Global mapping of c-Myc binding sites and target gene networks in human B cells

Abstract

The protooncogene MYC encodes the c-Myc transcription factor that regulates cell growth, cell proliferation, cell cycle, and apoptosis. Although deregulation of MYC contributes to tumorigenesis, it is still unclear what direct Myc-induced transcriptomes promote cell transformation. Here we provide a snapshot of genome-wide, unbiased characterization of direct Myc binding targets in a model of human B lymphoid tumor using ChIP coupled with pair-end ditag sequencing analysis (ChIP-PET). Myc potentially occupies > 4,000 genomic loci with the majority near proximal promoter regions associated frequently with CpG islands. Using gene expression profiles with ChIP-PET, we identified 668 direct Myc-regulated gene targets, including 48 transcription factors, indicating that Myc is a central transcriptional hub in growth and proliferation control. This first global genomic view of Myc binding sites yields insights of transcriptional circuitries and cis regulatory modules involving Myc and provides a substantial framework for our understanding of mechanisms of Myc-induced tumorigenesis.

Fig. 1.

ChIP-PET analysis of Myc binding sites in P493 cells. (A) Human B cells harboring a tetracycline repressible c-Myc construct (P493 cells) exhibit a B lymphoid phenotype when cultured in the absence of tetracycline and express high levels of exogenous MYC as detected on the Western blot. (B) ChIP was performed on P493 cells by using a c-Myc polyclonal antibody. PETs from the cloned ChIP DNA fragments were concatenated for sequence analysis. PETs were then mapped to hg17 genome to localize Myc binding loci represented by overlapping clusters. A PET-3 cluster is shown as an example here that maps to the first intron of CDK4, a well known direct Myc target gene.

Fig. 2.

Validation of PET clusters as reliable readouts of Myc binding. (A) A known Myc binding site in the first intron of NPM is localized by a PET-4 cluster. This 86-bp overlap region contains two tandem canonical E boxes. (B) Myc ChIP DNA (blue) and control HGF ChIP DNA (red) from an independent ChIP experiment were subjected to ChIP-qPCR to validate clusters from different categories. The first two lanes are randomly picked PET-3+ and PET-2 clusters. The last three lanes are clusters picked from PET-2, PET-1, and genomic background regions proximal to CpG and promoter with E box. In parentheses are the numbers of validated sites vs. tested sites. (C) Percentages of PET-2 clusters with different binding features positive-validated by ChIP-qPCR.

Fig. 3.

Locations of Myc binding sites relative to gene structure and CpG island. (A) The locations of 593 reliable Myc binding loci (blue) and 156 E box containing loci (red) were displayed in relation to a gene structure model. The number of binding sites at each particular location is indicated at the top of the bar. (B) Number and percentage of 593 PET-3+ and 156 E box-containing Myc binding loci located within 1, 5, or 10 kb of CpG islands.

Fig. 4.

Myc and E2F1 collaborate to regulate the expression of a number of target genes. (A) Schematic view of Myc–Max and E2F1 binding to the same gene regulatory regions and leading to transcriptional activation of several key cellular processes. (B) Myc ChIP DNA (blue) and E2F1 ChIP DNA (red) from the same P493 cells were subjected to qPCR to validate same binding regions of several target genes. The fold of enrichment is calculated by comparing with negative control HGF ChIP DNA (yellow). (C) Expression of key target genes bound by both Myc and E2F1 were measured by RT-qPCR from P493 cells in the presence (blue) or absence (red) of tetracycline. (D) High Myc expression leads to increased E2F1 activity by up-regulating genes such as cyclins and CDK4. The cooperative binding of Myc and E2F1 followed by transcription activation of key downstream targets leads to the increase of DNA replication and cell cycle progression.