Tight regulation of developmental signaling is essential for controlling the cell growth and division required for proper formation of tissues and organs. Impaired cell cycle progression can be extremely detrimental; reduced cell cycles can lead to small, impaired organs and, conversely, uncontrolled cell proliferation can lead to tissue overgrowth and cancer formation. In Drosophila, cell growth and cell cycle progression are regulated by a number of key genes, which have been shown to control cell cycle in an analogous manner in all multicellular organisms. The mammalian c-Myc (referred to as Myc from here on) transcription factor and oncogene and its Drosophila orthologue, d-Myc, are both key regulators of cell growth and division [1,2]. A collection of genetic experiments, transcriptome analyses and genome binding studies in mammals and Drosophila have revealed that Myc proteins can bind to the promoters and potentially control the transcription of 10%–15% of all genes [2,3,4,5,6,7] (reviewed in [8]). Thus the regulatory targets of Myc and dMyc include genes from virtually every biochemical and regulatory pathway in the cell, including growth, metabolism, cell cycle progression, differentiation and apoptosis (reviewed in [8,9,10]).

In addition to regulating a wide range of genes, Myc targets can vary depending on the cell-type and developmental context, a complexity of function which has thwarted attempts to identify a universal transcriptome signature for Myc. In an effort to address these observations, two recent studies ([11,12] and reviewed in [13]) provide a potential explanation for the cell and context dependent variations between transcriptional signatures associated with Myc. Genome-wide ChIP-Seq analysis suggests that rather than driving its own transcriptional program, Myc behaves as a general amplifier of the cells transcriptional state at the time of Myc activation. By monitoring co-occupancy of the elongating form of RNA polymerase II and the presence of active chromatin marks, both studies observed that after overexpression, Myc protein is loaded quantitatively onto active promoters to enhance transcription. The observation that Myc overexpression does not increase its enrichment within the promoters of silent genes, suggested to the authors that elevation of Myc is not sufficient to activate transcription [11,12].

These new models suggest that Myc behaves as an amplifier of existing cellular states, however, Myc activation has been largely linked with cell proliferation and elevated levels of Myc are sufficient to drive cell growth and cell cycle progression [1,8,14]. Moreover, the capacity of Myc to drive growth is critical to its oncogenic potential [15]. Indeed, many studies in mammals suggest that Myc is an instructive force, rather than a simple reinforcer of cell fate. In mammals, even physiological levels of Myc can drive quiescent cells to proliferate [8,14]. Extensive studies have shown that like mammalian Myc proteins, dMyc is not only essential but is also sufficient for accumulation of cellular mass or cell growth [1,2,16]. In Drosophila, dMyc regulates cell and organismal size; hypomorphic mutants of dMyc are small due to reduced cell growth [1], while null dMyc mutations lead to larval lethality due to a growth arrest [16]. Conversely, overexpression of dMyc produces larger flies [1,16]. A pertinent question then remains: how does Myc achieve this effect on growth?

Transcription of ribosomal DNA (rDNA) is required to produce functional ribosomes, which is one of the most fundamental rate-limiting steps for growth and DNA synthesis. Myc and dMyc are both key regulators of rDNA transcription, ribosome biogenesis and cell growth [1,2]. The Myc oncogene regulates cell growth via three RNA polymerases; RNA polymerase I, II and III [17]. Myc regulates a large number of RNA polymerase II-transcribed genes, many of which encode ribosomal proteins and translation factors [6]. Myc is also very efficient at activating transcription via RNA polymerase I (Pol I) [2,7] and III (Pol III) [18] to drive rDNA transcription, ribosome biogenesis and protein translation. Drosophila microrarrays revealed upregulation of Pol I transcripts in dMyc overexpressing cells, and an associated increase in rDNA transcription, ribosome biogenesis and cell growth [2]. The capacity to drive production of rRNA (ribosomal RNA) is central to mammalian Myc's powerful cell growth effects and oncogenic ability [15]. Small changes, either up or down, in Myc protein levels will modify growth and potentially result in cancer initiation and/or progression, emphasizing the requirement for extremely tight control of Myc expression [19].

