DNA methylation status of nuclear-encoded mitochondrial genes underlies the tissue-dependent mitochondrial functions

Abstract

Background: Mitochondria are semi-autonomous, semi-self-replicating organelles harboring their own DNA (mitochondrial DNA, mtDNA), and their dysregulation is involved in the development of various diseases. While mtDNA does not generally undergo epigenetic modifications, almost all mitochondrial proteins are encoded by nuclear DNA. However, the epigenetic regulation of nuclear-encoded mitochondrial genes (nuclear mt genes) has not been comprehensively analyzed.

Results: We analyzed the DNA methylation status of 899 nuclear mt genes in the liver, brain, and heart tissues of mouse, and identified 636 nuclear mt genes carrying tissue-dependent and differentially methylated regions (T-DMRs). These nuclear mt genes are involved in various mitochondrial functions and they also include genes related to human diseases. T-DMRs regulate the expression of nuclear mt genes. Nuclear mt genes with tissue-specific hypomethylated T-DMRs were characterized by enrichment of the target genes of specific transcription factors such as FOXA2 in the liver, and CEBPA and STAT1 in the brain.

Conclusions: A substantial proportion of nuclear mt genes contained T-DMRs, and the DNA methylation status of numerous T-DMRs should underlie tissue-dependent mitochondrial functions.

Figure 1

Nuclear mt genes with T-DMRs identified by D-REAM analysis. (A) Differential methylation levels of HpyCH4IV sites for which the relative hypomethylation levels were analyzed using the D-REAM analysis. For each HpyCH4IV site, the corresponding MATscore is indicated along the horizontal axis. In each tissue comparison, the lowest MATscore of the HpyCH4IV sites whose hypomethylation were confirmed using COBRA, was considered as a threshold value for detecting hypomethylated regions in the tissue comparison. Pale-colored circles indicate that the corresponding HpyCH4IV site was not included in the hypo T-DMRs (see text for definition). (B) Matrices show the differential level of DNA methylation of the liver-, brain-, and heart-hypo T-DMRs in the tissues relative to the respcective methylation level of hypo T-DMRs in the liver, brain, and heart, respectively. Each column represents a different hypo T-DMR. The names of nuclear mt genes containing hypo T-DMRs in their TSS flanking regions are displayed under each column. (C) Number of nuclear mt genes with T-DMRs. Matrix columns represent different nuclear mt genes. (D) Function of nuclear mt genes, nuclear mt genes with T-DMRs and nuclear mt genes with liver-hypo T-DMRs. Pie charts illustrate the percentage of genes that possess the indicated functions.

Figure 2

Expression of nuclear mt genes with hypo T-DMRs. (A) Boxplots show the expression ratio (log₂ ratio) of the nuclear mt genes. Tissues used for comparisons are shown under the plots. "All" indicates that the plot shows the expression ratio of entire nuclear mt genes. "U" and "D" indicate that the plot shows the expression ratio of nuclear mt genes with hypo T-DMRs in upstream regions and downstream regions, respectively. The number of probe sets representing the expression levels of corresponding genes are displayed under the plot. P-values obtained from the Wilcoxon test are indicated on the top of the plot. (B) Genes which were relatively highly expressed in the tissues where downstream T-DMRs were hypomethylated, according to gene expression array data. Relative gene expression was confirmed using real-time PCR and was normalized to the expression of ß-actin (Actb). Bar graphs show the results of real-time PCR. "ND" indicates that the expression was not detected under the experimental condition used. Experiments were performed in biological duplicates, and all amplification were performed in triplicates. Error bars show standard deviations. For the Otc and Acsl6 genes, methylation levels of downstream hypo T-DMRs were examined by COBRA, and the electrophoresis images obtained by MultiNA microchip electrophoresis system are shown.

Figure 3

Concentration of liver-hypo T-DMRs in downstream regions of nuclear mt genes. (A) Bar graphs show the ratio of genes with downstream hypo T-DMRs in nuclear mt genes with hypo T-DMRs. (B) Bar graphs show the ratio of nuclear mt genes in all genes and genes with hypo T-DMRs. (C) The vertical axis of the histograms indicate the average number of liver-hypo T-DMRs per gene. Gene sets represented in histograms are indicated on the top-left of each graph.