Molecular Studies of Eukaryotic Gene Regulation

真核基因调控的分子研究

• 批准号: 8157166
• 负责人: bruce paterson
• 金额: $ 111.06万
• 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/8157166
• 关键词: Gene Expression Regulation; Molecular

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 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 using the injection of nautilus dsRNA to induce gene silencing by RNAi indicated loss of nautilus function resulted in a severe embryonic muscle loss or disruption. 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 the nautilus cDNA 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. 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 undertaken a transcriptome analysis of mutant and wild-type embryos using the Solexa 1G Genomic Analyzer, a so-called deep sequence approach. Genes involved in determining the myogenic field in the mesoderm,in establishing the muscle founder and fusion competent myoblast populations,in regulating cell fusion, and in establishing muscle identity are measurably down regulated in the nautilus null. Expression patterns for genes involved in myotube positioning are also altered in the null. By contrast, certain genes representing muscle structural proteins, actin-binding proteins, ion channels, excitation-contraction coupling components, calcium binding proteins, and synaptic vesicle movement are mis-regulated and are expressed at somewhat higher levels in the nautilus null embryo. More that 2000 genes are unaffected in the mutant. Trends apparent in the transcriptome analysis have identified groups of genes that are negatively affected in the null, consistent with their roles in myogenesis. These genes may be direct targets for nautilus regulation and this will be determined by ChIP-Seq. Since nautilus is expressed in only 0.1% of the cells in the embryo, stringent ChIP conditions must be employed to identify target genes. In order to capture gene sequences that interact with nautilus, we have generated a fly line with the highest affinity tag known joined to the carboxy terminus of the engodenous nautilus gene, a peptide sequence that can be biotinylated by E. coli biotin ligase expressed from the targeting vector. The selectivity of the biotin-avidin capture in ChIP is being evaluated using a known nautilus target gene, the 8-miR locus discussed below. Once the proper conditions are established, we will perform ChIP-Seq (Solexa 1G Genomiic Analyzer) to identify nautilus target DNA sequences. In addition, recent advances in gene targeting have enabled us to introduce an AttP site in the nautilus gene to study important DNA sequences involved in promoter function, nautilus TF activity, miRNA binding and enhancer function. Small 21bp RNAs known as micro RNAs (miRNAs) play a key role in gene regulation in development and disease. We have recently identified two miRNAs that regulate post transcriptional expression of nautilus in the embryo and the adult. The miRNA binding sites are conserved in the 3'UTR of the nautilus gene in multiple species of Drosophila. The nautilus 3'UTR alone can regulate reporter expression in response to the ectopic expression of these miRNAs in S2 cells. A profile of miRNA expression in the nautilus null revealed that the 8-miR locus, aka the 309-locus, a genomic region encoding 9 microRNAs, regulates the post transcriptional expression of greater than 4000 genes and is under the direct control of nautilus via two E-boxes in the 309-locus promoter. miR3 in the locus fine tunes nautilus expression in the embryo in a negative feedback loop. Loss of the 8-miR locus impacts miR-1 and miR-184 levels, essential micro RNAs for myogenesis and egg laying, respectively. Deletion of the 8-miR cluster or ectopic expression of miR-3 decrease Dmef2 RNA levels, a transcription factor required for muscle formation. Ectopic miR-3 expression also targets the miR-310 locus encoding 7 micro RNAs, four of which target the 3'UTR of Dmef2. The convergence of these miRNA regulatory pathways points to a previously unappreciated complexity in gene regulation with clear implications for development and disease. AttP site insertion into the 8-miR locus as well now enables us to analyze each micro RNA in the cluster. In our efforts to gain insight into the molecular basis of RNAi-induced gene silencing, we identified a novel mechanism in Drosophila that appeared to involve an RNA-dependent RNA polymerase (RdRP) activity in RNA target degradation. siRNAs, produced by the Dicer RNase III-related enzymes in response to the trigger dsRNA, were shown to act as primers to convert the target mRNA into new dsRNA which was then degraded again by Dicers in a cycle of amplification and degradation. This was one of the first biochemical results that could partially explain the potency of the silencing mechanism since very few molecules of dsRNA were able to inactivate hundreds of target mRNA molecules. RdRP is a highly conserved component in RNAi in C. elegans and lower eukaryotes and plays a role in heterochromatin maintenance as well. We identified the the Drosophila RdRP protein as elongator subunit 1, D-elp1, a highly conserved noncanonical RdRP present from S. pombe to humans. D-elp1 is involved in RNAi and transposon suppression and interacts with other key components of the RNAi machinery. A manuscript describing this important finding was published recently (Lipardi and Paterson, PNAS 2009). Importantly,a mutation in the human homologue of D-elp1 produces a truncated protein correlated with the neurological disease Familial Dysautonomia (FD)that affects predominately the Ashkenazi Jewish population. A fly model of the mutation is being generated using a targeted AttP site in the gene. We intend to study the FD phenotype and identify the RdRP active site in D-elp1 as well as define domains essential for its role in RNAi.

