Identification and validation of MYCN/c-MYC target genes in neuroblastoma cell lines. (a) Repression of endogenous c-MYC by targeted expression of a MYCN transgene in SH-EPMYCNcells defines MYCN/c-MYC-regulated genes. MYCN and c-MYC protein levels were monitored in a time series after removing tetracycline in exponentially growing SH-EPMYCNcells that stably express a tetracycline-regulated MYCN transgene. Mean and standard deviation of the relative mRNA levels of MYC, DKC1 and PTMA are given from two time series experiments as measured by a customized neuroblastoma oligo microarray. (b) Hierarchical clustering of MYCN- and c-MYC binding to 140 target gene promoters as measured by ChIP-chip in 6 neuroblastoma cell lines. ChIP-chip results of 140 MYCN/c-MYC target genes from 5 neuroblastoma cell lines that preferentially express either high levels of MYCN (SH-EPMYCN, IMR5/75 (approximately 75 copies of MYCN) and Kelly (approximately 100-120 copies of MYCN)) or c-MYC (SJ-NB12 and SY5Y). Additionally, as an intermediate type, parental SH-EP cells were analyzed. SH-EP cells preferentially express c-MYC, but also low levels of MYCN. ChIP-chip experiments were performed with a monoclonal antibody against human MYCN and a polyclonal antibody against human c-MYC for each neuroblastoma cell line. A cut-off for positive binding was set for both transcription factors to >4-fold enrichment for one and >2-fold enrichment of at least one of the two neighboring probes. MYCN/c-MYC-binding is color-coded as follows: blue, c-MYC binding; red, MYCN/c-MYC binding; dark red, MYCN binding; light yellow, lack of MYCN/c-MYC binding. Hierarchical clustering was used to group neuroblastoma cell lines according to their MYCN/c-MYC-binding pattern. Differentiation between MYCN and c-MYC-binding was mainly achieved through the monoclonal MYCN antibody. The polyclonal antibody against c-MYC also gave positive binding signals for a large set of analyzed target gene promoters in neuroblastoma cell lines with high MYCN that lack c-MYC expression (SH-EPMYCN, IMR5/75 and Kelly).