An integrated database of genes responsive to the Myc oncogenic transcription factor: identification of direct genomic targets

Abstract

We report a database of genes responsive to the Myc oncogenic transcription factor. The database Myc Target Gene prioritizes candidate target genes according to experimental evidence and clusters responsive genes into functional groups. We coupled the prioritization of target genes with phylogenetic sequence comparisons to predict c-Myc target binding sites, which are in turn validated by chromatin immunoprecipitation assays. This database is essential for the understanding of the genetic regulatory networks underlying the genesis of cancers.

Figure 1

Expression of MYC and direct Myc target genes in treated (+ tet) and untreated (- tet) P493 cells. (a) Left panel reflects Myc protein expression by western blot analysis. Alpha-tubulin is shown as a loading control. (b) Real-time RT-PCR using SYBR Green to detect mRNA levels of indicated genes in both treated (white bars) and untreated (black bars) P493 cells. 18S RNA was used as an internal standard. MYC and NPM1 are shown as positive controls.

Figure 2

Regulograms of Myc targets analyzed by genomic sequence comparison and ChIP. Human and mouse sequences are aligned in each regulogram which depicts in colored regions the sequences with greater than 50% identity. Within these regions, the percentage of sequence identity is represented on the top line and the number of conserved transcription factor binding site 'hits' is represented by the bottom line. The genomic region analyzed by ChIP is mapped below each regulogram. Red boxes represent exons, with the first box always being exon 1. Vertical bars with asterisks indicate locations of conserved canonical E-boxes (5'-CACGTG-3'). Blue bars under each map represent fragments analyzed by ChIP. Ex, exon.

Figure 3

Chromatin immunoprecipitation of Myc targets in P493 cells. Each graph represents real-time PCR amplification of the A and B region of each gene using anti-Myc, anti-HGF and no antibody precipitated chromatin as template. Chromatin was precipitated from P493 cells that were either untreated or treated for 72 hours with tet. Bars represent the percentage of total input DNA for each ChIP sample.

Figure 4

Regulogram and ChIP of CDC25A. (a) This represents the regulogram and genomic map for CDC25A. Exons 1 to 3 are depicted by the three red bars and the arrow indicates the transcriptional start site. All canonical and non-canonical Myc-binding sites are mapped onto this region. The single canonical site is illustrated in red and the conserved non-canonical 5'-CATGTG-3' site in intron 1 is indicated with an asterisks. Blue bars under map represent fragments analyzed by ChIP. (b) A graph of ChIP results from the four genomic regions indicated on the map is shown.