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Regulation of Nuclear Epigenome by Mitochondrial DNA Heteroplasmy

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Regulation of Nuclear Epigenome by Mitochondrial DNA Heteroplasmy Regulation of nuclear epigenome by mitochondrial DNA heteroplasmy Piotr K. Kopinskia,b,c, Kevin A. Janssend,e, Patrick M. Schaeferc, Sophie Trefelyf,g, Caroline E. Perryc, Prasanth Potluric, Jesus A. Tintos-Hernandezc, Larry N. Singhc, Kelly R. Karchd,e, Sydney L. Campbellf, Mary T. Doang, Helen Jiangg, Itzhak Nissime, Eiko Nakamaru-Ogisoe, Kathryn E. Wellenf, Nathaniel W. Snyderg, Benjamin A. Garciad,e, and Douglas C. Wallacec,h,1 aHoward Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA 19104; bPerelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; cCenter for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104; dEpigenetics Institute, University of Pennsylvania, Philadelphia, PA 19104; eDepartment of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; fDepartment of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; gA.J. Drexel Autism Institute, Drexel University, Philadelphia, PA 19104; and hDepartment of Pediatrics, Division of Human Genetics, The Children’s Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 Contributed by Douglas C. Wallace, May 27, 2019 (sent for review April 23, 2019; reviewed by Yidong Bai, Scott W. Ballinger, and Victor Faundez) Diseases associated with mitochondrial DNA (mtDNA) mutations are cardiomyopathy, lactic acidosis, and stroke-like episodes, pre- highly variable in phenotype, in large part because of differences in the senting separately or when in aggregate referred to as the percentage of normal and mutant mtDNAs (heteroplasmy) present MELAS syndrome. At 90% to 100% mutant, individuals manifest within the cell. For example, increasing heteroplasmy levels of the perinatal lethal diseases such as Leigh syndrome. Leu(UUR) mtDNA tRNA nucleotide (nt) 3243A > G mutation result succes- To investigate the molecular basis of the relationship between sively in diabetes, neuromuscular degenerative disease, and perinatal the mtDNA heteroplasmy levels and shifts in patient phenotype, lethality. These phenotypes are associated with differences in mitochon- we prepared transmitochondrial cybrids with the same nucleus, drial function and nuclear DNA (nDNA) gene expression, which are re- but with different heteroplasmic percentages of the tRNALeu(UUR) capitulated in cybrid cell lines with different percentages of m.3243G mutant mtDNAs. Using metabolic tracing, histone mass spectrometry, m.3243G mutation. Cell biological and biochemical character- and NADH fluorescence lifetime imaging microscopy in these cells, we ization of these cybrid cell lines revealed that the physiological now show that increasing levels of this single mtDNA mutation cause parameters of the cybrids mirrored those previously observed in profound changes in the nuclear epigenome. At high heteroplasmy, the patients with the same ranges of m.3243G heteroplasmy. mitochondrially derived acetyl-CoA levels decrease causing decreased Transcriptional analysis by RNA sequencing revealed that cybrids histone H4 acetylation, with glutamine-derived acetyl-CoA compensat- ing when glucose-derived acetyl-CoA is limiting. In contrast, Significance α-ketoglutarate levels increase at midlevel heteroplasmy and are in- versely correlated with histone H3 methylation. Inhibition of mitochon- Mitochondrial DNA (mtDNA) mutations have been associated drial protein synthesis induces acetylation and methylation changes, and with common metabolic and degenerative disease phenotypes, restoration of mitochondrial function reverses these effects. mtDNA het- + implying a bioenergetic etiology for these diseases. For example, eroplasmy also affects mitochondrial NAD /NADH ratio, which corre- tRNALeu(UUR) > + the mtDNA m.3243A G mutation manifests as di- lates with nuclear histone acetylation, whereas nuclear NAD /NADH abetes, neurodegenerative disease, or lethal pediatric disease, ratio correlates with changes in nDNA and mtDNA transcription. Thus, depending on the percentage of mutant mtDNAs within the cell mutations in the mtDNA cause distinct metabolic and epigenomic (heteroplasmy). Cultured cybrid cell lines harboring 3243G heter- changes at different heteroplasmy levels, potentially explaining tran- oplasmy levels corresponding to the different clinical phenotypes scriptional and phenotypic variability of mitochondrial disease. have distinct transcriptional profiles. Exhaustive metabolomic and histone posttranscriptional modification analysis of these 3243G mitochondria | epigenetics | metabolism | transcription | common diseases cybrids revealed that changes in mtDNA heteroplasmy cause changes in mitochondrial intermediates and redox state, which he mtDNA is maternally inherited and codes for 13 essential result in distinctive histone modification changes. Thus, changes in Tsubunits of the mitochondrial energy generating system, ox- the mitochondrial genotype change mitochondrial metabolism, idative phosphorylation (OXPHOS), plus the 2 rRNAs for the which change the epigenome and transcriptome, which induce mitochondrial, chloramphenicol (CP)-sensitive, ribosome and distinct clinical phenotypes. the 22 tRNAs required for mitochondrial mRNA translation. Mutations in the mtDNA tRNAs impair the biogenesis of all Author contributions: P.K.K. and D.C.W. designed research; P.K.K., K.A.J., P.M.S., S.T., mtDNA-encoded OXPHOS enzyme complexes, but particularly C.E.P., P.P., J.A.T.