Mechanisms of regulation of the human N-myc oncogene

The expression of oncogenes and tumor suppressor genes must be precisely regulated during normal growth and development. Inappropriate expression of these genes can occur through multiple mechanisms. The myc oncogenes, c-, L-, and N-myc, are differentially expressed during normal development, and deregulated expression occurs frequently in specific human malignancies. The N-myc gene is amplified and overexpressed in 30% of pediatric neuroblastomas. N-myc amplification defines a subset of patients with advanced disease and poor prognosis. C-myc expression is regulated at multiple levels, including transcription initiation, transcription attenuation, mRNA degradation, and protein degradation. Deregulation at each level has been documented in human malignancies. The mechanisms controlling N-myc expression are undefined. Lack of significant homology between the human c- and N-myc genes in putative regulatory regions and a difference in their tissue distributions imply that mechanisms of N-myc regulation may diverge from that of c-myc. To investigate the regulation of the human N-myc gene and to determine which mechanisms are disrupted in neuroblastoma, we investigated two features of N-myc expression: N-myc negative autoregulation, and tissue-specific N-myc expression. We defined the region of the N-myc promoter required for expression in human cell lines. We asked whether N-myc autoregulation is intact in neuroblastoma. We found that this feedback loop is disabled in amplified neuroblastoma but intact in single-copy lines, which produce insufficient N-Myc protein to activate the downstream autoregulatory effector(s). We localized the region mediating autoregulation to within 233 nucleotides upstream from the N-myc transcription initiation sites. Our findings imply that myc autoregulation may occur through interaction of Myc with components of the preinitiation complex. We asked by what mechanism(s) the restricted pattern of N-myc expression is regulated. We found that a major component of regulation is post-transcriptional. We localized a 116 base pair region within the first intron sufficient to mediate tissue-specific expression. This element can function in a cell type-specific manner independently of its native position in the N-myc gene, and appears to act by destabilizing pre-mRNA. The presence of a tissue-specific RNA instability determinant in the first intron introduces a novel component of regulation of the human N-myc gene.