Molecular Studies of Eukaryotic Gene Regulation

真核基因调控的分子研究

• 批准号: 6949796
• 负责人: bruce paterson
• 金额: --
• 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/6949796
• 关键词: Caenorhabditis elegans; Drosophilidae; RNA interference; affinity chromatography; cell cycle; cell growth regulation; cyclin dependent kinase; developmental genetics; embryology; enzyme activity; enzyme inhibitors; eukaryote; gene expression; genetic regulation; muscle proteins; myogenesis; phosphorylation; protein binding; protein protein interaction; retinoblastoma protein; tissue /cell culture; transcription factor

Tissue formation during development involves the determination, controlled proliferation and specific differentiation of cells in the embryo. Misregulation in any phase of this process can lead to failure in the development of the embryo, severe disease or uncontrolled cellular growth. Thus the study of gene regulation during development provides insight into areas important in human disease. Embryonic muscle formation in vertebrates and Drosophila (the fruit fly) provide excellent model systems in which to study the origin of one of the major tissues in higher organisms. The determination, proliferation, and differentiation of muscle cells during development in both vertebrates and invertebrates depend upon the function of the MyoD family of basic helix-loop-helix proteins, the muscle regulatory factors (MRFs). Determination of the first muscle precursor cells involves the activation of the MRFs in early mesoderm while gene expression characteristic of differentiated muscle remains repressed. Terminal differentiation is marked by the withdrawal of the myoblast from the cell cycle just prior to the activation of the muscle-specific genes and both processes involve the MRFs. Furthermore, greater than 90% of the genes expressed in the dividing muscle cell are shut down in a process that involves massive chromatin reorganization while the muscle-specific genes are activated. Cell cycle control during terminal differentiation is thought to involve the MRFs in a pathway that regulates the phosphorylation status of the retinoblastoma protein, Rb. (Project 1) We have recently shown that ectopically expressed MyoD binds directly to the G1 cyclin-dependent kinase cdk4 to inhibit cell growth and the phosphorylation of Rb. The cdk4-MyoD interaction also blocks the trans activation functions of MyoD by disrupting DNA-binding by the MyoD/E-protein heterodimer. Therefore, high levels of nuclear cdk4 block MyoD function in growing myoblasts while the loss of nuclear cdk4 in the absence of growth factors and mitogens allows MyoD to function. We have identified a 15 amino acid domain on MyoD responsible for the interaction with cdk4. Expression of this domain either as a fusion protein with GST or GFP inhibits the kinase activity of cdk4 in vitro and in vivo, blocking its ability to phosphorylate the retinoblastoma protein, Rb. This results in the cessation of cell growth and induces myoblast differentiation in the presence of mitogens. We have a patent application on the inhibitory activity of the 15 amino acid domain of MyoD on cdk4 kinase activity. We have recently made alanine substitutions in all the positions of the 15 amino acid cdk4-binding domain in order to map the critical residues for interaction. Single substitutions have a marginal affect on inhibitory and binding activity of the domain but two simultaneous substitutions reduce cdk4 binding and kinase inhibition for the various binding domain derivatives. The binding parameters are being determined uisng the