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子筛选中，我们回收了总共 77 个增强子和 22 个抑制子，分别促进或抑制线粒体生物发生。我们进一步追踪了之前未被注意到的由 CG1603 位点编码的锌指蛋白。 CG1603 的无效突变体是致命的。 CG1603 的组织特异性破坏极大地降低了 mtDNA 的稳态水平、mtDNA 维持和表达所需的核编码因子以及线粒体总体质量。 RNAseq 分析显示，CG1603 突变果蝇中近 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 CHIP-seq 数据，发现 CG1603 与保守的回文 8 bp 序列 TATCGATA 结合，该序列存在于超过 50% nu-Mito 基因和一些转录因子的转录起始点。这些结果表明 CG1603 是线粒体生物发生的主要调节因子，直接或间接控制 nu-Mito 基因的表达。有趣的是，负责表达 mtDNA 编码的 ETC 亚基的 ETC 基因和线粒体核糖体基因是 CG1603 突变果蝇中下调最明显的基因之一，这表明在 ETC 生物发生中存在协调核和线粒体基因组活动的潜在转录机制。我们使用投票排序算法对 49 个已确认命中的 CHIP 数据进行网络分析，并构建了调节线粒体生物发生的转录因子的调节网络。大多数这些转录因子由于其广泛的连接而被识别为强连接成分，并被分类为层次结构，表明共同调节的存在和复杂性。此外，网络分析确定 YL-1 是 CG1603 的上游调节因子，这得到了遗传研究的证实。项目2：周围组织的机械刺激激活果蝇分化生殖细胞的线粒体能量代谢在多细胞生命中，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转变。然而，这种代谢转变的潜在机制仍然很大程度上未知。我们之前发现了 JNK-Myc 信号级联在促进 ETC 生物发生中的作用，我们对 JNK 在 2B 区胚泡分化过程中的急剧和瞬时激活感兴趣。在 2B 区域，圆形 16 细胞囊肿被体细胞包裹，将囊肿压缩成单细胞层盘。研究表明，增加膜张力可以促进果蝇中肠肠干细胞的增殖和分化。有趣的是，线粒体生物发生的增加和从糖酵解到氧化磷酸化的代谢转变通常伴随干细胞分化，这表明机械应激和线粒体生物发生之间存在潜在联系。因此，我们假设 2B 区生殖细胞上的机械应力可能是激活 JNK 的发育线索，随后触发线粒体生物发生。我们证明周围的体细胞使 16 细胞分化囊肿变平，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激活的通道（类似跨膜通道）维持生殖细胞中的胞质 Ca2+ 浓度是必要的。持续的胞质 Ca2+ 触发 CaMKI-Fray-JNK 信号传递，导致分化囊肿中氧化磷酸化的转录激活。我们的研究结果证明了细胞力学和线粒体能量代谢之间的分子联系，这对哺乳动物中其他发育协调的代谢转变具有影响。项目 3：揭示果蝇精子发生过程中驱动父系线粒体 DNA 消除的新型核酸外切酶线粒体DNA的单亲遗传一度被认为是卵子和精子细胞质内容不同的被动结果。最近的研究证明了在不同物种的精子发生过程中去除线粒体 DNA (mtDNA) 的积极机制。然而，线粒体DNA清除或线粒体单亲遗传的生理意义仍然是个谜，参与这一过程的因素在很大程度上也是未知的。我们发现 POLDIP2（一种假定的线粒体核蛋白）在精子发生晚期（mtDNA 清除发生的阶段）高度富集。尽管 Poldip2 突变果蝇表现出正常的精子发生进程，但它们产生的成熟精子较少，且通常含有多个 mtDNA 拷贝，这表明 POLDIP2 在 mtDNA 清除中发挥着重要作用。我们发现 POLDIP2 是一种核酸外切酶，优先降解 ssDNA 和带切口的 dsDNA。 Poldip2 突变果蝇中线粒体靶向大肠杆菌核酸外切酶 III 的异位表达有效去除了残留的 mtDNA，并显着恢复了雄性生育能力，凸显了成熟精子细胞中持续存在 mtDNA 的有害后果。据我们所知，POLDIP2 代表了第一个被确定为专门增强线粒体基因组母系遗传的因子。这一发现为未来研究这种高度保守但神秘的 mtDNA 单亲遗传的生理意义和潜在机制开辟了道路。项目4：线粒体DNA单核苷酸多态性的可视化，以直接评估卵巢中的选择性遗传。我们提出了一种线粒体选择性遗传的新模型，即含有野生型基因组的健康线粒体将增殖得更加活跃，从而击败那些受到破坏性突变影响的线粒体。然而，尚未证明野生型 mtDNA 在异质生殖细胞中比突变基因组复制得更快、更稳健。一个主要的技术障碍是传统的 FISH 检测无法识别单核苷酸多态性 (SNP)。我们开发了一种检测果蝇卵巢线粒体 DNA 上 SNP 的新方法。关键程序包括 1) 将限制性内切酶与核酸外切酶相结合，在野生型和突变体 mtDNA 上产生不同的 3 末端突出端； 2)应用两个设计的圆形探针，其具有与这两个突出端结合的特定序列； 3)将环状探针中的正常核苷酸替换为锁定核酸，进一步提高特异性； 4)应用滚动循环放大来进一步放大模板。我们已经成功区分果蝇卵巢中野生型和突变型 mtDNA 的单个 SNP。我们现在正在测试突变基因组在限制条件下是否确实比野生型基因组复制得少。我们还将确定选择的确切时间，并评估各种环境和遗传条件下的选择遗传。项目 5：盘基网柄菌线粒体 tRNA 输入的遗传分析。我们之前利用高通量多重蛋白质定量和同源性分析来生成由 936 种蛋白质组成的高置信度线粒体蛋白质纲要。目前，我们正在线粒体上进行 tRNA-seq，以确定选择性从细胞质转运到线粒体的 tRNA 种类。我们还试图建立盘基网柄菌线粒体转化系统，将缺失的 tRNA 基因的诱导型 tRNA 表达盒插入线粒体基因组中。我们将使用这种合成的 tRNA 互补系统对盘基网柄菌中 tRNA 运输所需的基因进行候选筛选。所涉及组件的识别和表征