Myc Target Genes

Myc target genes can be generally classified in two ways: those that are repressed or induced by Myc or those that are direct vs indirect transcription targets. Identifying Myc targets has become a prodigious task because Myc regulates the expression of so many genes; indeed, a Web site has been established to keep track of all of these targets (http://www.myccancergene.org) (84). Currently, 1697 Myc target genes have been identified using biased approaches and unbiased tools, such as Serial Analysis of Gene Expression and microarray (85-94). Although already rather mind-blowing, recent experiments using chromatin immunoprecipitation and DamID assays actually indicate that the numbers of Myc targets is underestimated, and these studies are primarily focused on those targets having E-boxes in their promoter regulatory regions (95-100). That most of these target genes have been identified in response to c-Myc overexpression also raises the question of whether they are only relevant in scenarios such as cancer, where Myc is overexpressed. Indeed, early attempts to identify genes that lose their normal expression pattern in response to serum in cells deficient in c-Myc only identified cad, out of the few established target genes tested, to be dependent on c-Myc for its expression (101).

Germane to this review, Ornithine decarboxylase (Odc), S-adenosylmethionine decarboxylase (Amd1), and Spermidine synthase (Srm) are identified as targets in the

Myc Structure

Fig. 2. The Max network. The functional domains of Max, and of the Myc and Mad families of proteins are shown. The N-termini of Myc and Mad family proteins direct their interactions with numerous proteins controlling protein stability and activity (for Myc: Ras, Fbw7, and Skp2) and transactivation/transrepression (for Myc: TRRAP and TIPs; for Mad: Sin3a and Sin3b). The C-termini of these transcription factors harbor the bHLH-LZ domain responsible for DNA binding (the basic domain, b) and the obligate dimerization domain (HLH-LZ) required for interactions with Max. The number of residues in these proteins is also indicated. HLH, helix-loop-helix; LZ, leucine zipper; NLS, nuclear localization signal; PP, phosphorylation sites; SID, Sin3-interacting domain; TAD, transactivation domain.

Fig. 2. The Max network. The functional domains of Max, and of the Myc and Mad families of proteins are shown. The N-termini of Myc and Mad family proteins direct their interactions with numerous proteins controlling protein stability and activity (for Myc: Ras, Fbw7, and Skp2) and transactivation/transrepression (for Myc: TRRAP and TIPs; for Mad: Sin3a and Sin3b). The C-termini of these transcription factors harbor the bHLH-LZ domain responsible for DNA binding (the basic domain, b) and the obligate dimerization domain (HLH-LZ) required for interactions with Max. The number of residues in these proteins is also indicated. HLH, helix-loop-helix; LZ, leucine zipper; NLS, nuclear localization signal; PP, phosphorylation sites; SID, Sin3-interacting domain; TAD, transactivation domain.

Myc target gene database. Odc was one of the first Myc target genes identified (102-104) and is the only gene encoding a polyamine biosynthetic enzyme that has been characterized in regard to Myc (despite the presence of several E-boxes in Amdl and Srm; Fig. 3). This chapter, therefore, focuses on the regulation and role of Odc in Myc-induced tumorigenesis, yet Amdl and Srm are certainly important candidates that deserve further scrutiny as mediators of Myc-induced pathways.

1.2.1. Myc Induces Odc

The mouse Odc gene contains 12 exons (Fig. 3) and is essential for embryonic development (105). Odc is a direct target induced by Myc based on the following facts: (a) its promoter activity and transcript levels are elevated in cells overexpressing Myc (102-104); (b) Odc transcripts are induced by Myc in the absence of de novo protein synthesis—this comes from analyses of cells expressing MycER, a chimeric form of Myc that can be activated by tamoxifen, in which Myc is fused to the ligand-binding

Myc Cancer Gene
Fig. 3. Genomic structures of the genes encoding polyamine biosynthetic enzymes that are induced by Myc in B-cells of the Ep-Myc mouse (123). Note the presence of canonical/classical E-boxes in all three genes.

domain of the estrogen receptor (15,104); and (c) the Odc gene harbors two perfect (CACGTG), conserved, and functional E-boxes in intron 1 (102,103), to which Myc binding can be detected by chromatin immunoprecipitation assays (ChIP assays) and in electromobility shift assays (102,106). In addition, a third functional E-box was recently found upstream of the transcriptional start site of Odc (107).

Our understanding of the mechanism by which Myc transactivates Odc (and likely other targets) has recently undergone some revisions (108). Based on the fact that in quiescent or differentiating cells Myc levels are low and Mad levels are high, it was assumed that Mad family members antagonize Myc's ability to activate transcription (54,70,71,109-112). However, when one adds Mnt into the equation this model changes. As with Mad factors, Mnt is a transcriptional repressor and can inhibit Myc-induced cell growth and transformation (55,113). However, Mnt is coexpressed with Myc (55) and has even been identified as a Myc target in screens for Myc-responsive genes (91). When analyzing the Odc promoter by ChIP assay and electromobility shift assay, we recently discovered that the E-boxes of Odc are not occupied by Mad or Myc but by Mnt (106). Serum stimulation of quiescent fibroblasts resulted in a rapid appearance of Myc:Max complexes, which displaced Mnt:Max complexes from binding to the E-boxes of Odc intron 1, resulting in activation by relieving transrepression (Fig. 4). Interestingly, RNA interference-mediated knockdown of Mnt (106), and the targeted deletion of Mnt (114), recapitulated the biological effects of Myc overexpression in

Myc Cancer Gene

Fig. 4. Revised model of how Myc activates genes such as Odc. This model posits that Mnt:Max complexes bind the E-boxes of Odc. Mitogen stimulation leads to transient increases in Myc protein that then dimerizes with Max and displaces Mnt:Max complexes, resulting in induction of transcription by displacement or transactivation. However, removal of Mnt by knockdown or deletion is also sufficient to activate Odc, even in cells lacking Myc, suggesting that relief of transrepression is the mode of action, not classic transactivation (106).

Fig. 4. Revised model of how Myc activates genes such as Odc. This model posits that Mnt:Max complexes bind the E-boxes of Odc. Mitogen stimulation leads to transient increases in Myc protein that then dimerizes with Max and displaces Mnt:Max complexes, resulting in induction of transcription by displacement or transactivation. However, removal of Mnt by knockdown or deletion is also sufficient to activate Odc, even in cells lacking Myc, suggesting that relief of transrepression is the mode of action, not classic transactivation (106).

fibroblasts (Fig. 1); (i.e., Mnt loss enhanced cell proliferation and apoptosis, as well as transformation in conjunction with an activated Ras oncogene) (106). Because these effects of Mnt RNA interference were also observed in Myc-nuW fibroblasts, this suggests that the function of Myc is to antagonize Mnt-mediated transrepression of target genes, such as Odc (Fig. 4). This model has been supported by very recent studies conditionally codeleting c-Myc and Mnt in mouse embryo fibroblasts, which again leads to the induction of many other Myc targets and to the restoration of cell growth (115).

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