BiaCore and the imobilized 15 amino acid derivatives. We have also determined that the MyoD 15 amino acid domain binds to the other major G1 cyclin-dependent kinases, cdk6 and cdk2. cdk6 behaves like cdk4 during muscle differentiation in that cdk6 leaves the nucleus when mitogen levels are reduced but can be induced to re-enter myotube nuclei with the expression of a stable cyclin D1 in the cells. cdk6 phosphorylation of Rb is also inhibited by the MyoD binding domain. However, although cdk2 binds to the same 15 amino acid domain, phosphorylation of histone in vitro is not inhibited. All the in vitro kinase assays are performed using baculovirus produced cyclin D1/cdk4, cyclin D1/cdk6, and cyclin E/cdk2 purified by Flag-tag affinity chromatography. cdk4/6 kinases are inhibited by p16 and p21 while cdk2 activity is only blocked by p21. We suggest that in the dividing myoblast the G1 cdks can act to hold MyoD activity in check until the cell begins to exit the cell cycle as mitogen levels are lowered. Chromatin immunoprecipitation assays with MyoD antibody indicate MyoD is not associated with its target genes in the dividing myoblast eventhough MyoD is a nuclear protein. Mouse fibroblasts from cdk4 mutant mice are available (S. Rane NIH) that either have no ckd4 or have a cdk4 that does not bind p16, the cdk inhibitor. We would like to use these mutants to check if the MyoD inhibitory domain binds to the same regions as p16 and to see if myogenic conversion is enhanced due to a failure to regulate the MyoD-cdk4 interaction. Myogenic conversion by MyoD may be enhanced in cdk4-/- cells. In Drosophila we have also shown that MyoD (nautilus) expression defines a subset of mesodermal cells that are required to set up the muscle pattern in each hemisegment of the embryo. Ricin toxin ablation of nautilus positive cells, or injection of double stranded nautilus RNA into the embryo (RNA interference or RNA-i) alter normal muscle formation in the embryo and define nautilus as an essential gene for myogenesis in the fly. This study demonstrated the general utility of RNA-i ablation of gene function in Drosophila in the absence of a genetic mutation and is the method of choice for a rapid analysis of gene function. We have developed a Drosophila vector system to induce dsRNA in selective tissues at particular times during development and this is under analysis. Preliminary results using nautilus as a test gene suggest loss of nautilus function is a lethal that results in disruption of the normal muscle pattern, similar to the results obtained by direct injection of the dsRNA. Control transgenic flies with either the inducer gene or the target gene that produces the dsRNA are wild-type, confriming that the phenotype we observe is due to the induction of dsRNA for the target gene. We have recently started to use the newly developed gene targeting method (developed by Dr. Yikang Rong, NCI) to knock out genes involved in Drosophila myogenesis and RNAi(see below).As a first test of the method and to establish procedures we are targeting nautilus expression. In an effort to understand the molecular basis of RNAi in Drosophila we have recently uncovered a novel mechanism we have termed degradtive PCR that appears to involve an RNA-dependent RNA polymerase (RdRP) and the 21-25 nucleotide RNAs produced from the trigger dsRNA, called siRNAs for short interfering RNAs. The short RNAs serve as primers to convert the target RNA into dsRNA which is then degraded by an RNase III-related enzyme, called Dicer, to produce new primers while degrading the target RNA in the