Loss of transcriptional attenuation in N-myc is associated with progression towards a more malignant phenotype

The modulation of RNA polymerase II processivity through the untranslated N-myc first exon represents an important mechanism governing N-myc mRNA levels during normal development. In this study, we employed the rat embryo fibroblast (REP) cooperation assay as a functional means to (i) map N-myc first exon sequences involved in the regulation of N-myc gene expression and specifically their contribution to controlling a block to transcriptional elongation and (ii) determine whether this transcriptional control mechanism plays a role in governing the oncogenic activity of N-myc. Transfection of N-myc expression constructs harboring various deletions within the untranslated first exon revealed that a region encoding a potential stem-loop structure followed by a thymine stretch (stem-loop/T region) was required for efficient transcriptional attenuation. The increase in transcriptional read-through associated with all deletions involving the stem-loop/T region also resulted in a more aggressive malignant phenotype as evidenced by a significant enhancement in the subcloning efficiency of N-myc/ras transformed foci. Most significantly, when cell lines generated from overexpression of the intact N-myc expression construct were selected for anchorage-independent growth, a strong block to transcriptional elongation was completely eliminated in all cases examined. Since the subcloning efficiency of transformed foci and the capacity of permanently established cell lines for anchorage-independent growth are direct correlates of more advanced stages of malignant transformation, our findings suggest that loss of transcriptional attenuation represents an important genetic event in the progression of N-myc-induced cellular transformation.