Common Inherited Variation in Mitochondrial Genes Is Not Enriched for Associations with Type 2 Diabetes or Related Glycemic Traits

Mitochondrial dysfunction has been observed in skeletal muscle of people with diabetes and insulin-resistant individuals. Furthermore, inherited mutations in mitochondrial DNA can cause a rare form of diabetes. However, it is unclear whether mitochondrial dysfunction is a primary cause of the common form of diabetes. To date, common genetic variants robustly associated with type 2 diabetes (T2D) are not known to affect mitochondrial function. One possibility is that multiple mitochondrial genes contain modest genetic effects that collectively influence T2D risk. To test this hypothesis we developed a method named Meta-Analysis Gene-set Enrichment of variaNT Associations (MAGENTA; http://www.broadinstitute.org/mpg/magenta). MAGENTA, in analogy to Gene Set Enrichment Analysis, tests whether sets of functionally related genes are enriched for associations with a polygenic disease or trait. MAGENTA was specifically designed to exploit the statistical power of large genome-wide association (GWA) study meta-analyses whose individual genotypes are not available. This is achieved by combining variant association p-values into gene scores and then correcting for confounders, such as gene size, variant number, and linkage disequilibrium properties. Using simulations, we determined the range of parameters for which MAGENTA can detect associations likely missed by single-marker analysis. We verified MAGENTA''s performance on empirical data by identifying known relevant pathways in lipid and lipoprotein GWA meta-analyses. We then tested our mitochondrial hypothesis by applying MAGENTA to three gene sets: nuclear regulators of mitochondrial genes, oxidative phosphorylation genes, and ∼1,000 nuclear-encoded mitochondrial genes. The analysis was performed using the most recent T2D GWA meta-analysis of 47,117 people and meta-analyses of seven diabetes-related glycemic traits (up to 46,186 non-diabetic individuals). This well-powered analysis found no significant enrichment of associations to T2D or any of the glycemic traits in any of the gene sets tested. These results suggest that common variants affecting nuclear-encoded mitochondrial genes have at most a small genetic contribution to T2D susceptibility.     

A Genome-Wide Map of Mitochondrial DNA Recombination in Yeast

In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome maintenance.     

Bioenergetics of the Calf Muscle in Friedreich Ataxia Patients Measured by 31P-MRS Before and After Treatment with Recombinant Human Erythropoietin

Friedreich ataxia (FRDA) is caused by a GAA repeat expansion in the FXN gene leading to reduced expression of the mitochondrial protein frataxin. Recombinant human erythropoietin (rhuEPO) is suggested to increase frataxin levels, alter mitochondrial function and improve clinical scores in FRDA patients. Aim of the present pilot study was to investigate mitochondrial metabolism of skeletal muscle tissue in FRDA patients and examine effects of rhuEPO administration by phosphorus 31 magnetic resonance spectroscopy (31P MRS). Seven genetically confirmed FRDA patients underwent 31P MRS of the calf muscles using a rest-exercise-recovery protocol before and after receiving 3000 IU of rhuEPO for eight weeks. FRDA patients showed more rapid phosphocreatine (PCr) depletion and increased accumulation of inorganic phosphate (Pi) during incremental exercise as compared to controls. After maximal exhaustive exercise prolonged regeneration of PCR and slowed decline in Pi can be seen in FRDA. PCr regeneration as hallmark of mitochondrial ATP production revealed correlation to activity of complex II/III of the respiratory chain and to demographic values. PCr and Pi kinetics were not influenced by rhuEPO administration. Our results confirm mitochondrial dysfunction and exercise intolerance due to impaired oxidative phosphorylation in skeletal muscle tissue of FRDA patients. MRS did not show improved mitochondrial bioenergetics after eight weeks of rhuEPO exposition in skeletal muscle tissue of FRDA patients.

Trial Registration

EU Clinical Trials Register 2008-000040-13     

Unusual Regulation of Splicing of the Cholinergic Locus in Caenorhabditis elegans

The essential neurotransmitter acetylcholine functions throughout the animal kingdom. In Caenorhabditis elegans, the acetylcholine biosynthetic enzyme [choline acetyltransferase (ChAT)] and vesicular transporter [vesicular acetylcholine transporter (VAChT)] are encoded by the cha-1 and unc-17 genes, respectively. These two genes compose a single complex locus in which the unc-17 gene is nested within the first intron of cha-1, and the two gene products arise from a common pre-messenger RNA (pre-mRNA) by alternative splicing. This genomic organization, known as the cholinergic gene locus (CGL), is conserved throughout the animal kingdom, suggesting that the structure is important for the regulation and function of these genes. However, very little is known about CGL regulation in any species. We now report the identification of an unusual type of splicing regulation in the CGL of C. elegans, mediated by two pairs of complementary sequence elements within the locus. We show that both pairs of elements are required for efficient splicing to the distal acceptor, and we also demonstrate that proper distal splicing depends more on sequence complementarity within each pair of elements than on the sequences themselves. We propose that these sequence elements are able to form stem-loop structures in the pre-mRNA; such structures would favor specific splicing alternatives and thus regulate CGL splicing. We have identified complementary elements at comparable locations in the genomes of representative species of other animal phyla; we suggest that this unusual regulatory mechanism may be a general feature of CGLs.     

