Single-cell analysis reveals context-dependent, cell-level selection of mtDNA.

Heteroplasmy occurs when wild-type and mutant mitochondrial DNA (mtDNA) molecules co-exist in single cells1. Heteroplasmy levels change dynamically in development, disease and ageing2,3, but it is unclear whether these shifts are caused by selection or drift, and whether they occur at the level of cells or intracellularly. Here we investigate heteroplasmy dynamics in dividing cells by combining precise mtDNA base editing (DdCBE)4 with a new method, SCI-LITE (single-cell combinatorial indexing leveraged to interrogate targeted expression), which tracks single-cell heteroplasmy with ultra-high throughput. We engineered cells to have synonymous or nonsynonymous complex I mtDNA mutations and found that cell populations in standard culture conditions purge nonsynonymous mtDNA variants, whereas synonymous variants are maintained. This suggests that selection dominates over simple drift in shaping population heteroplasmy. We simultaneously tracked single-cell mtDNA heteroplasmy and ancestry, and found that, although the population heteroplasmy shifts, the heteroplasmy of individual cell lineages remains stable, arguing that selection acts at the level of cell fitness in dividing cells. Using these insights, we show that we can force cells to accumulate high levels of truncating complex I mtDNA heteroplasmy by placing them in environments where loss of biochemical complex I activity has been reported to benefit cell fitness. We conclude that in dividing cells, a given nonsynonymous mtDNA heteroplasmy can be harmful, neutral or even beneficial to cell fitness, but that the 'sign' of the effect is wholly dependent on the environment.

Circulating N-lactoyl-amino acids and N-formyl-methionine reflect mitochondrial dysfunction and predict mortality in septic shock.

INTRODUCTION: Sepsis is a highly morbid condition characterized by multi-organ dysfunction resulting from dysregulated inflammation in response to acute infection. Mitochondrial dysfunction may contribute to sepsis pathogenesis, but quantifying mitochondrial dysfunction remains challenging. OBJECTIVE: To assess the extent to which circulating markers of mitochondrial dysfunction are increased in septic shock, and their relationship to severity and mortality. METHODS: We performed both full-scan and targeted (known markers of genetic mitochondrial disease) metabolomics on plasma to determine markers of mitochondrial dysfunction which distinguish subjects with septic shock (n = 42) from cardiogenic shock without infection (n = 19), bacteremia without sepsis (n = 18), and ambulatory controls (n = 19) - the latter three being conditions in which mitochondrial function, proxied by peripheral oxygen consumption, is presumed intact. RESULTS: Nine metabolites were significantly increased in septic shock compared to all three comparator groups. This list includes N-formyl-L-methionine (f-Met), a marker of dysregulated mitochondrial protein translation, and N-lactoyl-phenylalanine (lac-Phe), representative of the N-lactoyl-amino acids (lac-AAs), which are elevated in plasma of patients with monogenic mitochondrial disease. Compared to lactate, the clinical biomarker used to define septic shock, there was greater separation between survivors and non-survivors of septic shock for both f-Met and the lac-AAs measured within 24 h of ICU admission. Additionally, tryptophan was the one metabolite significantly decreased in septic shock compared to all other groups, while its breakdown product kynurenate was one of the 9 significantly increased. CONCLUSION: Future studies which validate the measurement of lac-AAs and f-Met in conjunction with lactate could define a sepsis subtype characterized by mitochondrial dysfunction.

Nuclear genetic control of mtDNA copy number and heteroplasmy in humans.

