Progressive increase in mtDNA 3243A>G heteroplasmy causes abrupt transcriptional reprogramming

Abstract

Variation in the intracellular percentage of normal and mutant mitochondrial DNAs (mtDNA) (heteroplasmy) can be associated with phenotypic heterogeneity in mtDNA diseases. Individuals that inherit the common disease-causing mtDNA tRNA(Leu(UUR)) 3243A>G mutation and harbor ∼10-30% 3243G mutant mtDNAs manifest diabetes and occasionally autism; individuals with ∼50-90% mutant mtDNAs manifest encephalomyopathies; and individuals with ∼90-100% mutant mtDNAs face perinatal lethality. To determine the basis of these abrupt phenotypic changes, we generated somatic cell cybrids harboring increasing levels of the 3243G mutant and analyzed the associated cellular phenotypes and nuclear DNA (nDNA) and mtDNA transcriptional profiles by RNA sequencing. Small increases in mutant mtDNAs caused relatively modest defects in oxidative capacity but resulted in sharp transitions in cellular phenotype and gene expression. Cybrids harboring 20-30% 3243G mtDNAs had reduced mtDNA mRNA levels, rounded mitochondria, and small cell size. Cybrids with 50-90% 3243G mtDNAs manifest induction of glycolytic genes, mitochondrial elongation, increased mtDNA mRNA levels, and alterations in expression of signal transduction, epigenomic regulatory, and neurodegenerative disease-associated genes. Finally, cybrids with 100% 3243G experienced reduced mtDNA transcripts, rounded mitochondria, and concomitant changes in nuclear gene expression. Thus, striking phase changes occurred in nDNA and mtDNA gene expression in response to the modest changes of the mtDNA 3243G mutant levels. Hence, a major factor in the phenotypic variation in heteroplasmic mtDNA mutations is the limited number of states that the nucleus can acquire in response to progressive changes in mitochondrial retrograde signaling.

Keywords: RNA-Seq; electron microscopy; epigenetic; mitochondrial disease; mtDNA variant.

Fig. 1.

The mtDNA mutation 3243A>G alters cellular energetics, mitochondrial morphology, and cell size. (A) Experimental paradigm whereby increasing levels of heteroplasmy for the mtDNA mutation 3243A>G tRNA^Leu(UUR) were introduced into 143B(TK⁻) ρ^o cells. (B) Linear regression of mtDNA heteroplasmy and complex IV subunit COII protein levels. (C) Abundance of electron transport chain protein subunits of each complex (CI–CIV) probed by Western blotting of a constituent protein (20 kDa, 30 kDa, core II, COII, F1α) for complex I (CI), complex II (CII), complex III (CIII), complex IV (CIV), and complex V (CV) at different heteroplasmy levels. (D) Cybrid respiration rates fitting a nonlinear regression (third order polynomial) with maximal oxygen consumption revealing a threshold for respiratory deficiency above 60% 3243G heteroplasmy. (E) Confocal microscopy, and (F) electron microscopy (EM) representative images of 0%, 90% and ρ^o cell lines with pseudocolored (green) mitochondria. (G) Quantification of mitochondrial aspect ratio (length-to-width ratio) and roundness from confocal images. (H) Effect of heteroplasmy on cell size measured from electron micrographs and expressed as cross-sectional area, and (I) mtDNA copy number expressed as mtDNA/nDNA ratio (red line) and normalized to cell volume (green line). Significance is based on 95% (P < 0.05) or 99% (P < 0.01) confidence intervals (C.I.). Data for G and H are in means ± 95% C.I. and for I in means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA and Dunnett’s post hoc test.

Fig. 2.

Representative electron micrographs of cybrid cell lines. Shown are lower magnification (Upper) and higher magnification (Lower) micrographs of cybrid cell lines harboring different percentages of 3243G heteroplasmy. M, mitochondria; N, nucleus; yellow arrowheads, endoplasmic reticulum.

Fig. 3.

Regulation of the mitochondrial transcriptome in heteroplasmic cell lines. (A) Map of the mitochondrial genome and its 37 genes, showing origins of replication and promoters for the mtDNA light (L, inner) and heavy (H, outer) strands: replication origins O_L and O_H, and promotors P_L, P_H1, and P_H2, respectively. Bar graphs show transcript levels of each gene at different heteroplasmy levels relative to 0% 3243G. Histograms are means ± SEM. (B) Relative abundance of nuclear- and mitochondrial-encoded transcripts in whole transcriptome analysis for each 3243G heteroplasmy level. (C) Expression level for the 13 mtDNA-encoded messenger RNA (mRNA) genes. (D) Relative abundance of mitochondrial ribosomal (rRNA), mRNA, and transfer RNA (tRNA) molecules with increasing heteroplasmy. *P < 0.01, **P < 0.001 relative to 0% by multiple two-tailed t tests. All data are from RNA sequencing, n = 3 runs per sample.

Fig. 4.

Biphasic induction of metabolic and stress response genes, signaling pathways, and of the epigenetic machinery. (A) Cumulative expression levels relative to 0% for genes encoding each enzymatic step of glycolysis in the various 3243G mutant cell lines. (B) Relative transcript levels for the adenine nucleotide translocator (ANT) isoforms. (C) Enrichment for gene ontology (GO) terms associated with gene families involved in transcriptional activity, expressed relative to 0% mutant. (D) Relative expression levels for the mitochondrial heat shock proteins (HSPs). (E) Expression levels of ER/cytoplasmic HSPs. (F) Expression levels of the Ca²⁺-sensitive signaling proteins calmodulin kinases (CAMK) and associated kinases (CAMKK). (G) Expression levels of cellular phosphatases showing inverse trend relative to kinases. (H) Expression of DNA methyltransferases (DNMTs). (I) Ratio of DNMT isoforms 3A/B and 1/3A. (J) Expression level of methyl-CpG binding protein 2 (Mecp2) and methyl-DNA binding domain (MBD1 and MBD2) genes. (K) Expression level of selected functional histone variants including the linker histone cluster 1 H4L (H1H4L); histone 2 variant MacroH2A2, which replaces conventional H2A in nucleosome and acts as transcriptional repressor; and histone cluster 2 variant H4B (H2H4B). Each gene is normalized to 1 in the 0% cell line and expressed in relative terms for cell lines of different heteroplasmy levels. Data for B and D–I are means ± SEM, n = 3 per cell line.

Fig. 5.

Multiphasic reprogramming of nuclear gene expression. (A) Gene set enrichment analysis (GSEA) of transcriptional and signaling pathways from whole transcriptome data showing differential gene transcription phases of cybrids in the heteroplasmy ranges of 0%, 20–30%, 50–90%, and 100% 3243G plus ρ^o cells (log P values relative to 0% cell line). (B) Expression levels of 211 genes that are up-regulated at 20–30% 3243G but strongly repressed >100-fold at 60–100% 3243G, enriched for transmembrane and G-protein–coupled receptor systems (see SI Appendix, Fig. S12 for details). (C) Principal component analysis (PCA) showing the striking discontinuity in gene expression profiles of 0% (normal), 20–30% (diabetes and autism), 50–90% (degenerative diseases), 100% (perinatal diseases and Leigh syndrome), and ρ^o (mitochondrial null). (D) Pathway analysis comparing the 3243G cybrid transcript profiles to those from public databases (databases shown in gray), log P significance values.