Mitochondrial DNA Variants Can Mediate Cellular Methylation Status and Expression of Inflammation, Angiogenesis and Signaling Genes

Abstract

 

Purpose

 

Individuals can be classified by their mitochondrial (mt) DNA into haplogroups representing different geographic and/or racial origins. Studies have shown that mtDNA variants are associated with risk for age-related macular degeneration (AMD). Using transmitochondrial cybrids, which possess identical nuclei but mtDNA from different individuals, it has been shown that cybrids containing mitochondria with the H haplogroup (protective for AMD) or J haplogroup (high risk for AMD) have differential expression of non-energy related genes. Epigenetic changes have been correlated with severity of AMD and some studies have demonstrated that mitochondria and methylation status are interconnected. The present study uses cybrids with H versus J haplogroup mtDNA to determine differences in methylation status and responses to 5-aza-2’-deoxycytidine (5-aza-dC, a methylation inhibitor).

 

Methods

 

Cybrids were created by fusing Rho0 ARPE-19 cells (depleted of mtDNA) with platelets isolated from haplogroup H (n=7) or J (n=6) individuals. Cultures were analyzed for total global DNA methylation, transcription levels for five methylation genes (MAT2B, MBD4, DNMT1, DNMT3A and DNMT3B) and sequencing of the entire mtDNA. In some cultures, methylation was blocked by treatment with 5-aza-dC and gene expression levels were measured.

 

Results

 

J cybrids showed significantly higher global methylation (5-methyC%, p=0.02) and expression levels for the MAT2B gene (1.51-fold, p = 0.002) but lower levels for DNMT1 (0.24-fold, p = 0.0001), DNMT3A (0.3-fold, p<0.0001), DNMT3B (0.27-fold, p < 0.001) and MBD2 (0.4-fold, p=0.001) compared to the H cybrids. The untreated-H and -J cybrids showed differential expression levels for nuclear-encoded genes (CHF, EFEMP1, VEGFA and NF-kB) and mtDNA-encoded genes (MT-ND1 and MT-ND6) but the expression levels became equal after 5-aza-dC treatment. Sequencing of the entire mtDNA suggested that differences in epigenetic status found in cybrids were due to haplogroup profiles rather than rare variants or private single nucleotide polymorphisms (SNPs).

 

Conclusions

 

Our findings indicate that mtDNA representing different haplogroup populations can mediate methylation profiles and transcription for inflammation, angiogenesis and signaling pathways, which are important in AMD and other common diseases.