Molecular Studies of Eukaryotic Gene Regulation

真核基因调控的分子研究

• 批准号: 8937631
• 负责人: bruce paterson
• 金额: $ 43.35万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/8937631
• 关键词: 3' Untranslated Regions; Ablation; Affect; Avidin; Binding Sites; Biological Models; Biotin; Boxing; Buffers; C-terminal; Cell Cycle; Cell Lineage; Cell fusion; Cell physiology; Cells; Chromatin; Complementary DNA; Complex; Coupled; DNA; DNA Sequence; Data; Defect; Development; Diagnosis; Disease; Dorsal; Double-Stranded RNA; Drosophila genus; Ectopic Expression; Embryo; Embryonic Development; Enhancers; Excision; Failure; Feedback; Female sterility; Gene Activation; Gene Expression Profile; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Genome; Genomics; Heart; Heat-Shock Response; Homeostasis; Hour; Informatics; Injection of therapeutic agent; Lead; Mesoderm; Mesoderm Cell; MicroRNAs; Molecular; Molecular Profiling; Muscle; Muscle Cells; Muscle Fibers; Myoblasts; Myogenic Regulatory Factors; Nautilus; Output; Pathway interactions; Pattern; Phenotype; Play; Population; Porifera; Process; Proteins; Publishing; RNA; RNA Interference; Reading; Regulation; Regulatory Pathway; Reporter; Repression; Ricin; Role; Sampling; Signal Transduction; Site; Small RNA; Specific qualifier value; Staging; Tissues; Toxin; Transgenes; Transgenic Organisms; Untranslated Regions; Withdrawal; Work; base; cancer type; chromatin remodeling; fly; human disease; insight; muscle form; mutant; myogenesis; nerve supply; notch protein; novel; promoter; research study; transcription factor; uncontrolled cell growth

We have made substantial progress in understanding the role of nautilus in Drosophila myogenesis. The highly organized and segmentally reiterated muscle pattern in the Drosophila embryo is prefigured by the arrangement of a sub-population of mesodermal cells called founder myoblasts. We had shown earlier that the expression of nautilus, the only MyoD-related gene in Drosophila, is initiated at stage 9 in a stereo-specific pattern in a subset of mesodermal cells that become incorporated into every somatic muscle in the embryo. Targeted ricin toxin ablation of these cells resulted in the loss of embryonic muscle. We now know that at stage 11 these same cells begin to express a later founder cell-specific marker, duf (rP298LacZ) thus nautilus is the earliest marker for the critical founder myoblast population. We inactivated the nautilus gene using both homology-directed gene targeting and a novel gal4-inducible nautilus RNAi transgene to determine if any aspect of founder cell function required nautilus gene activity. An earlier study in our lab, using the injection of nautilus dsRNA to induce gene silencing by RNAi, indicated loss of nautilus function resulted in a range of phenotypes from no muscle disruption to severe embryonic muscle loss and disruption (30% of the embryos). Both gene targeting and the gal4-inducable nautilus RNAi resulted in a range of defects that included severe embryonic muscle disruption, reduced viability and female sterility. All these phenotypes were rescued by a hsp70 nautilus cDNA transgene in the absence of heat shock in independent transgenic lines. More importantly, the highly organized founder cell pattern that is needed to establish the proper embryonic muscle organization was disrupted in nautilus null embryos prior to MHC expression and the disruption prefigured the subsequent embryonic muscle defects observed at later stages in development. Tinman, a marker for mesodermal cells that give rise to the