POST TRANSCRIPTIONAL CONTROL OF THE HUMAN N MYC GENE

描述：（改编自研究者摘要）N-myc癌基因 在器官形成和不适当表达中起着至关重要的作用，通常是由于 在人类肿瘤中观察到基因扩增。N-myc扩增是 儿童常见肿瘤的一个最不利预后特征 神经母细胞瘤携带N-myc扩增拷贝的肿瘤患者， 即使采用骨髓移植等积极治疗， 移植与此形成鲜明对比的是， 治愈率现在接近的其他形式癌症儿童的结果 75- 90%我们的实验室正在研究人类N-myc基因的调控， 开发新的沉默治疗方法的长期目标 在扩增的肿瘤中表达。我们已经证明，5 'N-myc启动子序列 在迄今为止检查的所有细胞类型中， 是否检测到内源性来源的N-myc转录物。但我们 在第一个内含子内定义了一个116个碱基对的区域， 仅在具有内源性N-myc基因活性的细胞中“特异性”表达。 内含子组织特异性元件（TSE）独立发挥调节细胞增殖的作用。 异源启动子（SV 40），其模式与天然基因相同 这表明它在指导N-myc的表达中起着重要作用 基因我们已经证明，这一过程涉及转录后机制 我们的数据表明，TSE的功能是使N-myc前体mRNA不稳定， 非神经母细胞瘤细胞类型。我们现在已经确定， 其特征在于TSE-蛋白复合物的迁移率在N-myc 表达和不表达的细胞系表明复合物的形成可能 调节N-myc mRNA水平。本建议旨在进一步了解 TSE发挥作用的机制是将N-myc表达靶向特定的 细胞类型。具体目标是1.定位更多序列， TSE内负责N-myc调控的核苷酸， 报道基因测定，2.直接评估TSE在调节N-myc中的作用 前mRNA半衰期，3.利用RNA-蛋白质条带表征反式作用蛋白 位移测定、UV交联和RNA足迹法，以及4.分离蛋白质 介导TSE调节的N-myc RNA水平。一旦TSE蛋白的成分 复合物被鉴定，复合物形成的抑制剂可以被开发， 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基因模式的深入了解 调节，它在调节时间和空间方面的作用 N-myc基因在个体发育过程中的表达以及可能的鉴定 其他基因受此途径调控。

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.