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.
| Kim, Marianne K H; Carroll, William L (2004) Autoregulation of the N-myc gene is operative in neuroblastoma and involves histone deacetylase 2. Cancer 101:2106-15 |
| Raetz, Elizabeth A; Kim, Marianne K H; Moos, Philip et al. (2003) Identification of genes that are regulated transcriptionally by Myc in childhood tumors. Cancer 98:841-53 |
