POST TRANSCRIPTIONAL CONTROL OF THE HUMAN N MYC GENE

描述：(改编自研究人员摘要)N-myc癌基因 在器官发生和不适当的表达中起着关键作用，通常是由于 基因扩增，是在人类肿瘤中观察到的。N-myc扩增是 常见儿童肿瘤的单一最不利预后特征 神经母细胞瘤。肿瘤中含有N-myc扩增拷贝的患者 即使采用骨髓等积极治疗，预后也很差 移植。这与中国经济的显著改善形成鲜明对比。 治愈率接近的其他形式癌症儿童的预后 75%-90%。我们实验室正在研究人类N-myc基因的调控。 开发治疗沉默的新方法的长期目标 在扩增的肿瘤中表达。我们已经证明了5‘N-myc启动子序列 迄今为止检查的所有细胞类型中的直接混杂表达，而不考虑 是否检测到内源性N-myc转录本。然而，我们有 在第一个内含子内定义了一个116个碱基对的区域，该区域指导着组织 仅在具有内源性N-myc基因活性的细胞中表达。 内含子组织特异性元件(TSE)独立发挥作用，调节 异源启动子(SV40)，其模式与天然基因相同 提示IS在指导N-myc基因表达中起重要作用。 吉恩。我们已经证明，这个过程涉及转录后机制。 我们的数据表明，TSE具有破坏N-myc前-mRNA在脑内的稳定作用 非神经母细胞瘤细胞类型。我们现在已经确定并初步确定了 鉴定了TSE-蛋白质复合体，其迁移率在N-myc之间不同 表达和不表达的细胞株表明复合体的形成可能 调节N-myc基因表达水平。这项建议旨在进一步了解 TSE将N-myc表达靶向于特定基因的机制 单元类型。具体目标是1.定位更多的序列和 TSE内负责N-myc调控的核苷酸使用功能性 记者分析，2.直接评估东京证交所在调控N-myc中的作用 前信使核糖核酸半衰期，3.用RNA-蛋白条带鉴定反式作用蛋白 移位分析、UV交联和RNA足迹，以及4.分离蛋白质 调节TSE调节的N-myc RNA水平。一旦TSE-蛋白质的成分 确定了络合物，络合物形成的抑制剂可以开发成 在扩增的肿瘤中下调表达。此外，更好地了解 N-myc TSE途径将提供对一种新的基因模式的洞察 调节，它在调节时间和空间上所起的作用 N-myc基因在个体发育过程中的表达及其可能的鉴定 受该途径调控的其他基因。

DESCRIPTION: (Adapted from the investigator's abstract) The N-myc oncogene plays a crucial role in organogenesis and inappropriate expression, usually due to gene amplification, is observed in human tumors. N-myc amplification is the single most adverse prognostic feature in the common childhood tumor neuroblastoma. Patients whose tumors harbor amplified copies of N-myc have a dismal prognosis even with aggressive therapy such as bone marrow transplantation. This is in sharp contrast to the dramatic improvement in outcome for children with other forms of cancer where cure rates now approach 75-90%. Our laboratory is studying the regulation of the human N-myc gene with the long-term goal of developing novel therapeutic approaches to silence expression in amplified tumors. We have shown that 5'N-myc promoter sequences direct promiscuous expression in all cell types examined to date, regardless of whether endogenously derived N-myc transcripts are detected. However, we have defined a 116 base pair region within the first intron that directs "tissue specific" expression only in cells with activity of the endogeneous N-myc gene. The intron tissue specific element (TSE) functioned independently to regulate a heterologous promoter (SV40) in a pattern that was identical to the native gene suggesting that is plays an important role in directing expression of the N-myc gene. We have shown that this process involves a post-transcriptional mechanism and our data indicates that the TSE functions to destabilize N-myc pre-mRNA in non-neuroblastoma cell types. We have now identified and initially characterized a TSE-protein complex whose mobility differs between N-myc expressing and non-expressing cell lines suggesting that complex formation may modulate N-myc mRNA levels. This proposal seeks to understand further the mechanism by which the TSE functions to target N-myc expression to specific cell types. The specific aims are to 1. Localize further sequences and nucleotides within the TSE responsible for N-myc regulation using functional reporter assays, 2. Assess directly the role of the TSE in regulating N-myc pre-mRNA half-life, 3. Characterize trans-acting proteins by RNA-protein band shift assays, UV cross-linking and RNA-footprinting, and 4. Isolate proteins that mediate TSE-regulated N-myc RNA levels. Once the components of TSE-protein complexes are identified, inhibitors of complex formation can be developed to downregulate expression in amplified tumors. Moreover a better understanding of the N-myc TSE pathway will provide insight into a novel mode of gene regulation, the role that it plays in regulating the temporal and spatial expression of the N-myc gene during ontogeny and the possible identification of other genes regulated by this pathway.