Molecular Studies of Eukaryotic Gene Regulation

真核基因调控的分子研究

• 批准号: 6761448
• 负责人: B M 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/6761448
• 关键词: Caenorhabditis elegans; Drosophilidae; cell cycle; cell growth regulation; cyclin dependent kinase; developmental genetics; embryology; enzyme inhibitors; eukaryote; gene induction /repression; genetic promoter element; genetic regulation; myogenesis; phosphoproteins; 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) provides an excellent model system 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. 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 recently determined that the MyoD 15 amino acid domain also 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 prufied 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 yet the protein is nuclear. This MyoD interaction with cell cycle related proteins may be a more general mechanism with regard to other tissue-specific bHLH transcriptions factors and this is being examined with NeuroD2, a neurogenic bHLH protein. 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. 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 the inducer gene alone that does not produce dsRNA are under study to confrim that the phenotype observed is due to the induction of dsRNA for the target gene. Unfortunately, similar treatment of mammalian cells with dsRNA larger that 30 nucleotides induces cell death via apoptosis. 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 results explains the underlying 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 Dicer and expressed the full-length cDNA in baculovirus to produce active enzyme. We have identified other components of the RNAi system in Drosophila by gene comparison using genetic studies from C.elegans, Arabidopsis, and neurospora. Dicer as well as these associated proteins are being used as bait to characterize protein complexes capable of performing various steps in RNAi. (Project 2) Determination of the myoblast in the mesoderm involves the activation of MyoD and MyoD responsive downstream target genes. We have been studying the activation of the single MyoD gene, nautilus, in Drosophila and have determined that the nautilus promoter is activated predominately by DMEF2, a Drosophila SRF homolog, and twist, a major determinant of the mesoderm. We can induce Schneider cells to activate a partial myogenic program by expressing daughterless in these cells. This myogenic conversion is potentiated by the co-expression of DMEF2 and nautilus. The cells exit the cell cycle, become multinucleated and express muscle-specific myosin similar to embryonic muscle. Myogenic conversion of Schneider cells by daughterless is dependent upon the endogenous expression of very low levels of nautilus and DMEF2, two markers that establish Schneider cells are of mesodermal origin. Inhibition of the endogenous gene function for nautilus and DMEF2 by RNA interference established these gene products were essential for myogenic conversion of Schneider cells by daughterless. Quantitative RT-PCR has shown that Schneider cells express 100-1000 fold less daughterless than nautilus. Raising the levels of daughterless protein by ectopic expression allow sufficient levels of the nautilus/daughterless heterodimer to form to activate the myogenic program. This work defined conditions for the application of RNAi to cultured Drosophila cells and established a myogenic system in which to analyze nautilus-responsive genes by micro array analysis. We have established a daughterless inducible KC cell line using the metallothionine promoter system which converts to a muscle phenotype upon induction. This system is being used to identify nautilus target genes by microarray. RNA interference is being used to explore the role of other factors in the myogenic process as well. We hope to analyze genes that are differentially expressed between the non-myogenic and myogenic state to identify early target genes for myogenic conversion. We have recently begun to culture primary embryonic Drosophila muscle and hope to use RNAi to explore the role of genes identified in the microarray studies on the differentiation of the primary cultures.

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一种出色的模型系统，可以研究高等生物体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。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。我们最近确定MYOD 15氨基酸结构域还与其他主要的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。所有体外激酶测定均使用杆状病毒D1/CDK4，细胞周期蛋白D1/CDK6和细胞周期蛋白E/CDK2进行的所有体外激酶测定。 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与其在分裂成肌细胞中的靶基因无关，但该蛋白质是核的。对于其他组织特异性BHLH转录因子，这种与细胞周期相关蛋白的MYOD相互作用可能是一种更通用的机制，并且正在使用神经源性BHLH蛋白进行NeuroD2进行检查。 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基因功能的一般效用是果蝇中基因功能的一般效用，并且是快速分析的选择方法。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。仅研究未产生dsRNA的诱导剂基因的对照转基因果蝇正在研究中，即观察到观察到的表型是由于靶基因诱导dsRNA所致。不幸的是，用较大的dsRNA对哺乳动物细胞的类似处理，30个核苷酸通过细胞凋亡诱导细胞死亡。 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。结果解释了RNAi和转录基因沉默背后的潜在机制。我们正在尝试识别果蝇的RDRP。我们还克隆了果蝇dicer，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，拟南芥和Neurospora的遗传研究来比较果蝇中RNAi系统的其他成分。 DICER以及这些相关的蛋白质被用作诱饵，以表征能够在RNAi中执行各种步骤的蛋白质复合物。 （项目2）中胚层中的成肌细胞的确定涉及Myod和Myod响应式下游靶基因的激活。我们一直在研究果蝇中单个myod基因nautilus的激活，并确定nautilus启动子由果蝇SRF同源物DMEF2主要激活，并且是中胚层的主要决定因素。我们可以通过在这些细胞中表达女儿来诱导施耐德细胞来激活部分肌源程序。 DMEF2和Nautilus的共表达增强了这种肌源性转化。细胞退出细胞周期，变得多核并表达类似于胚胎肌肉的肌肉特异性肌球蛋白。施耐德细胞的肌源性转化是由无生成的肌源性转化，取决于非常低水平的nautilus和dmef2的内源性表达，这是两个建立施耐德细胞的标记是中胚层的。通过RNA干扰，对Nautilus和DMEF2的内源基因功能的抑制作用确定了这些基因产物对于通过无子女对Schneider细胞的肌源性转化至关重要。定量RT-PCR表明，施耐德细胞的表达100-1000倍的女儿比Nautilus少。通过异位表达提高无子蛋白的水平，使足够水平的Nautilus/无女无二聚体形成以激活肌源性程序。这项工作定义了RNAi在培养的果蝇细胞中应用的条件，并建立了一个肌原系统，在该系统中，通过微阵列分析来分析鹦鹉螺响应基因。我们使用金属噻硫代启动子系统建立了无女诱导的KC细胞系，该系统在诱导后转化为肌肉表型。该系统被用于通过微阵列识别Nautilus靶基因。 RNA干扰也用于探索其他因素在肌源过程中的作用。我们希望分析非肌源性和肌源性状态之间差异表达的基因，以鉴定早期靶基因的肌源性转化率。我们最近开始培养原发性胚胎果蝇肌肉，并希望使用RNAi探索在微阵列研究中鉴定的基因在分化原发性培养物中的作用。