Direct recruitment of N-myc to target gene promoters

Mol Carcinog. 2000 Oct;29(2):76-86.

Abstract

The N-myc gene is amplified in 20-25% of human neuroblastomas, and this amplification serves as a poor prognostic factor. However, few genes have been determined to be direct targets of N-myc. Our current studies focused on identifying N-myc target genes, especially those affected in cells such as neuroblastomas that have high levels of N-myc protein. To pursue this goal, we performed differential expression screens with cell-culture systems containing high versus low levels of N-myc. The design of our experiments was such that we should identify genes both upregulated and downregulated by N-myc. Accordingly, we identified 22 genes upregulated by N-myc and one gene downregulated by N-myc. However, only five of these genes responded to increased N-myc levels in more than one system. Further analysis of the regulation of these genes required determining whether they were direct or indirect targets of N-myc. Therefore, we used a formaldehyde crosslinking and immunoprecipitation procedure to determine whether N-myc was bound to the promoters of these putative target genes in living cells. We found that low levels of N-myc were bound to the promoters of the telomerase and prothymosin genes in neuroblastoma cells having low amounts of N-myc but that the amounts of N-myc bound to these promoters greatly increased with overexpression of N-myc. However, the amount of max bound to the promoters was high before and after induction of N-myc. Therefore, our studies suggest that N-myc competes with other max partners for binding to target promoters. Our use of the chromatin immunoprecipitation assay suggests a molecular explanation for the consequences of amplification of the N-myc gene in neuroblastomas.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Line
  • Fibroblasts / metabolism
  • Fibroblasts / physiology
  • Gene Expression Profiling
  • Gene Expression Regulation, Neoplastic
  • Genes, myc
  • Humans
  • Neuroblastoma / genetics
  • Neuroblastoma / metabolism
  • Oligonucleotide Array Sequence Analysis
  • Promoter Regions, Genetic / genetics
  • Promoter Regions, Genetic / physiology*
  • Proto-Oncogene Proteins c-myc / genetics
  • Proto-Oncogene Proteins c-myc / metabolism
  • Proto-Oncogene Proteins c-myc / physiology*
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Skin / cytology
  • Transcriptional Activation / physiology*

Substances

  • Proto-Oncogene Proteins c-myc
  • RNA, Messenger