A novel role for mitochondria in regulating epigenetic modification in the nucleus

Abstract

Epigenetic modification in the nuclear genome plays a key role in human tumorigenesis. In this paper, we investigated whether changes in the mtDNA copy number frequently reported to vary in a number of human tumors induce methylation changes in the nucleus. We utilized the Restriction Landmark Genomic Scanning (RLGS) to identify genes that undergo changes in their methylation status in response to the depletion and repletion of mtDNA. Our study demonstrates that depletion of mtDNA results in significant changes in methylation pattern of a number of genes. Furthermore, our study suggests that methylation changes are reversed by the restoration of mtDNA in cells otherwise lacking the entire mitochondrial genome. These studies provide the first direct evidence that mitochondria regulate epigenetic modification in the nucleus that may contribute to tumorigenesis.

Figure 1

Mitochondrial COXII expression in rho⁰ and cybrid cells. Western blot analysis of expression of subunit II of respiratory chain enzyme complex IV (cytochrome c oxidase: COX II) in cultured cells lacking mtDNA (rho⁰ cells) derived either chemically (A) or genetically after transfection of parental cells with HSV UL12.5 gene (B) characterized by absence of mtDNA encoded COXII expression. Repopulation of 143B rho⁰ cells with human platelet mitochondria resulted in reexpression of COX II expression level in cybrids cells. The tubulin antibody was used as a loading control.

Figure 2

Aberrant CpG island methylation in rho⁰ cells. Representative examples of RLGS analysis are shown comparing three parental cell lines to their corresponding rho⁰ cell lines. RLGS spots of interest are shown by solid-line circles to indicate presence of the spot (lack of methylation), while dashed-line circles indicate absence of a spot (methylation). (A) MCF12A cell-line showing spots 1 and 2 exhibiting hypermethylation in the rho⁰ cell line and spot 3 exhibiting hypomethylation in the rho⁰ cell line. (B) MDA-MB-435 cell line showing spot 1 and 2 exhibiting hypermethylation in the rho⁰ cell line. (C) 143B cell line showing RLGS spot 2E58 exhibiting hypomethylation in the rho⁰ cell line. (D) Bisulfite-sequencing of 2E58 from both the 143B parental and rho⁰ cell lines. Four clones each were sequenced across a 285 bp region containing 34 CpG dinucleotides. Each horizontal line of circles represents the sequencing data from an individual clone. Each circle represents one CpG: closed circle = methylation; open circle = no methylation.

Figure 3

Figure 3. (A–C) Reversal of aberrant CpG island methylation in cybrid cell-line. (A) Representative examples of RLGS analysis are shown comparing spot 4B15 in the 143B parental, genetically engineered rho⁰ cell line, and cybrid cell lines. The dashed-line circle indicates methylation of 4B15 in the parental line, while the solid-line circles indicate partial hypomethylation of 4B15 in both the rho⁰ and cybrid lines. (B) Smoothed-line graph of Mass Array Quantitative Methylation Analysis (MAQMA) data for 4B15. The level of methylation at each informative CpG dinucleotide is shown. CpGs 3, 4 and 9 were non-informative because of inappropriate fragment size for MALDI-TOF detection. The left panel shows the results on control DNAs with the indicated ratio of in vitro methylated (IVM) DNA. The right panel shows the results of the three cell-lines, with both the rho⁰ and the cybrid lines showing hypomethylation relative to the parental line. (C) Representative examples of RLGS analysis comparing spot 5C32 in the 143B parental, genetically rho⁰ and cybrid cell-lines. The dashed-line circle indicates partial methylation of 5C32 in both the parental and cybrid lines, while the solid-line circle indicates hypomethylation of 4B15 in the rho⁰ cell line. Figure 3. (D and E) Reversal of aberrant CpG island methylation in cybrid cell-line. (D) Smoothed-line graph of MAQMA data for 5C32. The level of methylation at each of 20 CpG dinucleotides is shown. The top panel shows the results on control DNAs with the indicated ratio of in vitro methylated (IVM) DNA. The bottom panel shows the results of the three cell-lines, with the rho⁰ cell line showing hypomethylation relative to the parental line and cybrid lines, except at CpGs 9–11. (E) Histograph analysis of MAQMA data. To obtain a single value for each locus in each cell-line, the average level of methylation at which all informative CpG dinucleotides for 4B15 and 5C32 were calculated is shown.

Figure 3

Figure 3. (A–C) Reversal of aberrant CpG island methylation in cybrid cell-line. (A) Representative examples of RLGS analysis are shown comparing spot 4B15 in the 143B parental, genetically engineered rho⁰ cell line, and cybrid cell lines. The dashed-line circle indicates methylation of 4B15 in the parental line, while the solid-line circles indicate partial hypomethylation of 4B15 in both the rho⁰ and cybrid lines. (B) Smoothed-line graph of Mass Array Quantitative Methylation Analysis (MAQMA) data for 4B15. The level of methylation at each informative CpG dinucleotide is shown. CpGs 3, 4 and 9 were non-informative because of inappropriate fragment size for MALDI-TOF detection. The left panel shows the results on control DNAs with the indicated ratio of in vitro methylated (IVM) DNA. The right panel shows the results of the three cell-lines, with both the rho⁰ and the cybrid lines showing hypomethylation relative to the parental line. (C) Representative examples of RLGS analysis comparing spot 5C32 in the 143B parental, genetically rho⁰ and cybrid cell-lines. The dashed-line circle indicates partial methylation of 5C32 in both the parental and cybrid lines, while the solid-line circle indicates hypomethylation of 4B15 in the rho⁰ cell line. Figure 3. (D and E) Reversal of aberrant CpG island methylation in cybrid cell-line. (D) Smoothed-line graph of MAQMA data for 5C32. The level of methylation at each of 20 CpG dinucleotides is shown. The top panel shows the results on control DNAs with the indicated ratio of in vitro methylated (IVM) DNA. The bottom panel shows the results of the three cell-lines, with the rho⁰ cell line showing hypomethylation relative to the parental line and cybrid lines, except at CpGs 9–11. (E) Histograph analysis of MAQMA data. To obtain a single value for each locus in each cell-line, the average level of methylation at which all informative CpG dinucleotides for 4B15 and 5C32 were calculated is shown.