Direct recruitment of N-myc to target gene promoters.
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.
Mac
S M
SM
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
D'Cunha
C A
CA
Farnham
P J
PJ
D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism / Q000502:physiology; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D016259:Genes, myc; D006801:Humans; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000166:cytology; D015533:Transcriptional Activation / Q000502:physiology*
D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger