ReviewThe c-Myc target gene network
Introduction
The c-MYC proto-oncogene is one of the most frequently activated oncogenes and is estimated to be involved in 20% of all human cancers, affecting about 100,000 US cancer deaths per year (http://www.myccancergene.org) [1], [2]. It is therefore critical that the function of c-MYC is well delineated. Despite its initial ambiguous functional definition as an oncoprotein implicated in DNA replication, RNA splicing or transcription, c-Myc has emerged foremost as a transcription factor. In particular, the discoveries that c-Myc contains an N-terminal transactivation domain and requires the bHLH (basic helix–loop–helix) partner protein Max to bind specific DNA sequences solidified its role in transcription (Fig. 1) [1], [3], [4], [5], [6], [7], [8]. In fact, recent estimates suggest that c-Myc could regulate as many as 15% of genes in genomes from flies to humans. c-Myc regulates transcription through several mechanisms, including recruitment of histone acetylases, chromatin modulating proteins, basal transcriptional factors and DNA methyltransferase [9], [10], [11], [12], [13], [14], [15], [16]. Hence, c-Myc targets can be classified into distinct subgroups whose regulation may involve some or all mechanisms through which c-Myc affects transcription. Moreover, the cis-regulatory modules for these subgroups are likely to contain binding sites for other specific transcription factors that cooperate with c-Myc, such that a module containing binding sites for c-Myc and transcription factor X may regulate the subgroup Xi of target genes. For example, one may envision that there are c-Myc target genes (such as “housekeeping” genes) whose histones are pre-acetylated via the binding of transcription factor X prior to c-Myc binding. The association of c-Myc with the promoters of these targets would then recruit additional factors to enhance rates of transcription. In contrast, a hypothetical subset of c-Myc target genes may exist that require c-Myc to bind first and initiate histone acetylation before other transcription factors proceed to execute the final steps of transcriptional activation. These targets would thus require c-Myc in order to allow for any transcription to occur.
In addition to transactivation, the mechanisms of c-Myc-mediated trans-repression are beginning to be defined. The identification of the Mad family of proteins (Mad1/Mad2/Mxi1, Mad3, and Mad4) has allowed further insight into the dynamics of protein interactions that regulate c-Myc's function. When bound to Max, Mad family members bind consensus enhancer box (E-box) sequences and compete for binding with c-Myc/Max heterodimers. Mad/Max dimers repress transcription by recruiting the chromatin-modifying co-repressor complex containing Sin3, N-CoR, and the class I histone deacetylases HDAC1 and 2 to the promoters of target genes. Recruitment of this complex results in deacetylation of histone tails and a closed chromatin conformation, thus preventing the transcriptional activation that occurs through E-boxes [17], [18]. In contrast to Max, which is ubiquitously expressed, the Mad/Mnt proteins are induced during terminal differentiation [19]. Consistent with these findings, chromatin immunoprecipitation experiments revealed a switch from c-Myc/Max binding to Mad/Max binding during cellular differentiation [20], [21]. Recent studies combining ChIP and CpG island microarrays have demonstrated that Max bound to targets that were activated and repressed by c-Myc [22]. Moreover, a functional Myc:Max interaction was shown to be essential for repression of gene targets. These findings suggest that Max may in fact play a more universal role in transcriptional regulation than previously thought.
Through direct interaction with the transcriptional activator Miz-1, Myc binds to and interferes with Miz-1 mediated transactivation thereby causing trans-repression of specific Miz-1 target genes [23], [24], [25], [26]. One study demonstrated that c-Myc/Max dimers bind the p15INK4B promoter (at the INR initiator element, which recruits the basal transcriptional machinery to TATA-lacking promoters) and repress transcription by disrupting the association of Miz1 with the p300 coactivator [26]. Another report found that c-Myc is recruited to the p21 promoter by Miz1. This interaction prevented p21 induction by p53, resulting in the initiation of apoptosis over cell cycle arrest [27]. Recently, c-Myc has been shown to serve as a molecular bridge between Miz-1 bound to the p21 promoter and the DNA methylase Dnmt3a to mediate methylation and transcriptional silencing of p21 [16]. Another possible mechanism of trans-repression may involve the interaction of c-Myc with CAAT box binding proteins, such as NF-Y. This appears to be the case with the transcriptional repression of PDGFR-β and perhaps of collagen genes [28], [29], [30], [31]. Taken together, these studies provide evidence that c-Myc can inhibit transcription by directly interfering with other factors that activate gene expression.