-H., K.R.K., S.L.C., M.T.D., H.J., I.N., E.N.-O., N.W.S., and B.A.G. performed research; P.K.K., K.A.J., P.M.S., L.N.S., K.E.W., N.W.S., B.A.G., and D.C.W. analyzed data; compromise complex I (NADH:CoQ oxidoreductase), which and P.K.K. and D.C.W. wrote the paper. incorporates 7 of the 13 mtDNA encoded polypeptides (1). Reviewers: Y.B., University of Texas Health Science Center at San Antonio; S.W.B., Univer- The overall prevalence of mtDNA mutations is about 1 in sity of Alabama at Birmingham; and V.F., Emory University. 5,000 (2, 3). This high prevalence of mtDNA diseases results The authors declare no conflict of interest. from the high mtDNA mutation rate, which is reflected in 1 in This open access article is distributed under Creative Commons Attribution-NonCommercial- 200 cord bloods harboring 1 of the 10 most common pathogenic NoDerivatives License 4.0 (CC BY-NC-ND). mtDNA mutations (4, 5) and 1 in 8 adults harboring a disease- Data deposition: Histone Post-Translational Modification Data: The current Chorus mass associated mutation at ≥1% heteroplasmy (6). spectrometry database is labeled project 1569. It is publicly shared at the URL https:// One of the most common pathogenic mtDNA mutations is the chorusproject.org/pages/dashboard.html#/projects/public/1569/experiments. Leu(UUR) tRNA m.3243 A > G mutation, which causes starkly See Commentary on page 15763. different clinical phenotypes depending on the heteroplasmy 1To whom correspondence may be addressed. Email: [email protected]. levels (1). At 20% to 30% m.3243G mutant, patients com- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. monly present with type 1 or type 2 diabetes or manifest au- 1073/pnas.1906896116/-/DCSupplemental. tism. At 50% to 80% mutant, patients present with myopathy, Published online June 28, 2019. 16028–16035 | PNAS | August 6, 2019 | vol. 116 | no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1906896116 Downloaded by guest on September 30, 2021 A R−value 1 + SEE COMMENTARY 0 Lactate (L) P/L Citrate aKG Succinyl-CoA Succinate Fumarate Malate Glutamate (E) Acetyl-CoA Pyruvate (P) - 1 Isocitrate aKG/suc Glutamine (Q) Q/E ac aKG ~ H3K9me3 H3 K9 me2 2. 0.2 R = -0.45 me3 p < 0.05 K14 ac 2 aKG/suc (ratio) ac K18 me1 0.1 K23 ac 1 ac me1 K27 me2 H3K9me3 (fraction) me3 0.0 0 me1 K36 0255075100 me3 A3243G (%) K27 me2 H3.3 K36 me3 K5 ac Acetyl-CoA ~ H4K16ac H4 K8 ac K12 ac1. R = 0.57 100 K16 ac 0.3 p < 0.01 [pmol] Acetyl-CoA ac 75 K9 H2A.1 me1 GENETICS ac 0.2 K13 50 me1 K15 ac 0.1 T16 ac 25 H4K16ac (fraction) K7 ac H2A.V K11 ac 0.0 0 K7 ac 0255075100 H2A.Z K11 ac A3243G (%) B 2 Relative + 1 Fraction C p-value p-value Histone Modification 0 (adjusted) 1 H3_27_40 K27ac 0.0010 0.0418 - 2 H4_4_17 K5acK16ac 0.0004 0.0418 H4_4_17 K8acK16ac 0.0003 0.0418 H4 K5acK8acK12ac H4_4_17 K8acK12acK16ac 0.0011 0.0418 H4 K5ac H2AZ_1_19 K4ac 0.0008 0.0418 H4 K5acK8acK16ac H4_4_17 K12acK16ac 0.0013 0.0434 H4 K5acK12acK16ac H4 K8acK12acK16ac H3_27_40 unmod 0.0018 0.0495 H4 K8acK16ac H4 K5acK16ac H4_4_17 K5acK12ac 0.0020 0.0495 H4 K12acK16ac H4_4_17 K5acK8acK16ac 0.0029 0.0570 H4 K5acK12ac H4_4_17 K5acK12acK16ac 0.0028 0.0570 H4 K5acK8ac H4 K16ac H33_27_40 K27ac 0.0033 0.0599 H4_4_17 unmod 0.0040 0.0666 H2AZ_1_19 K7ac 0.0049 0.0746 H3_18_26 K23me1 0.0093 0.1307 H4_4_17 K5ac 0.0105 0.1307 H4 K5acK8acK12acK16ac H4_4_17 K16ac 0.0103 0.1307 H3_9_17 K14ac 0.0138 0.1618 H2AZ_1_19 unmod 0.0196 0.2167 H4 unmodified H33_27_40 unmod 0.0307 0.2671 A3243G (%) 0 0 0 100 100 100 H14_25_32 unmod 0.0296 0.2671 Fig. 1. MtDNA heteroplasmy modulates metabolites and histone modifications. (A) Seven mtDNA m.3243A > G heteroplasmic cell lines plus the ρ0 parent cells were cultured in parallel for metabolite (j = 15) and histone modification (i = 107) quantification by liquid chromatography tandem mass spectrometry. Spearman correlation coefficient was calculated for each histone modification (unmod = unmodified peptide) against each metabolite (blue = positive, red = negative, and blank = no cor- relation), with the color intensity corresponding to correlation strength (significance = P < 0.05). (Example #1, blue box) Positive association between acetyl-CoA levels and histone H4 acetylation of lysines 8 (K8) and 16 (K16) across heteroplasmy levels. (Example #2, red box) Negative association between αKG/succinate ratio and histone H3 lysine 9 di- (me2) and trimethylation (me3) across heteroplasmy levels. Each point represents a measurement and lines represent local regression of the mean (R-software loess), n = 3. (B) Heat map of histone acetylation modifications of 0% and 100% m.3243G lines (n = 3), showing a dramatic effect of mtDNA genotype on the histone epigenome.
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