dorsal vessel or heart, was expressed normally in the nautilus null embryo. Although nautilus does not specify the myogenic cell lineage, it has a cell autonomous role in establishing the correct muscle organization in the embryo through its regulation of the founder cell pattern. This work has been published recently in PNAS (Wei et al). We are currently carrying out experiments to identify nautilus target genes. To identify nautilus target genes we have used two approaches. First we have just completed a transcriptome comparison between mutant and wild-type embryos using the Solexa HiSeq2000 Genomic Analyzer with more that 30Gb of filtered reads for each sample that are currently under analysis by NCI informatics and other collaborators. Nautilus is expressed in only 0.1% of the cells in the embryo (800 cells). In order to capture gene loci that interact with nautilus, we used gene targeting to generated a fly line to express C-terminal biotinylated nautilus protein. The selectivity of the biotin-avidin capture has been evaluated using a recently identified nautilus target gene, the miR-309 micro RNA cluster. Biotin selected DNA from 9-13 hour wild-type and nau-biotin embryos has been isolated and sequenced on the Solexa 1G Genomic Analyzer to give 16-17 million filtered, good quality reads. We have preliminary data on 3000 peaks across the genome that are under analysis with the help of Dr. Sameet Mehta, an informatics expert in Dr. Shiv Grewal's lab. We have also targeted an AttP site into the nautilus gene in order to determine the role of possible regulatory DNA sequences in the promoter and 3' UTR. We have also identified six intra-genic enhancer regions that modulate the nautilus expression pattern in the embryo using insulated enhancer reporter transgenes. Selective removal of these regions will determine their impact on muscle formation. micro RNAs (miRNAs) play a key role in gene regulation in development and disease. A miRNA expression profile in the nautilus null revealedthat expression from the miR-309 locus is down regulated and is dependent upon two E-boxes in the miR-309 promoter/enhancer. miR-3 in the locus fine tunes nautilus expression in the embryo in a negative feedback loop involving the nau 3'-UTR. Deletion of the miR-309 cluster or ectopic expression of miR-3 also decrease Dmef2 RNA levels, a transcription factor required for muscle formation. Ectopic miR-3 expression enhances output from the miR-310 locus encoding 7 micro RNAs, four of which target the 3'UTR of the essential myogenic regulatory factor, Dmef2. The convergence of these miRNA regulatory pathways points to a previously unappreciated complexity in nautilus gene regulation of Drosophila myogenesis and the complex miRNA circuitry buffering the myogenic transcriptome. Targeted deletion of the mirs in the miR-309 cluster using an attP site as well as removal of the mir-3 binding site in the nau 3'-UTR via the nau attP are under way. We have used a specific mir-3 sponge transgene to determine the effects of mir-3 reduction during development. Loss or gain of mir-3 result in severe muscle disruption in 10-15% of the embryos. Importantly, nautilus direct regulation of the miR-309 cluster and mir-3 output, coupled to mir-3 activation of the miR-310 cluster which regulates dMef2, the notch pathway and Jak-Stat signaling, all affect Drosophila myogenesis. Transcriptome buffering by microRNA circuits under the control of tissue-sspecific transcription factors give insight to cellular homeostasis and a basis for disease mechanisms.

