Mitochondrial DNA genetics inheritance

线粒体DNA遗传学遗传

基本信息

批准号：
10929153
负责人：
Hong Xu
金额：
$ 253.75万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10929153
关键词：
Adenosine Triphosphate Algorithms Automobile Driving Binding Biogenesis Cell Differentiation process Cell physiology Cells ChIP-seq Classification Complex Cues Cyst Cytoplasm Cytosol DNA cassette Data Development Dictyostelium Disease Drosophila genus Ectopic Expression Electron Transport Encapsulated Energy Metabolism Enhancers Escherichia coli Exhibits Exodeoxyribonuclease III Exonuclease Fluorescent in Situ Hybridization Future Genes Genetic Genetic Diseases Genetic Transcription Genetic study Genome Germ Cells Glycolysis Goals Health Homeostasis Intestines Investigation Ion Channel Life Link MAPK8 gene Maintenance Mammals Mechanical Stimulation Mechanical Stress Mechanics Membrane Metabolic Methods Midgut Mitochondria Mitochondrial DNA Mitochondrial Diseases Mitochondrial Inheritance Mitochondrial Proteins Modeling Molecular Mutation Nuclear Nucleotides Outcome Ovary Oxidative Phosphorylation Pathway Analysis Physiological Procedures Process Proliferating Proteins Publishing Regulation Research Residual state Ribosomes Role Signal Transduction Single Nucleotide Polymorphism Single-Stranded DNA Somatic Cell Sorting Specificity Spermatogenesis Structure System Testing Time Tissues Transcriptional Activation Transfer RNA Visualization Zinc Fingers cell behavior design ds-DNA egg fly follow-up genetic analysis human disease locked nucleic acid male fertility mitochondrial DNA mutation mitochondrial genome mutant novel preference restriction enzyme sperm cell stem cell differentiation stem cell proliferation stem cells tool transcription factor transcription regulatory network transcriptome sequencing transmission process