Molecular Evolution of Cytochrome c Oxidase in High-Performance Fish (Teleostei: Scombroidei)

The 13 peptides encoded by vertebrate mitochondrial DNA (mtDNA) are essential subunits of oxidative phosphorylation (OXPHOS) enzymes. These genes normally experience purifying selection and also coevolve with nuclear-encoded subunits of OXPHOS complexes. However, the role of positive selection on mtDNA evolution is still unclear, as most examples of intergenomic coevolution appear to be the result of compensation by nuclear-encoded genes for mildly deleterious mtDNA mutations, and not simultaneous positive selection in both genomes. Organisms that have experienced strong selective pressures to increase aerobic capacity or adapt to changes in thermal environment may be better candidates in which to examine the impact of positively selected changes on mtDNA evolution. The tuna (suborder Scombroidei, family Scombridae) and billfish (suborder Scombroidei, families Xiphiidae and Istiophoridae) are highly aerobic fish with multiple specializations in muscle energetics, including a high mitochondrial content and regional endothermy. We examined the role of positively selected mtDNA substitutions in the production of these unique phenotypes. Focusing on a catalytic subunit of cytochrome c oxidase (COX II), we found that the rate ratio of nonsynonymous (d(N); amino acid changing)-to-synonymous (d(S); silent) substitutions was not increased in lineages leading to the tuna but was significantly increased in the lineage preceding the billfish. Furthermore, there are a number of individual positively selected sites that, when mapped onto the COX crystal structure, appear to interact with other COX subunits and may affect OXPHOS function and regulation in billfish.     

State Transitions in the TORC1 Signaling Pathway and Information Processing in Saccharomyces cerevisiae

TOR kinase complex I (TORC1) is a key regulator of cell growth and metabolism in all eukaryotes. Previous studies in yeast have shown that three GTPases—Gtr1, Gtr2, and Rho1—bind to TORC1 in nitrogen and amino acid starvation conditions to block phosphorylation of the S6 kinase Sch9 and activate protein phosphatase 2A (PP2A). This leads to downregulation of 450 Sch9-dependent protein and ribosome synthesis genes and upregulation of 100 PP2A-dependent nitrogen assimilation and amino acid synthesis genes. Here, using bandshift assays and microarray measurements, we show that the TORC1 pathway also populates three other stress/starvation states. First, in glucose starvation conditions, the AMP-activated protein kinase (AMPK/Snf1) and at least one other factor push the TORC1 pathway into an off state, in which Sch9-branch signaling and PP2A-branch signaling are both inhibited. Remarkably, the TORC1 pathway remains in the glucose starvation (PP2A inhibited) state even when cells are simultaneously starved for nitrogen and glucose. Second, in osmotic stress, the MAPK Hog1/p38 drives the TORC1 pathway into a different state, in which Sch9 signaling and PP2A-branch signaling are inhibited, but PP2A-branch signaling can still be activated by nitrogen starvation. Third, in oxidative stress and heat stress, TORC1-Sch9 signaling is blocked while weak PP2A-branch signaling occurs. Together, our data show that the TORC1 pathway acts as an information-processing hub, activating different genes in different conditions to ensure that available energy is allocated to drive growth, amino acid synthesis, or a stress response, depending on the needs of the cell.     

Coordination of Recombination with Meiotic Progression in the Caenorhabditis elegans Germline by KIN-18, a TAO Kinase That Regulates the Timing of MPK-1 Signaling

Meiosis is a tightly regulated process requiring coordination of diverse events. A conserved ERK/MAPK-signaling cascade plays an essential role in the regulation of meiotic progression. The Thousand And One kinase (TAO) kinase is a MAPK kinase kinase, the meiotic role of which is unknown. We have analyzed the meiotic functions of KIN-18, the homolog of mammalian TAO kinases, in Caenorhabditis elegans. We found that KIN-18 is essential for normal meiotic progression; mutants exhibit accelerated meiotic recombination as detected both by analysis of recombination intermediates and by crossover outcome. In addition, ectopic germ-cell differentiation and enhanced levels of apoptosis were observed in kin-18 mutants. These defects correlate with ectopic activation of MPK-1 that includes premature, missing, and reoccurring MPK-1 activation. Late progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator of MPK-1 signaling, KSR-2. However, the acceleration of recombination events observed in kin-18 mutants is largely MPK-1-independent. Our data suggest that KIN-18 coordinates meiotic progression by modulating the timing of MPK-1 activation and the progression of recombination events. The regulation of the timing of MPK-1 activation ensures the proper timing of apoptosis and is required for the formation of functional oocytes. Meiosis is a conserved process; thus, revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans would help to elucidate TAO kinase’s role in germline development in higher eukaryotes.     

