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. We have taken advantage of previously developed mitochondrial genetics mosaic system to create mtDNA deficiency in developing eye, which allowed us to carry modifier genetic screen for nuclear factors controlling mitochondrial biogenesis. From the primary RNAi screen of 631 transcription factors annotated in fly genome, we recovered total 78 enhancers and 19 suppressors, which promote or inhibit mitochondrial biogenesis, respectively. We further validated their roles in mitochondrial biogenesis by directly assessing mitochondria mass, morphology and positioning (SDHB-GFP), and mtDNA contents (TFAM-GFP). Among positive hits, a previously unnoted Zinc finger protein, encoded by CG1603 locus, was essential for the maintenance of both mitochondrial mass and mtDNA. Null mutant of CG1603 was lethal, died at the first instar larva stage. Tissue specific knock down of CG1603 greatly diminished mitochondrial genome, nuclear encoded factors required for mtDNA maintenance and expression, as well as the overall mitochondrial mass. RNAseq analyses revealed that more than 2/3 of nuclear encoded mitochondrial genes were down-regulated in CG1603 mutant flies. Interestingly, per published CHIP-seq data, CG1603 binds to the gene regions of a few other transcription factors that are positive hits in the primary RNAi screen, besides many mitochondrial genes, suggesting a complex transcriptional network regulating mitochondrial biogenesis. We are now performing additional analyses, trying to construct a regulatory network among all hits emerged from the primary RNAi screen and to illustrate connections and the hierarchical organization of these factors. Project 2: Genetic analyses of mitochondrial regulation on lipid homeostasis in both Drosophila and mouse models. Our genetic analyses revealed that 15% mitochondrial proteins that are highly conserved between fruit flies and mammals were not essential genes. Ubiquitous knockdown of these genes in flies have no obvious fitness defect, judging by their viability, fertility and locomotor activity. Among these genes, there is a mitochondrial AAA protein (ATPases Associated with diverse cellular Activities) encoded by CG12149 locus. CG12149 mutant flies were completely healthy, and surprisingly, showed higher basal respiration rate and lower respiratory quotient, suggesting that mutant flies have higher mitochondrial activity and consume more fatty acids compared to wild type animals. Indeed, mutant flies had markedly increased ETC activity, but reduced TAG levels and less lipid droplets in tissues. Most mitochondrial AAA proteins are mitochondrial carriers, chaperones or proteases. CG12149 is localized to the mitochondrial matrix, but its biochemical activity and actual function are completely unknown. We are performing quantitative proteomics, trying to identify changes on mitochondrial proteome in mutant flies, which would shed light on the function of CG12149, and help us formulate a hypothesis of how CG12149 regulates lipid homeostasis. Intrigued by a possible role of CG12149 in lipid metabolism and the potential impact of this regulation on human health, we are expanding our study into a mammalian model. We generated a knockout mouse line deleting the mouse homolog of CG12149. Consistent with the phenotypes observed in mutant flies, homozygous KO mice showed reduced amount of white fat. We also noticed severe nurturing defects of KO mice: homozygous knockout mothers did not feed new born, which resulted in maternal effect neonatal lethal. We are now further characterizing the metabolic defects in KO mice, and trying to understand the neurological basis of nurturing phenotypes. Project 3: MDIS, a mitochondrial DNA exonuclease enforces uniparental inheritance of mitochondrial genome Mitochondrial genome is exclusively transmitted through maternal linage in animals. The uniparental inheritance 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. Using proximity-labeling based proteomic screen, we recovered a putative mitochondrial nucleoid protein MDIS (mitochondrial DNA in sperm), encoded by CG12162 locus. MDIS is highly enriched in Drosophila testis, and specifically in late spermatogenesis stages, when the mtDNA clearance takes place. MDIS null flies are male semi-sterile, otherwise generally healthy. Spermatogenesis progresses normally in MDIS flies but produces immotile mature sperms that often contain multiple copies of mtDNA nucleoids, indicating that MDIS is required for mtDNA clearance. In vitro assay suggests that MDIS is an exonuclease that degrades both single-stranded DNA and double-stranded DNA with overhangs. Importantly, ectopic expression of a mitochondrially targeted E.coli exonuclease III in MDIS flies, effectively removes mtDNA remnants and largely restores male fertility, demonstrating that failure to eliminate mtDNA causes immotile sperms and male infertility. Surprisingly, we observed pronounced nuclear DNA fragmentations in mature sperms of MDIS flies, suggesting a potential detrimental consequence of persisting mtDNA in mature sperms. Our results demonstrate that MDIS play essential roles in preventing the transmission of paternal mtDNA to the offspring, and in safeguarding the nuclear genome integrity. MDIS, to our knowledge, represents the first-ever identified factor that specially enforces maternal inheritance of mitochondrial genomes. This will allow future studies to understand physiological significances and underlying mechanisms of this highly conserved, yet mysterious phenomenon. Project 4: Visualization of single nucleotide polymorphism on mtDNA to directly assess the selective inheritance in ovary. We previously demonstrated a selective inheritance limiting the transmission of deleterious mtDNA mutations in Drosophila. We also revealed that mtDNA replication depends on active respiration in ovaries. 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 proved yet that wild type mtDNA replicate faster and more robust than the mutant genome in a heteroplasmic germ cells. A major technical hurdle is that conventional FISH assay fails to discern the single nucleotide polymorphism. We modified the CRISPR/Cas9-mediated proximity ligation assay (CasPLA), by applying restriction enzyme digestion to space out wild type mtDNA together with a dumbbell template DNA mediated rolling circle amplification, besides the sequence specific sgRNA probes. We have successfully detected single-nucleotide polymorphism on mtDNA in Drosophila ovaries using this improved CasPLA. We are now applying this assay in a heteroplasmic line containing both wild type and a conditional lethal mtDNA allele, to quantify the relative abundance of the mutant genome in ovary. We will examine whether the mutant genome is indeed replicated less than wild type under the restriction condition. This assay will also allow us to determine the exact stage when the selection takes place and assess the selection inheritance under various environmental and genetic conditions.