process. This result sheds light on the role of the siRNAs in RNAi and may explain the potentcy of the mechanism behind RNAi and post transcriptional gene silencing. We are in the process of trying to identify the RdRP from Drosophila. We have also cloned Drosophila Dicers 1 and 2 and expressed the full-length cDNAs in baculovirus to produce active enzymes. We have identified other components of the RNAi system in Drosophila by gene comparison using genetic studies from C.elegans and this has led to the identification of other RNAse III-related proteins, RNA-binding proteins, Argonaute family members, and members of the nonsense mediated decay proteins or smgs. Using RNAi to knock out these proteins we have established their role in the initial processing of the trigger dsRNA versus targeting. We have established that Dicer-1 participates in the initial processing step as well as siRNA-mediated targeting and suggest Dicer-1 is in the RISC. We have also shown that Dicer-1 can cleave dsRNA processively from the ends in the absence of ATP or any NTPs, contrary to published ideas.

发育过程中的组织形成涉及胚胎中细胞的测定，受控的增殖和特异性分化。在此过程的任何阶段，不正体都会导致胚胎发展，严重疾病或不受控制的细胞生长的失败。因此，开发过程中基因调节的研究提供了对人类疾病重要领域的见解。脊椎动物和果蝇（果蝇）中的胚胎肌肉形成提供了出色的模型系统，可以在其中研究高等生物中主要组织之一的起源。脊椎动物和无脊椎动物在发育过程中肌肉细胞的确定，增殖和分化取决于基本螺旋 - 环螺旋蛋白的MYOD家族的功能，即肌肉调节因子（MRFS）。第一肌肉前体细胞的测定涉及MRF在早期中胚层中的激活，而分化肌肉的基因表达特征仍然受到抑制。末端分化是在肌肉特异性基因激活之前从细胞周期中撤出细胞周期的标志性的，并且两个过程都涉及MRF。此外，在分裂肌肉细胞中表达的90％以上的基因在涉及大规模染色质重组的过程中被关闭，而激活了肌肉特异性基因。终末分化过程中的细胞周期控制被认为涉及MRF中的途径，该途径调节视网膜细胞瘤蛋白的磷酸化状态RB。 （项目1）我们最近表明，异位表达的MYOD直接与G1 Cyclin依赖性激酶CDK4结合，以抑制RB的细胞生长和磷酸化。 CDK4-MYOD相互作用还通过破坏MyOD/E蛋白异二聚体DNA结合来阻止MYOD的反式激活函数。因此，高水平的核CDK4阻止了肌细胞在生长肌细胞中的功能，而在没有生长因子和有丝分裂剂的情况下核CDK4的损失使MyOD可以发挥作用。我们已经在MyoD上确定了一个15个氨基酸结构域，负责与CDK4相互作用。该结构域作为具有GST或GFP的融合蛋白的表达抑制了CDK4在体外和体内的激酶活性，从而阻断了其磷酸化视网膜母细胞瘤蛋白RB的能力。这导致停止细胞生长，并在有丝分裂剂的存在下诱导肌细胞分化。我们对MYOD的15个氨基酸结构域对CDK4激酶活性的抑制活性有专利应用。最近，我们在15个氨基酸CDK4结合结构域的所有位置中进行了丙氨酸取代，以绘制关键残基的相互作用。单个取代对域的抑制性和结合活性具有边际影响，但两个同时取代降低了CDK4结合和对各种结合结构域衍生物的激酶抑制作用。结合参数被确定为双子虫，并取代15个氨基酸衍生物。我们还确定MYOD 15氨基酸结构域与其他主要的G1 Cyclin依赖性激酶CDK6和CDK2结合。 CDK6在肌肉分化过程中的表现类似于CDK4，当CDK6降低有丝分裂原水平时，CDK6留下了核，但可以通过细胞中稳定的细胞周期蛋白D1的表达来诱导重新输入肌管核。 RB的CDK6磷酸化也受到MYOD结合结构域的抑制。但是，尽管CDK2与相同的15个氨基酸结构域结合，但体外组蛋白的磷酸化并未抑制。所有体外激酶分析均使用杆状病毒D1/CDK4，细胞周期蛋白D1/CDK6和细胞周期蛋白E/CDK2通过FLAG-TAG亲和色谱纯化。 CDK4/6激酶被p16和p21抑制，而CDK2活性仅被p21阻断。我们建议，在分裂成肌细胞中，G1 CDK可以用来控制MYOD活性，直到随着丝分裂原水平降低细胞开始退出细胞周期。用MYOD抗体的染色质免疫沉淀测定表明，Myod与其在成肌细胞事件中的靶基因无关，尽管MYOD是核蛋白质。来自CDK4突变小鼠的小鼠成纤维细胞可用（NIH S. rane NIH），该小鼠没有CKD4或具有不结合p16（CDK抑制剂）的CDK4。我们想使用这些突变体检查MYOD抑制域是否与p16相同的区域结合，并查看由于未能调节MYOD-CDK4相互作用而增强了肌源性转化率。在CDK4 - / - 细胞中，Myod的肌原转化可能会增强。 在果蝇中，我们还表明，Myod（Nautilus）表达定义了中胚层细胞的子集，这些细胞是在胚胎的每个半段中建立肌肉模式所需的。 Ricin毒素阳性细胞的消融，或将双束的Nautilus RNA注射到胚胎中（RNA干扰或RNA-I）改变了胚胎中的正常肌肉形成，并将Nautilus定义为蝇中肌生成的必要基因。这项研究表明，在没有遗传突变的情况下，RNA-I基因功能消融在果蝇中的一般效用，是快速分析基因功能的选择方法。我们已经开发了果蝇载体系统，以在发育过程中的特定时间诱导选择性组织中的dsRNA，这正在进行中。使用Nautilus作为测试基因的初步结果表明，Nautilus功能的丧失是一种致命的，导致正常肌肉模式破坏，类似于直接注射DSRNA获得的结果。用诱导剂基因或产生dsRNA的靶基因对照转基因蝇是野生型的，这与我们观察到的表型有关，是由于靶基因诱导dsRNA所致。我们最近开始使用新开发的基因靶向方法（由NCI Yikang Rong博士开发）来淘汰果蝇肌发生和RNAi涉及的基因（见下文）。作为该方法的首次测试，并确定了我们靶向Nautilus表达的程序。 为了了解果蝇中RNAi的分子基础，我们最近发现了一种新型机制，我们称为降解PCR，该机制似乎涉及RNA依赖性RNA聚合酶（RDRP）和21-25个核苷酸RNA，并从触发dsrna中产生了trigger dsrna，称为sirnas sirnas，用于简短的分解。短RNA用作将靶RNA转换为dsRNA的底漆，然后由RNase III相关酶（称为DICER）降解，以产生新的引物，同时在此过程中降低靶RNA。该结果阐明了siRNA在RNAi中的作用，可以解释RNAi和转录基因沉默背后的机制的有效性。我们正在尝试识别果蝇的RDRP。我们还克隆了果蝇1和2的克隆，并在杆状病毒中表达了全长cDNA以产生活性酶。我们已经使用C.Elegans的遗传研究来比较果蝇中RNAi系统的其他组件，这导致了其他RNase III相关蛋白，RNA结合蛋白，Argonaute家族成员以及无义介导的衰减蛋白或SMG的成员的鉴定。使用RNAi敲除这些蛋白质，我们已经确定了它们在触发dsRNA的初始处理中与靶向的初始处理中的作用。我们已经确定DICER-1参与初始处理步骤以及siRNA介导的靶向，并建议DICER-1在RISC中。我们还表明，在没有ATP或任何NTP的情况下，DICER-1可以从末端进行裂解，与已发表的思想相反。