Mitochondrial DNA genetics inheritance

National Heart, Lung, And Blood Institute

Investigators

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Abstract

Project 1: System genetic analyses of the transcriptional hierarchy controlling mitochondrial biogenesis and maintenance. From the ongoing genetic modifier screen, we recovered total 77 enhancers and 22 suppressors that promote or inhibit mitochondrial biogenesis, respectively. We further followed up on a previously unnoted Zinc finger protein, encoded by CG1603 locus. Null mutant of CG1603 was lethal. Tissue specific disruption of CG1603 greatly diminished the steady state level of mtDNA, nuclear encoded factors required for mtDNA maintenance and expression, and the overall mitochondrial mass. RNAseq analyses revealed that nearly 70% nuclear encoded mitochondrial genes (nu-Mito genes) were downregulated in CG1603 mutant flies. We re-analyzed the published CHIP-seq data and found that CG1603 bound to a conserved, palindromic, 8-bp sequence, TATCGATA, which presents at the transcription starting points of over 50% nu-Mito genes and a few transcription factors. These results suggest that CG1603 is a master regulator of mitochondrial biogenesis, either directly or indirectly controlling the expression of nu-Mito genes. Interestingly, ETC genes and mitochondrial ribosomal genes, which are responsible for the expression of mtDNA encoded ETC subunits were among the most downregulated genes in CG1603 mutant flies, suggesting a potential transcriptional mechanism coordinating the nuclear and mitochondrial genome activities in ETC biogenesis. We performed network analysis using the votex sort algorithm on the CHIP data of 49 confirmed hits, and constructed a regulatory network of transcription factors that regulate mitochondrial biogenesis. Most of these TFs were identified as strongly connected components due to their extensive connections and were classified into a hierarchical structure, suggesting the existence and complexity of co-regulation. Furthermore, the network analyses identified YL-1 as an upstream regulator of CG1603, which was confirmed by the genetic studies. Project 2: Mechanical stimulation from the surrounding tissue activates mitochondrial energy metabolism in Drosophila differentiating germ cells In multicellular lives, the differentiation of stem cells and progenitor cells is often accompanied by a transition from glycolysis to mitochondrial oxidative phosphorylation. However, the underlying mechanism of this metabolic transition remains largely unknown. We previously identified a JNK-Myc signaling cascade in promoting ETC biogenesis, we are intrigued by the sharp and transient activation of JNK in differentiating follicles at region 2B germarium. In region 2B, the round 16-cell cyst is encapsulated by somatic cells, which compress the cyst into a single-cell layer disc. It has been shown that increased membrane tension can promote Drosophila midgut intestine stem cell proliferation and differentiation. Interestingly, increased mitochondrial biogenesis and a metabolic shift from glycolysis to oxidative phosphorylation often accompany the stem cell differentiation, suggesting a potential link between mechanical stress and mitochondrial biogenesis. We therefore hypothesize that mechanical stress on the region 2B germ cell might be the developmental cue activating JNK, which subsequently triggers mitochondrial biogenesis. We demonstrate that the surrounding somatic cells flatten the 16-cell differentiating cyst, resulting in an increase of the membrane tension of germ cells inside the cyst. This mechanical stress is necessary to maintain cytosolic Ca2+ concentration in germ cells through a mechanically activated channel, Transmembrane channel-like. The sustained cytosolic Ca2+ triggers a CaMKI-Fray-JNK signaling relay, leading to the transcriptional activation of oxidative phosphorylation in differentiating cysts. Our findings demonstrate a molecular link between cell mechanics and mitochondrial energy metabolism, with implications in other developmentally orchestrated metabolic transitions in mammals. Project 3: Unveiling a Novel Exonuclease Driving Paternal Mitochondrial DNA Elimination in Drosophila Spermatogenesis The uniparental inheritance of mtDNA was once regarded as a passive outcome of distinct cytoplasmic contents of eggs and sperms. Recent studies demonstrated active mechanisms to remove mitochondrial DNA (mtDNA) during spermatogenesis in various species. However, the physiological significance of mtDNA clearance, or mitochondria uniparental inheritance in general remains a mystery and the factors involved in this process are largely unknown. We found that POLDIP2, a putative mitochondrial nucleoid protein, was highly enriched in late spermatogenesis stages, where the mtDNA clearance takes place. Although Poldip2 mutant flies exhibited normal spermatogenesis progression, they produced fewer mature sperms that frequently contained multiple copies of mtDNA, indicating an essential role of POLDIP2 in mtDNA clearance. We discovered that POLDIP2 was an exonuclease with a preference for degrading ssDNA and nicked dsDNA. Ectopic expression of a mitochondrially targeted E.coli exonuclease III in Poldip2 mutant flies effectively removed residual mtDNA and substantially restored male fertility, highlighting the detrimental consequences of persisting mtDNA in mature sperm cells. To our knowledge, POLDIP2 represents the first factor identified to specifically enforce maternal inheritance of mitochondrial genomes. This discovery opens avenues for future investigations into the physiological significance and underlying mechanisms of this highly conserved yet enigmatic uniparental inheritance of mtDNA. Project 4: Visualization of single nucleotide polymorphism on mtDNA to directly assess the selective inheritance in ovary. We proposed a new model of mitochondrial selective inheritance that healthy mitochondria containing wild type genome would proliferate much more vigorous and hence out-compete those afflicted by damaging mutations. However, it has not been demonstrated that wild type mtDNA replicates faster and more robust than the mutant genome in heteroplasmic germ cells. A major technical hurdle is that the conventional FISH assay fails to discern the single nucleotide polymorphism (SNP). We developed a novel method to detect SNP on mtDNA in drosophila ovary. Critical procedures include 1) combining restriction enzyme with an exonuclease to generate distinct 3 end overhangs on wild type and mutant mtDNA; 2) applying two designed circular probes with specific sequence bound to these two overhangs; 3) replacing normal nucleotides to locked nucleic acid in circular probes to further increase specificity; 4) applying rolling cycle amplification to further amplify the template. We have successfully distinguished wild type and mutant mtDNA with a single SNP in Drosophila ovaries. We are now testing whether the mutant genome is indeed replicated less than wild type under the restrictive condition. We will also determine the exact time of the selection and assess the selection inheritance under various environmental and genetic conditions. Project 5: Genetic analyses of mitochondrial tRNA import in Dictyostelium. We previously utilized high-throughput multiplexed protein quantitation and homology analyses to generate a high-confidence mitochondrial protein compendium consisting of 936 proteins. Currently, we are performing the tRNA-seq on mitoplast, to determine tRNA species that are selectively transported from cytosol to mitochondria. We are also trying to establish a Dictyostelium mitochondria transformation system, to insert an inducible tRNA expression cassette of the missing tRNA genes into the mitochondria genome. We will use this synthetic tRNA complementary system to perform a candidate screen for genes required for tRNA transport in Dictyostelium. Identification and characterization of the components involved