Regulation of myc-family gene expression in development of normal and neoplastic cells

Abstract

Differential developmental expression of myc-family genes (c-, N- and L-myc) is governed by a complex regulatory strategy that is executed at the levels of promoter utilization, transcriptional attenuation (block to transcriptional elongation), and mRNA stability. Through nuclear run-on assays, I have demonstrated that transcriptional attenuation appears to be the principal mechanism by which steady-state N-myc mRNA levels are down-regulated during postnatal brain development. The site of transcriptional attenuation in the N-myc gene was localized to sequences in the 5{dollar}\sp\prime{dollar} UT region (first exon) encoding a potential stem-loop structure followed by a thymine tract.;Employing the myc/ras rat embryo fibroblast (REF) cooperation assay, I have shown that deletion of the first exon/intron region of N-myc, as well as of c- and L-myc, results in a significant increase in the oncogenic potential. Analysis of N-myc/ras-transformed cell lines demonstrated (i) fewer transfected N-myc gene copies and overall higher level of steady-state N-myc mRNA with the first exon/intron deleted N-myc expression construct and (ii) the presence of a significant block to transcriptional elongation in the first exon of the complete N-myc expression construct.;Systematic deletion analysis in the N-myc first exon demonstrated that the region containing a potential stem-loop structure followed by a thymine stretch is required for efficient transcriptional attenuation in transfected N-myc expression constructs. Deletion-induced transcriptional read-through was found to be consistent with a substantial increase in subcloning efficiency. In addition, the soft agar selection of N-myc/ras-transformed cell lines transfected with the intact N-myc expression construct was associated invariably with a complete loss of the block to transcriptional elongation. 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 a key genetic event in the progression, rather than establishment, of N-myc induced tumorigenesis in cell culture transformation assays.