Characterization of the c-MYC-regulated transcriptome by SAGE: identification and analysis of c-MYC target genes

Abstract

To identify target genes of the oncogenic transcription factor c-MYC, serial analysis of gene expression (SAGE) was performed after adenoviral expression of c-MYC in primary human umbilical vein endothelial cells: 216 different SAGE tags, corresponding to unique mRNAs, were induced, whereas 260 tags were repressed after c-MYC expression (P < 0.05). The induction of 53 genes was confirmed by using microarray analysis and quantitative real-time PCR: among these genes was MetAP2/p67, which encodes an activator of translational initiation and represents a validated target for inhibition of neovascularization. Furthermore, c-MYC induced the cell cycle regulatory genes CDC2-L1, Cyclin E binding protein 1, and Cyclin B1. The DNA repair genes BRCA1, MSH2, and APEX were induced by c-MYC, suggesting that c-MYC couples DNA replication to processes preserving the integrity of the genome. MNT, a MAX-binding antagonist of c-MYC function, was up-regulated, implying a negative feedback loop. In vivo promoter occupancy by c-MYC was detected by chromatin immunoprecipitation for CDK4, Prohibitin, MNT, Cyclin B1, and Cyclin E binding protein 1, showing that these genes are direct c-MYC targets. The c-MYC-regulated genes/tags identified here will help to define the set of bona fide c-MYC targets and may have potential therapeutic value for inhibition of cancer cell proliferation, tumor-vascularization, and restenosis.

Figure 1

Analysis of c-MYC-induced genes by qPCR. Serum-starved HUVEC were infected with Ad-MYC, Ad-MADMYC, or Ad-GFP adenovirus. For restimulation, serum was added 4 h after infection. RNA was isolated 12 h after adenoviral infection or restimulation with serum. For details, see Material and Methods. (A) qPCR determination of fold induction of NTF2 mRNA after c-MYC expression (primer efficiency: NTF2, E = 1.93; ELF1α, E = 2.05). (B) c-MYC dependence of serum-induced gene activation. The dominant-negative c-MYC mutant MADMYC was used to inactivate endogenous c-MYC function.

Figure 2

In vivo binding of c-MYC to promoter sequences. (A) Maps of c-MYC-induced genes indicating the positions of E-boxes (black rectangles). Exons are represented with ORFs (gray rectangles). Arrows indicate the transcription start site (TSS), which corresponds to base pair +1. Horizontal brackets represent qPCR amplicons. (B) Chromatin immunoprecipitation assay of c-MYC-regulated genes. Each result is the average of at least three independent experiments. For details see Material and Methods.

Figure 3

Analysis of c-MYC-regulated genes identified in HUVEC in a human B cell line. P493-6 cells, which harbor a c-MYC gene under control of a tetracycline-responsive element (12), were cultured in the presence of tetracycline (0.1 μg/ml). After 72 h, tetracycline was removed for the indicated periods to induce ectopic c-MYC and total RNA was isolated. exp., cells asynchronously growing in absence of tetracycline for 3 days. (A) Northern blot analysis. Total RNA (4 μg) was loaded per lane. After transfer, the membrane was hybridized with the indicated probes. As a loading control, the ethidium bromide (EtdBr) stained gel is shown. (B) qPCR analysis. Total RNA was isolated 12 h after removal of tetracycline. The average of two determinations is presented.

Figure 4

Requirement of c-Myc for normal induction of BRCA1, MSH2, and PHB after serum stimulation. c-Myc^+/+ and c-Myc^−/− RAT1A cells were maintained in DMEM containing 0.25% calf serum for 48 h. After stimulation with 8% calf serum, total RNA was isolated at the indicated time points. Northern blot analysis was performed with the indicated probes. Total RNA was stained with ethidium bromide (EtdBr).