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.

Direct examination of histone acetylation on Myc target genes using chromatin immunoprecipitation.

Overexpression of c-Myc can lead to altered transcriptional regulation of cellular genes and to neoplastic transformation. Although DNA binding is clearly required, the mechanism by which recruitment of c-Myc to target promoters results in transcriptional activation is highly debated. Much of this controversy comes from the difficulty in clearly defining a true Myc target gene. We have previously determined that cad is a bona fide Myc target gene and thus now use the cad promoter as a model to study Myc function. Others have shown that Myc can interact indirectly with histone acetylases and have suggested that Myc mediates transcriptional activation by causing an increase in the levels of acetylated histones on target promoters. To directly test this model, we employed a chromatin immunoprecipitation assay to examine the levels of acetylated histones on the cad promoter. Although Myc was bound to the cad promoter in S phase but not in G(0) phase, we found high levels of acetylated histones on the promoter in both stages. We also examined acetylated histones on the cad promoter before and after differentiation of U937 cells. Although the levels of c-Myc bound to the cad promoter were greatly reduced after differentiation, we saw high levels of acetylated histones on the cad promoter both before and after differentiation. Finally, we found that a 30-fold change in binding of N-Myc to the telomerase promoter did not result in a concomitant change in histone acetylation. Thus, recruitment of a Myc family member to a target promoter does not necessarily influence the amount of acetylated histones at that promoter. Further investigations are in progress to define the role of Myc in transcriptional activation.