Mitochondrial DNA (mtDNA) is a maternally inherited, high-copy-number genome required for oxidative phosphorylation1. Heteroplasmy refers to the presence of a mixture of mtDNA alleles in an individual and has been associated with disease and ageing. Mechanisms underlying common variation in human heteroplasmy, and the influence of the nuclear genome on this variation, remain insufficiently explored. Here we quantify mtDNA copy number (mtCN) and heteroplasmy using blood-derived whole-genome sequences from 274,832 individuals and perform genome-wide association studies to identify associated nuclear loci. Following blood cell composition correction, we find that mtCN declines linearly with age and is associated with variants at 92 nuclear loci. We observe that nearly everyone harbours heteroplasmic mtDNA variants obeying two principles: (1) heteroplasmic single nucleotide variants tend to arise somatically and accumulate sharply after the age of 70 years, whereas (2) heteroplasmic indels are maternally inherited as mixtures with relative levels associated with 42 nuclear loci involved in mtDNA replication, maintenance and novel pathways. These loci may act by conferring a replicative advantage to certain mtDNA alleles. As an illustrative example, we identify a length variant carried by more than 50% of humans at position chrM:302 within a G-quadruplex previously proposed to mediate mtDNA transcription/replication switching2,3. We find that this variant exerts cis-acting genetic control over mtDNA abundance and is itself associated in-trans with nuclear loci encoding machinery for this regulatory switch. Our study suggests that common variation in the nuclear genome can shape variation in mtCN and heteroplasmy dynamics across the human population.

Hypoxia extends lifespan and neurological function in a mouse model of aging.

There is widespread interest in identifying interventions that extend healthy lifespan. Chronic continuous hypoxia delays the onset of replicative senescence in cultured cells and extends lifespan in yeast, nematodes, and fruit flies. Here, we asked whether chronic continuous hypoxia is beneficial in mammalian aging. We utilized the Ercc1 Δ/- mouse model of accelerated aging given that these mice are born developmentally normal but exhibit anatomic, physiological, and biochemical features of aging across multiple organs. Importantly, they exhibit a shortened lifespan that is extended by dietary restriction, the most potent aging intervention across many organisms. We report that chronic continuous 11% oxygen commenced at 4 weeks of age extends lifespan by 50% and delays the onset of neurological debility in Ercc1 Δ/- mice. Chronic continuous hypoxia did not impact food intake and did not significantly affect markers of DNA damage or senescence, suggesting that hypoxia did not simply alleviate the proximal effects of the Ercc1 mutation, but rather acted downstream via unknown mechanisms. To the best of our knowledge, this is the first study to demonstrate that "oxygen restriction" can extend lifespan in a mammalian model of aging.

Nuclear genetic control of mtDNA copy number and heteroplasmy in humans.

Human mitochondria contain a high copy number, maternally transmitted genome (mtDNA) that encodes 13 proteins required for oxidative phosphorylation. Heteroplasmy arises when multiple mtDNA variants co-exist in an individual and can exhibit complex dynamics in disease and in aging. As all proteins involved in mtDNA replication and maintenance are nuclear-encoded, heteroplasmy levels can, in principle, be under nuclear genetic control, however this has never been shown in humans. Here, we develop algorithms to quantify mtDNA copy number (mtCN) and heteroplasmy levels using blood-derived whole genome sequences from 274,832 individuals of diverse ancestry and perform GWAS to identify nuclear loci controlling these traits. After careful correction for blood cell composition, we observe that mtCN declines linearly with age and is associated with 92 independent nuclear genetic loci. We find that nearly every individual carries heteroplasmic variants that obey two key patterns: (1) heteroplasmic single nucleotide variants are somatic mutations that accumulate sharply after age 70, while (2) heteroplasmic indels are maternally transmitted as mtDNA mixtures with resulting levels influenced by 42 independent nuclear loci involved in mtDNA replication, maintenance, and novel pathways. These nuclear loci do not appear to act by mtDNA mutagenesis, but rather, likely act by conferring a replicative advantage to specific mtDNA molecules. As an illustrative example, the most common heteroplasmy we identify is a length variant carried by >50% of humans at position m.302 within a G-quadruplex known to serve as a replication switch. We find that this heteroplasmic variant exerts cis -acting genetic control over mtDNA abundance and is itself under trans -acting genetic control of nuclear loci encoding protein components of this regulatory switch. Our study showcases how nuclear haplotype can privilege the replication of specific mtDNA molecules to shape mtCN and heteroplasmy dynamics in the human population.

