Genome-wide characterization of mitochondrial DNA methylation in human brain

Abstract

Background: There is growing interest in the role of DNA methylation in regulating the transcription of mitochondrial genes, particularly in brain disorders characterized by mitochondrial dysfunction. Here, we present a novel approach to interrogate the mitochondrial DNA methylome at single base resolution using targeted bisulfite sequencing. We applied this method to investigate mitochondrial DNA methylation patterns in post-mortem superior temporal gyrus and cerebellum brain tissue from seven human donors.

Results: We show that mitochondrial DNA methylation patterns are relatively low but conserved, with peaks in DNA methylation at several sites, such as within the D-LOOP and the genes MT-ND2, MT-ATP6, MT-ND4, MT-ND5 and MT-ND6, predominantly in a non-CpG context. The elevated DNA methylation we observe in the D-LOOP we validate using pyrosequencing. We identify loci that show differential DNA methylation patterns associated with age, sex and brain region. Finally, we replicate previously reported differentially methylated regions between brain regions from a methylated DNA immunoprecipitation sequencing study.

Conclusions: We have annotated patterns of DNA methylation at single base resolution across the mitochondrial genome in human brain samples. Looking to the future this approach could be utilized to investigate the role of mitochondrial epigenetic mechanisms in disorders that display mitochondrial dysfunction.

Keywords: 5-Methylcytosine (5mC); Brain; DNA Methylation; Mitochondria; epigenetics; mtDNA.

Figure 1

The mitochondrial genome is characterized by specific regions of methylation, predominantly in a CpH context. (A) Shown is the mean number of sites with >1% methylation in the STG (blue) and CER (red) in either a CpG or CpH context. Error bars represent the standard error of the mean (SEM). (B) Graph showing mean % methylation at each cytosine in the mitochondrial genome in STG (top panel) and CER (bottom panel) samples, created using UCSC browser (http://genome.ucsc.edu).

Figure 2

Validation of mtDNA methylation in the D-Loop using pyrosequencing. Box plots showing % methylation levels at site ChrM:545 in the seven donors used in the study, when profiled using targeted bisulfite sequencing (Seq) and pyrosequencing (Pyro) in the STG (blue) and CER (red). Boxplots represent the median (central line) and interquartile range (perimeter), with plus sign denoting the mean, whiskers showing the minimum and maximum value, and individual datapoints shown as black circles (STG) or triangles (CER). The dashed black horizontal line represents the reported minimum threshold for detecting methylation using pyrosequencing (4.3%).