A Genome-Wide RNAi Screen for Enhancers of par Mutants Reveals New Contributors to Early Embryonic Polarity in Caenorhabditis elegans

The par genes of Caenorhabditis elegans are essential for establishment and maintenance of early embryo polarity and their homologs in other organisms are crucial polarity regulators in diverse cell types. Forward genetic screens and simple RNAi depletion screens have identified additional conserved regulators of polarity in C. elegans; genes with redundant functions, however, will be missed by these approaches. To identify such genes, we have performed a genome-wide RNAi screen for enhancers of lethality in conditional par-1 and par-4 mutants. We have identified 18 genes for which depletion is synthetically lethal with par-1 or par-4, or both, but produces little embryo lethality in wild type. Fifteen of the 18 genes identified in our screen are not previously known to function in C. elegans embryo polarity and 11 of them also increase lethality in a par-2 mutant. Among the strongest synthetic lethal genes, polarity defects are more apparent in par-2 early embryos than in par-1 or par-4, except for strd-1(RNAi), which enhances early polarity phenotypes in all three mutants. One strong enhancer of par-1 and par-2 lethality, F25B5.2, corresponds to nop-1, a regulator of actomyosin contractility for which the molecular identity was previously unknown. Other putative polarity enhancers identified in our screen encode cytoskeletal and membrane proteins, kinases, chaperones, and sumoylation and deubiquitylation proteins. Further studies of these genes should give mechanistic insight into pathways regulating establishment and maintenance of cell polarity.     

Too Much of a Good Thing: The Unique and Repeated Paths Toward Copper Adaptation

Copper is a micronutrient essential for growth due to its role as a cofactor in enzymes involved in respiration, defense against oxidative damage, and iron uptake. Yet too much of a good thing can be lethal, and yeast cells typically do not have tolerance to copper levels much beyond the concentration in their ancestral environment. Here, we report a short-term evolutionary study of Saccharomyces cerevisiae exposed to levels of copper sulfate that are inhibitory to the initial strain. We isolated and identified adaptive mutations soon after they arose, reducing the number of neutral mutations, to determine the first genetic steps that yeast take when adapting to copper. We analyzed 34 such strains through whole-genome sequencing and by assaying fitness within different environments; we also isolated a subset of mutations through tetrad analysis of four lines. We identified a multilayered evolutionary response. In total, 57 single base-pair mutations were identified across the 34 lines. In addition, gene amplification of the copper metallothionein protein, CUP1-1, was rampant, as was chromosomal aneuploidy. Four other genes received multiple, independent mutations in different lines (the vacuolar transporter genes VTC1 and VTC4; the plasma membrane H+-ATPase PMA1; and MAM3, a protein required for normal mitochondrial morphology). Analyses indicated that mutations in all four genes, as well as CUP1-1 copy number, contributed significantly to explaining variation in copper tolerance. Our study thus finds that evolution takes both common and less trodden pathways toward evolving tolerance to an essential, but highly toxic, micronutrient.     

Wide-Ranging Effects of the Yeast Ptc1 Protein Phosphatase Acting Through the MAPK Kinase Mkk1

The Saccharomyces cerevisiae type 2C protein phosphatase Ptc1 is required for a wide variety of cellular functions, although only a few cellular targets have been identified. A genetic screen in search of mutations in protein kinase–encoding genes able to suppress multiple phenotypic traits caused by the ptc1 deletion yielded a single gene, MKK1, coding for a MAPK kinase (MAPKK) known to activate the cell-wall integrity (CWI) Slt2 MAPK. In contrast, mutation of the MKK1 paralog, MKK2, had a less significant effect. Deletion of MKK1 abolished the increased phosphorylation of Slt2 induced by the absence of Ptc1 both under basal and CWI pathway stimulatory conditions. We demonstrate that Ptc1 acts at the level of the MAPKKs of the CWI pathway, but only the Mkk1 kinase activity is essential for ptc1 mutants to display high Slt2 activation. We also show that Ptc1 is able to dephosphorylate Mkk1 in vitro. Our results reveal the preeminent role of Mkk1 in signaling through the CWI pathway and strongly suggest that hyperactivation of Slt2 caused by upregulation of Mkk1 is at the basis of most of the phenotypic defects associated with lack of Ptc1 function.