Comprehensive characterization of tissue-specific chromatin accessibility in L2 Caenorhabditis elegans nematodes.

Recently developed single-cell technologies allow researchers to characterize cell states at ever greater resolution and scale. Caenorhabditis elegans is a particularly tractable system for studying development, and recent single-cell RNA-seq studies characterized the gene expression patterns for nearly every cell type in the embryo and at the second larval stage (L2). Gene expression patterns give insight about gene function and into the biochemical state of different cell types; recent advances in other single-cell genomics technologies can now also characterize the regulatory context of the genome that gives rise to these gene expression levels at a single-cell resolution. To explore the regulatory DNA of individual cell types in C. elegans, we collected single-cell chromatin accessibility data using the sci-ATAC-seq assay in L2 larvae to match the available single-cell RNA-seq data set. By using a novel implementation of the latent Dirichlet allocation algorithm, we identify 37 clusters of cells that correspond to different cell types in the worm, providing new maps of putative cell type-specific gene regulatory sites, with promise for better understanding of cellular differentiation and gene regulation.

MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations.

The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.

PREDICTD PaRallel Epigenomics Data Imputation with Cloud-based Tensor Decomposition.

The Encyclopedia of DNA Elements (ENCODE) and the Roadmap Epigenomics Project seek to characterize the epigenome in diverse cell types using assays that identify, for example, genomic regions with modified histones or accessible chromatin. These efforts have produced thousands of datasets but cannot possibly measure each epigenomic factor in all cell types. To address this, we present a method, PaRallel Epigenomics Data Imputation with Cloud-based Tensor Decomposition (PREDICTD), to computationally impute missing experiments. PREDICTD leverages an elegant model called "tensor decomposition" to impute many experiments simultaneously. Compared with the current state-of-the-art method, ChromImpute, PREDICTD produces lower overall mean squared error, and combining the two methods yields further improvement. We show that PREDICTD data captures enhancer activity at noncoding human accelerated regions. PREDICTD provides reference imputed data and open-source software for investigating new cell types, and demonstrates the utility of tensor decomposition and cloud computing, both promising technologies for bioinformatics.

The ModERN Resource: Genome-Wide Binding Profiles for Hundreds of Drosophila and Caenorhabditis elegans Transcription Factors.

To develop a catalog of regulatory sites in two major model organisms, Drosophila melanogaster and Caenorhabditis elegans, the modERN (model organism Encyclopedia of Regulatory Networks) consortium has systematically assayed the binding sites of transcription factors (TFs). Combined with data produced by our predecessor, modENCODE (Model Organism ENCyclopedia Of DNA Elements), we now have data for 262 TFs identifying 1.23 M sites in the fly genome and 217 TFs identifying 0.67 M sites in the worm genome. Because sites from different TFs are often overlapping and tightly clustered, they fall into 91,011 and 59,150 regions in the fly and worm, respectively, and these binding sites span as little as 8.7 and 5.8 Mb in the two organisms. Clusters with large numbers of sites (so-called high occupancy target, or HOT regions) predominantly associate with broadly expressed genes, whereas clusters containing sites from just a few factors are associated with genes expressed in tissue-specific patterns. All of the strains expressing GFP-tagged TFs are available at the stock centers, and the chromatin immunoprecipitation sequencing data are available through the ENCODE Data Coordinating Center and also through a simple interface (http://epic.gs.washington.edu/modERN/) that facilitates rapid accessibility of processed data sets. These data will facilitate a vast number of scientific inquiries into the function of individual TFs in key developmental, metabolic, and defense and homeostatic regulatory pathways, as well as provide a broader perspective on how individual TFs work together in local networks and globally across the life spans of these two key model organisms.