.
• This technology holds promise for understanding and potentially treating aging-associated diseases linked to mitochondrial dysfunction.