Mitochondrial DNA variants can mediate methylation status of inflammation, angiogenesis and signaling genes

Abstract

Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/or racial origins of populations. The H haplogroup is protective against age-related macular degeneration (AMD), while the J haplogroup is high risk for AMD. In the present study, we performed comparison analyses of human retinal cell cybrids, which possess identical nuclei, but mtDNA from subjects with either the H or J haplogroups, and demonstrate differences in total global methylation, and expression patterns for two genes related to acetylation and five genes related to methylation. Analyses revealed that untreated-H and -J cybrids have different expression levels for nuclear genes (CFH, EFEMP1, VEGFA and NFkB2). However, expression levels for these genes become equivalent after treatment with a methylation inhibitor, 5-aza-2'-deoxycytidine. Moreover, sequencing of the entire mtDNA suggests that differences in epigenetic status found in cybrids are likely due to single nucleotide polymorphisms (SNPs) within the haplogroup profiles rather than rare variants or private SNPs. In conclusion, our findings indicate that mtDNA variants can mediate methylation profiles and transcription for inflammation, angiogenesis and various signaling pathways, which are important in several common diseases.

Figure 1.

J cybrids show increased levels of global DNA methylation. The levels of methylated DNA (5-mC%) were quantified in J (n = 3) and H (n = 3) cybrids, cultured under identical conditions. The H cybrids had a lower 5-mC% mean value (0.007 ± 0.001) compared with the J cybrids (0.022 ± 0.0053, P = 0.02). Samples were run in duplicate and the experiment was repeated twice. Statistical significance is denoted by *P < 0.05.

Figure 2.

Schematic of the acetylation and methylation enzymes affecting transcription. Upper panel shows the active transcription state for chromatin with unmethylated CpG sites on the DNA and acetylated histone sites. Bottom panel shows the inactive transcription state for chromatin with methylated CpG sites and non-acetylated histone H3 lysine residues. HATs, histone acetyltransferase; HDACs, histone deacetylase; DNMTs, DNA (cystosine-5) methyltransferase; MBDs, methyl-CpG binding domain protein 2; H3, histone lysine residue; M, methylated CpG site.

Figure 3.

Schematic summarizing epigenetic profiles of H versus J cybrids. The H and J cybrids have different levels of total global methylation and expression of acetylation and methylation-related genes. Untreated H and J cybrids show significantly different transcription levels for CFH (P < 0.0001), EFEMP1 (P = 0.015), NFkB2 (P = 0.03) and VEGFA (P = 0.0006). After 48 h treatment with 5-aza-dC, a methylation inhibitor, then the gene expression levels are equivalent in the H and J cybrid cultures; CFH (P < 0.42, EFEMP1 (P = 0.2), NFkB2 (P = 0.71) and VEGFA (P = 0.3). Rho0, lacking mtDNA; 5-aza-dC, 5-aza-2′-deoxycytidine; CFH, Complement factor H; EFEMP1, EGF containing fibulin-like extracellular matrix protein1; VEGFA, vascular endothelial growth factor A; NFkB2, nuclear factor of kappa light polypeptide gene enhancer in B-cells 2.