Genomic targets of the human c-Myc protein

Abstract

Supplemental material is available at http://www.genesdev.org.

The behavior of mammalian cells is modulated by many extracellular stimuli, which trigger a variety of intracellular signaling pathways. Those signals induce expression of primary, or immediate early (IE) genes. Several IE genes encode transcription factors that, in turn, regulate secondary transcriptional responses (Winkles 1998). The c-myc proto-oncogene is such an IE gene. In normal cells, c-myc expression is generally induced by mitogens and suppressed by growth-inhibitory signals. Oncogenic activation of c-myc occurs by direct gene alterations, such as translocation or amplification, or by mutations in upstream signaling pathways. These accidents commonly result in deregulated and/or elevated expression of c-myc and its product, the Myc protein. c-myc is structurally related to two other genes, L- and N-myc, which are also overexpressed in specific tumor types (for reviews, see Garte 1993; Henriksson and Lüscher 1996; Grandori et al. 2000; Oster et al. 2002).

The changes in cellular behavior imparted by abnormal Myc expression have been studied extensively in cultured cells and transgenic animals. Myc has generally been associated with the promotion of cellular growth and proliferation, desensitization to growth-inhibitory stimuli, blockade of cell differentiation, cellular immortalization, and oncogenic transformation, as well as sensitization to apoptosis-inducing signals. Myc has also been linked to the regulation of various metabolic pathways, the induction of DNA damage, and genomic instability (for reviews, see Henriksson and Lüscher 1996; Amati et al. 1998; Grandori et al. 2000; Oster et al. 2002). In addition, Myc is likely to promote tumorigenesis in vivo through the induction of angiogenesis and tumor-cell invasiveness (Pelengaris et al. 1999, 2002; Brandvold et al. 2000; Ngo et al. 2000). Conversely, loss of Myc function has been associated with defects in cell growth, proliferation, and/or apoptosis, and is likely to profoundly impair the response of cells to their environment (e.g., Mateyak et al. 1997; Johnston et al. 1999; Bates et al. 2000; de Alboran et al. 2001; Douglas et al. 2001; Trumpp et al. 2001). In summary, the pathological effects of Myc overexpression are likely to result from multiple biological activities, reflecting the normal roles of Myc in coordinating cellular physiology with extracellular stimuli.

Myc is a transcription factor of the basic helix–loop–helix–leucine zipper (bHLH–LZ) family that can activate or repress gene expression. Activation occurs via dimerization with the bHLH–LZ partner Max and direct binding to the DNA sequence CACGTG, called the E-box (Blackwood and Eisenman 1991; Prendergast et al. 1991; Amati et al. 1992; Kretzner et al. 1992). Repression, instead, occurs through functional interference with transcription factors, such as Miz-1, that bind different DNA sequences (Oster et al. 2002). Both transcriptional activities of Myc appear to be critical for its biological function. Most relevant to the present work, dimerization with Max and binding to the E-box are essential for Myc to promote cell cycle progression, apoptosis, and cellular transformation (Amati et al. 1998). Thus, in order to understand the function of Myc, we need to identify its target genes, and in particular the E-box elements that Myc binds in the cellular genome.

Identification of Myc-regulated genes has generally relied on experimental activation of Myc followed by monitoring of changes in mRNA levels (Grandori et al. 2000; Oster et al. 2002). More than 10 investigative works reported the use of high-throughput screening based on cDNA microarrays or the SAGE assay, significantly expanding the list of genes that are up- or down-regulated by Myc (e.g., Schuldiner and Benvenisty 2001; Menssen and Hermeking 2002; Oster et al. 2002; Watson et al. 2002; O'Connell et al. 2003). Based on an updated online compilation (http://www.myc-cancer-gene.org/index.asp), this list now includes several hundred genes. It remains unclear, however, how many of these genes are direct targets of Myc. All studies based on mRNA expression have been hampered by the fact that a large fraction of Myc-target genes respond weakly, or even fail to respond to Myc activation, depending on the cell type or experimental conditions used. For example, several target genes that require Myc for induction by serum in Rat1 fibroblasts do not respond well to Myc alone in the same cells (Frank et al. 2001). Underlying this limitation, the lists of genes identified in high-throughput screens are only partially overlapping, and many genes were identified only once. Thus, we still possess a fragmentary picture of the loci that are directly targeted by Myc, and no accurate estimate of their numbers.

The primary criterion defining a direct target gene for any transcription factor is binding of the factor to regulatory DNA elements in cellular chromatin. This can be studied in live cells by chromatin immunoprecipitation (ChIP), which was used to demonstrate binding of Myc to several loci (Boyd and Farnham 1997; Bouchard et al. 2001; Frank et al. 2001; Xu et al. 2001; Zeller et al. 2001). Here, we have devised a large-scale, ChIP-based screen to identify E-box-containing genes that are bound by Myc in the human genome independently of their expression. The accompanying article (Orian et al. 2003) presents a large-scale analysis of Drosophila Myc-target loci. Both studies demonstrate the association of Myc with a very large population of genomic sites.

The 257 genes identified within the high-affinity group of Myc-targets (see Results) are listed and assigned to a functional category. The genes qualify as high-affinity targets in all cell lines tested, unless selectively indicated: ^{U-937, HL60, P493, T98G, WS1. Where applicable, the HUGO nomenclature is used (}http://www.gene.ucl.ac.uk/nomenclature). ^{Previously identified Myc-regulated genes (}http://www.myc-cancer-gene.org/index.asp).

Acknowledgments

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