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。较早的研究使用注射Nautilus dsRNA诱导RNAi诱导基因沉默表明Nautilus功能的丧失导致严重的胚胎肌肉损失或破坏。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。所有这些表型均由独立的转基因系中的nautilus cDNA营救。 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中通常表达的中胚层细胞的标志物。 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，我们使用Solexa 1G基因组分析仪对突变体和野生型胚胎进行了转录组分析，这是一种所谓的深层序列方法。在确定肌肉创始人和融合有效的成肌细胞群体，调节细胞融合以及确定肌肉身份时，在确定肌肉创始人和融合的肌肉创始人和融合中涉及的基因在nautilus null中可以降低。 NULL中涉及肌管定位的基因的表达模式也会改变。相比之下，某些代表肌肉结构蛋白，肌动蛋白结合蛋白，离子通道，激发诱导偶联成分，钙结合蛋白和突触囊泡运动的基因被错误调节，并且在Nautililus Null null Embryo中以较高的水平表达。更多的是2000个基因在突变体中不受影响。转录组分析中明显的趋势已经确定了在零中受到负面影响的基因组，这与它们在肌发生中的作用一致。这些基因可能是nautilus调节的直接靶标，这将由Chip-Seq确定。 由于Nautilus仅在胚胎中仅0.1％的细胞中表达，因此必须使用严格的芯片条件来识别靶基因。 为了捕获与Nautilus相互作用的基因序列，我们已经生成了一条具有最高亲和力标签的飞行线，已知连接到Engodenous Nautilus Gene的羧基末端，这是一种肽序列，一种可以由来自靶向载体表达的大肠杆菌生物素酶生物素化的肽序列。芯片中生物素 - 阿维丁捕获的选择性正在使用已知的Nautilus靶基因进行评估，下面讨论的8-MIR基因座。一旦建立了适当的条件，我们将执行CHIP-SEQ（Solexa 1G基因组分析仪）以识别Nautilus靶DNA序列。此外，基因靶向的最新进展使我们能够在Nautilus基因中引入ATTP位点，以研究涉及启动子功能，Nautilus TF活性，miRNA结合和增强子功能的重要DNA序列。 小型21bp RNA被称为微RNA（miRNA）在发育和疾病中的基因调节中起关键作用。我们最近确定了两个miRNA，这些miRNA调节了Nautilus在胚胎和成人中的转录后表达。 miRNA结合位点在果蝇多种种类的Nautilus基因的3'UTR中保守。单独的Nautilus 3'UTR可以根据S2细胞中这些miRNA的异位表达来调节报告基因的表达。 Nautilus null中miRNA表达的特征表明，8-MIR基因座（aka）（一个编码9个microRNA的基因组区域）（309-locus）调节了4000个基因的转录后表达，并且在309-Locus发起人中通过两个E-boxes的Nautilus直接控制。基因座中的miR3微调在负反馈回路中的胚胎中表达。 8-MIR基因座的丧失会影响miR-1和miR-184水平，分别用于肌发生和卵子的必需微RNA。 8-MIR簇的缺失或miR-3异位表达降低DMEF2 RNA水平，DMEF2 RNA水平是肌肉形成所需的转录因子。异位miR-3表达还靶向编码7个微RNA的miR-310基因座，其中四个靶向DMEF2的3'UTR。 这些miRNA调节途径的收敛表明基因调节中先前未批准的复杂性，对发育和疾病有明显的影响。现在，ATTP位点插入8-MIR基因座现在使我们能够分析群集中的每个微RNA。 为了深入了解RNAI诱导的基因沉默的分子基础，我们确定了果蝇中的一种新机制，该机制似乎涉及RNA靶标降解中RNA依赖性RNA聚合酶（RDRP）活性。由DICER RNase III相关酶响应触发dsRNA产生的siRNA被证明是将靶mRNA转化为新的dsRNA的引物，然后在扩增和退化的循环中，dicers再次将其转化为新的dsRNA。这是可以部分解释沉默机制的效力的第一个生化结果之一，因为很少有dsRNA分子能够使数百个靶mRNA分子失活。 RDRP是秀丽隐杆线虫和较低真核生物的RNAi中高度保守的成分，并且在异染色质维持中也起作用。我们将果蝇RDRP蛋白鉴定为伸长量亚基1，D-ELP1，这是一种从S. pombe到人类的高度保守的非规范性RDRP。 D-ELP1参与RNAi和转座子抑制，并与RNAi机械的其他关键组件相互作用。 一个描述这一重要发现的手稿最近发表（Lipardi and Paterson，PNAS，2009年）。重要的是，D-ELP1人类同源物的突变产生与神经系统疾病家族性障碍（FD）相关的截断蛋白，主要影响Ashkenazi犹太人人群。使用基因中的靶向ATTP位点生成突变的苍蝇模型。我们打算研究FD表型并确定D-ELP1中的RDRP活性位点，并定义对其在RNAi中作用至关重要的域。