我们在理解瑙蒂洛斯在果蝇肌发生中的作用方面取得了重大进展。果蝇胚胎中高度组织和分段的重复性肌肉模式是通过在称为创始人成肌细胞的中胚层细胞的亚群的排列来预先建立的。我们早些时候表明，果蝇中唯一与肌爱果石相关的基因的表达是在第9阶段以立体特异性模式在中胚层细胞中以立体特异性模式启动的，在中胚层细胞的一部分中，这些模式被纳入胚胎中的每个体细胞肌肉。这些细胞的靶向利环蛋白毒素消融导致胚胎肌肉的丧失。我们现在知道，在第11阶段，这些相同的细胞开始表达后来的创建者特异性标记DUF（RP298LACZ），因此Nautilus是关键创始人成肌细胞种群的最早标记。我们使用同源指导的基因靶向和一种新型的GAL4诱导的RNAi转基因灭活了Nautilus基因，以确定创建者细胞功能的任何方面是否需要Nautilus基因活性。在我们实验室中的一项较早的研究使用注射Nautilus dsRNA诱导RNAi诱导基因沉默，表明Nautilus功能的丧失导致一系列表型，从无肌肉破坏到严重的胚胎肌肉损失和破坏（30％的胚胎）。基因靶向和gal4诱导的Nautilus RNAi均导致一系列缺陷，包括严重的胚胎肌肉破坏，生存力降低和女性不育。在独立的转基因线中没有热休克的情况下，HSP70 Nautilus cDNA转基因挽救了所有这些表型。更重要的是，建立适当的胚胎肌肉组织所需的高度有组织的创始人细胞模式在Nautilus null胚胎中被破坏，然后在MHC表达之前被破坏，而破坏则预示着随后在发育中后期阶段观察到的随后的胚胎肌肉缺陷。廷曼（Tinman）是在nautilus null胚胎中通常表达的中胚层细胞的标记。尽管Nautilus并未指定肌原细胞谱系，但它通过调节创始人细胞模式在胚胎中建立正确的肌肉组织中具有自主作用。这项工作最近发表在PNAS（Wei等人）。我们目前正在进行实验以鉴定nautilus靶基因。为了识别nautilus靶基因，我们使用了两种方法。首先，我们刚刚使用solexa hiseq2000基因组分析仪完成了突变体和野生型胚胎之间的转录组比较，而NCI信息和其他协作者目前正在分析的每个样本中的30GB过滤读数。 Nautilus仅在胚胎（800个细胞）中仅0.1％的细胞中表达。为了捕获与Nautilus相互作用的基因基因座，我们使用基因靶向产生了飞行线以表达C末端生物素化的Nautilus蛋白。生物素 - 阿维丁蛋白捕获的选择性已使用最近鉴定的Nautilus靶基因MiR-309微RNA簇评估。生物素从9-13小时的野生型和Nau-Biotin胚胎中选择的DNA已在Solexa 1G基因组分析仪上分离并进行了测序，以进行16-1700万次过滤，质量良好的读数。我们拥有有关整个基因组3000个峰的初步数据，这些数据在Shiv Grewal博士实验室的信息专家Sameet Mehta博士的帮助下进行了分析。我们还将ATTP位点瞄准进入Nautilus基因，以确定启动子和3'UTR中可能的调节DNA序列的作用。我们还确定了使用绝缘增强剂报告基因转基因的六个基因内增强子区域，这些区域可调节胚胎中的Nautilus表达模式。选择性去除这些区域将决定它们对肌肉形成的影响。微RNA（miRNA）在发育和疾病中的基因调节中起关键作用。 Nautilus null中的miRNA表达谱揭示了MiR-309基因座的表达受到下调，并取决于miR-309启动子/增强子中的两个电子盒。 MiR-3中的miR-3在涉及Nau 3'-UTR的负反馈回路中，在胚胎中微调nautilus表达。 miR-309簇的缺失或miR-3的异位表达也降低了DMEF2 RNA水平，DMEF2 RNA水平是肌肉形成所需的转录因子。异位miR-3表达增强了编码7个微RNA的miR-310基因座的输出，其中四个靶向基本肌源性调节因子DMEF2的3'UTR。这些miRNA调节途径的收敛表明，果蝇肌发生的Nautilus基因调节中的复杂性和复杂miRNA回路缓冲了肌源性转录组。使用ATTP位点，通过Nau 3'-UTR中的miR-309簇中的miR靶向缺失，并通过Nau 3'-UTR中的miR-3结合位点进行了操作。我们已经使用了特定的miR-3海绵转基因来确定发育过程中miR-3降低的影响。 miR-3的损失或增益导致10-15％的胚胎严重肌肉破坏。重要的是，Nautilus对miR-309簇和miR-3输出的直接调节，与调节DMEF2，Notch途径和JAK-STAT信号传导的miR-3激活的miR-3激活相结合，都影响了果蝇肌生成。 MicroRNA电路在组织中特定转录因子的控制下对转录组缓冲可洞悉细胞稳态和疾病机制的基础。