In vivoregulation of single copy and amplified N-myc in human neuroblastoma cells

Abstract

Amplification with subsequent overexpression of N-myc is a recurrent genomic alteration of neuroblastoma cells. DMS-in-vivo-footprinting of the basal promoter of the human N-myc gene (positions −221 to +21) revealed three changes in promoter architecture that are clustered in a 50 bp region and included (1) protein binding to two overlapping E2F-sites, (2) extreme hypersensitivity of guanine −159, and (3) a short stretch of single stranded DNA with a single protected guanine. While in transient assays the basal promoter activated gene expression in all cell lines analysed, the changes at the endogenous promoter were restricted to neuroblastoma cells with strong N-myc expression. The hypersensitivity of guanine −159 could result from protein binding to a flanking, evolutionary conserved 5′-CCTCCC-3′-element, referred to as CT-box, that was bound in vitro in a Zn²⁺-requiring manner by a protein from nuclear extracts of neuroblastoma cells. Four copies of the CT-box in front of an N-myc minimal promoter (−60 to +18) activated expression of a reporter gene in transient transfections whereas four copies of the mutant element did not. Our data add N-myc to a growing list of mammalian genes with CT-boxes that bind proteins in a Zn²⁺-dependent manner. Moreover, the data suggest that a common mechanism controls N-myc expression in neuroblastomas irrespective of N-myc copy number, and that in cell lines with amplification all gene copies contribute to N-myc expression.