With transcriptional regulation as its acknowledged function, the search for physiological and pathological c-Myc target genes has intensified over the past decade [3], [4], [5], [6], [7], [32], [33], [34], [35], [36]. Searches for target genes have involved hypothesis-driven, low-throughput studies of candidate c-Myc target genes as well as medium-throughput studies to define a larger repertoire of c-Myc responsive genes through subtraction cloning methods. More recently, the field has rapidly adopted high-throughput microarray technologies for the discovery of c-Myc responsive genes [11], [22], [37], [38], [39], [40], [41], [42]. Despite impressive advances, major milestones still must be met to achieve a complete understanding of c-Myc responsive genes and how they relate to tumorigenesis.
Section snippets
Identification of c-Myc target genes
To understand the network of target genes regulated by c-Myc, it is crucial to determine whether c-Myc responsive genes are directly bound by c-Myc or whether the responsive genes are secondary events that require the activities of the direct target genes (i.e., indirect targets). Direct targets are defined as genes that are bound by c-Myc and respond to changes in c-Myc levels or c-Myc activity.
Until factors that alter the activity of c-Myc protein are better defined, most current models to
Myc target genes
What is the functional significance of a given c-Myc target gene? Only a fraction of genes appear to be universally regulated by c-Myc independent of cell type or species [64]. c-Myc responsive genes that appear recurrently in different cell types, systems and species can be identified from the c-Myc target gene database (http://www.myccancergene.org). In addition, the use of ChIP has further identified direct c-Myc targets among the genes that appear to respond to c-Myc regardless of the cell
Functions of c-Myc target genes
Although c-Myc is thought to influence up to about 15% of genes [10], [58], [69] and despite the functional range of specific genes altered, c-Myc consistently affects specific classes of genes that involve metabolism, protein biosynthesis, cell cycle regulation, cell adhesion and the cytoskeleton. The deregulated expression of c-Myc also induces genes that contribute to apoptosis under nutrient or growth factor deprivation; however, c-Myc target genes involved in apoptosis remain to be fully
An integrated database of Myc responsive genes
Given the diverse cell types and experimental systems used to study Myc target genes, how does the field begin to achieve a comprehensive accounting of Myc responsive target genes? To begin to glean a collective view of Myc responsive transcriptomes, we have launched a publicly accessible Myc target gene database (http://www.myccancergene.org) [64]. The database is searchable and provides the ability to prioritize the putative target genes according to the level of experimental evidence
The c-Myc transcriptional regulatory network
By studying the structure and behavior of transcriptional networks, biologists are beginning to understand the complex processes that control gene expression. The goal of this type of analysis is to better understand how relationships between molecules (in this case a transcription factor and its target genes) control cellular behavior (for example, the process of transformation). For a full appreciation of target genes, the wiring of the c-Myc target gene network will need to be delineated in
Conclusion and outlook
The intense interest in c-Myc function through the identification of its target genes has rallied the c-Myc research community to generate vast amounts of information over the last several years. Although thousands of c-Myc responsive genes have been identified and a general picture emerges for c-Myc function in regulating cell cycle progression, metabolism, ribosome biogenesis, and cell adhesion, the set of c-Myc target genes that distinguishes physiologic c-Myc function from pathologic,
Acknowledgements
We apologize for omissions due to space limitation. We thank J. Mendell and members of the Dang Laboratory for their comments. Our original work is supported by NCI grants CA51497 and CA57341. CVD is Johns Hopkins Family Professor in Oncology Research.
References (132)
- et al.
The myc oncogene: MarvelouslY complex
Adv Cancer Res
(2002) - et al.
A large scale genetic analysis of c-Myc-regulated gene expression patterns
J Biol Chem
(2003) - et al.
The novel ATM-related protein TRRAP is an essential cofactor for the c-Myc and E2F oncoproteins
Cell
(1998) - et al.
Myc recruits P-TEFb to mediate the final step in the transcriptional activation of the cad promoter
J Biol Chem
(2002) - et al.
Mad–Max transcriptional repression is mediated by ternary complex formation with mammalian homologs of yeast repressor Sin3
Cell
(1995) - et al.
Analysis of Myc bound loci identified by CpG island arrays shows that Max is essential for Myc-dependent repression
Curr Biol
(2003) - et al.
Transcriptional repression by Myc
Trends Cell Biol
(2003) - et al.
Identifying genes regulated in a Myc-dependent manner
J Biol Chem
(2002) - et al.
Skp2 regulates Myc protein stability and activity
Mol Cell
(2003) - et al.
Ras enhances Myc protein stability
Mol Cell
(1999)