Abstract
N-Myc is a member of the Myc family of transcription factors that have been shown to play a pivotal role in cell proliferation and differentiation. In this report, we have investigated the relationship between N-Myc and the developmental control gene Pax-3. Using transient transfection assays, we show that the Pax-3 promoter is activated by both N-Myc-Max and c-Myc-Max. Moreover, we show that Myc regulation of Pax-3 promoter activity is dependent upon a noncanonical E box site in the 5' promoter region of Pax-3. In addition, we show that ectopic expression of both N-Myc and c-Myc leads to increased expression of Pax-3 mRNA. Furthermore, we show that Pax-3 mRNA expression is cell cycle-regulated and that the 5' promoter region of Pax-3 (bp -1578 to +56) can direct cell cycle-dependent gene expression with kinetics similar to that of the endogenous transcript. Site-directed mutagenesis of the E box site within the Pax-3 promoter significantly altered the pattern of expression through the cell cycle. These results suggest that the Myc family of transcription factors may modulate Pax-3 expression in vivo.
Highlights
N-Myc is a member of the Myc family of transcription factors (c-Myc, N-Myc, L-Myc, B-Myc, and S-Myc) that are characterized by a basic DNA binding domain and dimerization domain composed of a helix-loop-helix and leucine zipper
We found that co-transfection of the Pax-3 promoter construct with either N-Myc or c-Myc led to the induction of Pax-3 promoter activity
When the amounts of expression vectors for N-Myc-Max and c-Myc-Max were increased in a fixed Myc/Max ratio, expression of the Pax-3 promoter was further increased (Fig. 1C), suggesting that members of the Myc family of transcription factors can activate Pax-3 promoter activity in vitro
Summary
N-Myc is a member of the Myc family of transcription factors (c-Myc, N-Myc, L-Myc, B-Myc, and S-Myc) that are characterized by a basic DNA binding domain and dimerization domain composed of a helix-loop-helix and leucine zipper. The Pax-3 E Box Motif Is Bound by Myc and Max—To determine whether N-Myc-Max or c-Myc-Max heterodimers can bind to the E box site from the Pax-3 promoter, whole cell extracts from COS-7 cells transiently transfected with expression vectors containing N-Myc, c-Myc, and Max were incubated with a radiolabeled oligonucleotide containing the Pax-3 E box sequence, and DNA binding was assessed by an electrophoretic mobility shift assay [34].
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