| The Myc family of oncogenes is involved in the genesis of many human cancers and amplification of one member, N-myc, is associated with a poor prognosis in the common childhood tumor neuroblastoma. Our laboratory has focused on post-transcriptional regulation of the N-myc gene through a unique region identified in intron 1, the Tissue Specific Element (TSE), as well as an autoregulatory pathway, whereby N-myc is regulated at the transcriptional level in a negative feedback loop. The first part of these studies describes preliminary data related to post-transcriptional regulation of the N-myc gene through the TSE. Mutation and deletion constructs of the TSE were made to better understand the mechanism of TSE action and to isolate TSE binding proteins. The second part of the studies was focused on elucidating N-myc autoregulatory mechanism. Previous observations indicated that N-myc autoregulation was disrupted in amplified neuroblastoma cell lines, but intact in single copy cell lines. However, in these studies the autoregulatory circuit is proved to be operative even in amplified cell lines. Based on the literature of c-myc regulation and our previous cDNA microarray data, several candidate genes—Mxi1 (Mad2), c-myc promoter binding protein (MBP-1), a c-Myc-interacting zinc finger protein (Miz), and HDAC2 (historic deacetylase 2) were evaluated for their involvement in N-myc autoregulation. In summary, Mxi1 showed consistently a modest effect in down-regulating the N- myc promoter in transient reporter assays. Expression of c-myc, Mxi1, and mHDAC2 genes resulted in a 3–4 fold decrease in endogenous N-myc level. Mxi1 and HDAC2 were up-regulated by N-Myc in a myc-inducible cell line and N-myc expressing cell lines. In addition, the down-regulation of the N-myc promoter was relieved in the presence of trichostatin A. Furthermore, increased association of HDAC2 with the N-myc promoter by chromatin immunoprecipitation was observed upon down-regulation of endogenous N-myc. In conclusion, these studies show that the autoregulatory circuit is intact in both amplified and single copy neuroblastoma cell lines, higher levels of trans-acting factors are needed to down-regulate N-myc expression in amplified cell lines, and HDAC2 is involved in N-myc autoregulatory pathway. |