项目摘要

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

项目1：控制线粒体生物发生和维持的转录层次结构的系统遗传分析。从正在进行的遗传修饰符筛选中，我们恢复了总计77个增强子和22个抑制器，分别促进或抑制线粒体生物发生。我们进一步跟进了先前未识别的锌指蛋白，该蛋白由CG1603基因座编码。 CG1603的无效突变体致命。 CG1603的组织特异性破坏大大降低了mtDNA的稳态水平，mtDNA维持和表达所需的核编码因子以及整体线粒体质量。 RNASEQ分析表明，在CG1603突变蝇中，将近70％的核编码线粒体基因（NU-MITO基因）下调。我们重新分析了已发表的芯片序列数据，发现CG1603与保守的，全文，8 bp序列Tatcgata结合，该序列呈现在超过50％NU-MITO基因的转录起点和一些转录因子上。这些结果表明，CG1603是直接或间接控制Nu-mito基因表达的线粒体生物发生的主要调节剂。有趣的是，负责MTDNA编码的亚基表达的基因和线粒体核糖体基因是CG1603突变体蝇中最下调的基因之一，这表明一种潜在的转录机制，该机制是核和线粒体基因组活性在等生物发生中的核和线粒体基因组活性。我们对49个确认的曲线数据的芯片数据进行了网络分析，并构建了调节线粒体生物发生的转录因子的调节网络。这些TF中的大多数由于其广泛的连接而被鉴定为紧密连接的组件，并被分类为层次结构，表明共同调节的存在和复杂性。此外，该网络分析将YL-1鉴定为CG1603的上游调节剂，该遗传研究证实了这一点。项目2：周围组织的机械刺激激活果蝇中的线粒体能量代谢在多细胞生活中，干细胞和祖细胞的分化通常伴随着从糖酵解到线粒体氧化磷酸化的过渡。但是，这种代谢过渡的基本机制在很大程度上仍然未知。我们先前鉴定出在促进生物发生中的JNK-MYC信号传导级联反应，我们对JNK在区分2B胚芽的卵泡中的急剧和短暂激活感兴趣。在区域2B中，圆形16细胞囊肿被体细胞封装，该细胞将囊肿压缩到单细胞层盘中。已经表明，增加的膜张力可以促进果蝇中肠干细胞增殖和分化。有趣的是，线粒体生物发生增加以及从糖酵解到氧化磷酸化的代谢转移通常伴随干细胞分化，这表明机械应激与线粒体生物发生之间存在潜在的联系。因此，我们假设对区域2b生殖细胞的机械应力可能是激活JNK的发育提示，随后会触发线粒体生物发生。我们证明，周围的体细胞会使16细胞区分囊肿扁平，从而导致囊肿内生殖细胞的膜张力增加。这种机械应力对于通过机械活化的通道跨膜通道样在生殖细胞中维持胞质Ca2+浓度是必不可少的。持续的胞质Ca2+触发了CAMKI-FRAY-JNK信号继电器，从而导致分化囊肿中氧化磷酸化的转录激活。我们的发现表明，细胞力学与线粒体能量代谢之间存在分子联系，对哺乳动物的其他发育策划的代谢转变产生了影响。项目3：揭示果蝇精子发生中的新核酸酶驱动父亲线粒体DNA消除 mtDNA的单亲遗传曾被视为卵和精子的不同细胞质含量的被动结果。最近的研究表明，在各种物种的精子发生过程中，在精子发生过程中去除线粒体DNA（mtDNA）的主动机制。但是，mtDNA清除率或线粒体单亲的生理意义一般仍然是一个谜，并且此过程中涉及的因素在很大程度上是未知的。我们发现Poldip2是一种推定的线粒体核苷蛋白，在晚期的精子发生阶段高度富集，在该阶段发生mtDNA清除率。尽管Poldip2突变蝇表现出正常的精子发生进展，但它们产生的成熟精子较少，这些精子经常包含多个MTDNA副本，表明Poldip2在mtDNA清除率中的重要作用。我们发现Poldip2是一种外核酸酶，偏爱降解ssDNA和划痕的dsDNA。线粒体靶向大肠杆菌外切核酸酶III的异位表达有效地消除了残留的mtDNA，并实质上恢复了男性生育力，突显了成熟精子细胞中持续的mtDNA的有害后果。据我们所知，POLDIP2代表了确定针对线粒体基因组孕产妇遗传的第一个因素。这一发现为未来对这种高度保守但神秘的单亲遗传的生理意义和潜在机制的研究开辟了途径。项目4：单核苷酸多态性在mtDNA上的可视化，直接评估卵巢中的选择性遗传。我们提出了一种新的线粒体选择性遗传模型，即含有野生型基因组的健康线粒体会增殖得更加有活力，因此远远超出了受损突变折磨的人。但是，尚未证明野生型mtDNA比在异质生殖细胞中的突变基因组更快，更健壮。一个主要的技术障碍是传统的鱼类测定法无法辨别单核苷酸多态性（SNP）。我们开发了一种新的方法来检测果蝇卵巢中mtDNA上的SNP。关键程序包括1）将限制酶与外切核酸酶结合起来，以在野生型和突变型mtDNA上产生不同的3端悬垂； 2）应用两个设计的圆形探针，其特定序列与这两个悬垂结合在一起； 3）将正常的核苷酸代替圆形探针中锁定的核酸，以进一步提高特异性； 4）应用滚动周期放大以进一步扩增模板。我们已经成功地区分了果蝇卵巢中的单个SNP的野生型和突变mtDNA。现在，我们正在测试在限制性条件下突变基因组是否确实比野生型要小于野生型。我们还将确定选择的确切时间，并评估在各种环境和遗传条件下的选择遗传。项目5：线粒体tRNA进口在dictyostelium中的遗传分析。我们先前利用高通量的多重蛋白定量和同源分析来产生由936个蛋白质组成的高信心线粒体蛋白汇编。目前，我们正在对线粒体上进行tRNA-seq，以确定从细胞质转移到线粒体的tRNA物种。我们还试图建立一个线粒体转化系统，以将缺失的TRNA基因的诱导型TRNA表达盒插入线粒体基因组中。我们将使用此合成tRNA互补系统来执行dictyostelium tRNA传输所需的基因的候选筛选。涉及组件的识别和表征