In addition to the ability to drive growth in Drosophila and mammals, the Myc protein is also required to couple growth with cell cycle progression. Like Myc, early in the G1 phase of the cell cycle, dMyc activates the genes required for DNA replication and progression through S phase [1,2,4,16,20,21,22]. Myc and dMyc activate the G1 to S-phase Cyclin/Cyclin-dependent-kinase complexes, CycE/Cdk2 and CycD/Cdk4, in order to trigger DNA replication and S-phase progression [23,24,25] (Figure 1A). As the loss of cell cycle control resulting from misregulation of Myc is associated with cancer [26,27], Myc must be tightly regulated during normal development [14,19,28,29]. The importance of maintaining a tight control of Myc levels is reflected by the multiple types of regulation observed for Myc and dMyc, including transcriptional initiation, RNA synthesis, translation and protein stability [8,30]. Therefore, the upstream signaling pathways and transcriptional, translational and proteolytic mechanisms that regulate Myc are of critical importance. In addition, the levels of Myc expression need to be responsive to growth and developmental signals to allow organ and tissue growth in response to nutrients and other external cues [1,20,31,32,33].

In mammals, one mechanism proposed for tight regulation of Myc levels and a quick response of Myc transcription to growth signals in vitro involves the presence of a paused, but transcriptionally engaged, RNA polymerase II (Pol II) within the Myc promoter [9,29,34,35]. Escape of Pol II from the promoter allows transcript elongation. Biochemical evidence has shown the movement of Pol II and elongation of the Myc transcript is dependent on the Transcription Factor IIH (TFIIH) complex and its DNA helicase subunit, XPB. Interactions with TFIIH/XPB helicase and two DNA structure-sensitive regulatory proteins called FUSE Binding Protein (FBP) and FBP Interacting Repressor (FIR) control the Pol II complex movement within the promoter of the Myc gene [35,36]. In this system, FBP and FIR act as dominant regulators of Myc: FBP is a potent activator of Myc, while FIR is required for repression of Myc transcription. FBP and FIR interact antagonistically and are essential for the transcriptional regulation of Myc transcription, and their actions are mediated by the XPB DNA helicase [9,29,34,35,37]. Consistent with a role for FIR in transcriptional repression of Myc, loss-of-function FIR mutations are associated with increased Myc mRNA levels in primary human colorectal cancer [38]. Mutations of XPB [35,36] and activation of FBP [39] have also been implicated in human cancer.

Evidence suggests Drosophila Hfp is the functional homolog of FIR, being essential for repression of dMyc transcription and, furthermore, cell growth in vivo [31,40]. ChIP experiments have revealed enrichment of Hfp proximal to the dMyc transcriptional start site (TSS). In addition, loss of Hfp results in enhanced cell growth, which depends on the presence of dMyc. Furthermore, Hfp physically and genetically interacts with the XPB helicase component of the TFIIH transcription factor complex, Hay, which is required for normal levels of dMyc expression, cell growth and cell cycle progression. Below we review how the Drosophila model has been used to increase our understanding of how Myc expression is correctly patterned and modulated in multicellular organisms in vivo, particularly in response to the Hfp transcriptional repressor.

The UAS-GAL4 system, derived from the yeast S. cereviase, has provided an invaluable genetic tool for studying the manipulation of gene expression during Drosophila development [79]. With a diverse variety of GAL4 drivers, temporal and tissue specific overexpression of a given UAS-transgene can be achieved in a specific tissue of interest. For example, cell cycle biologists combined the UAS-GAL4 system with the FLP/FRT system to generate the Actin<CD2<GAL4 “flip out” system in order to monitor cell growth and division in patches of tissue or “clones” over time (Figure 3A [80,81]). Gene expression is controlled by heatshock induced expression of the Flip recombinase (Flp) [82]. Flp will recognise the FRT sites in the Act<CD2<GAL4 cassette. The “flipping out” of the interruption cassette results in: (1) CD2 protein expression in the neighboring control clone and (2) the Actin promoter driving GAL4 in the clone, which can be positively marked with co-expression of a fluorescent transgene e.g. UAS-GFP or RFP with the UAS-transgene of interest (Figure 3A,B) [80,81,83]. This system is ideal for studying effects of manipulating gene expression on cell growth, proliferation, and signaling pathways in comparison to surrounding normal tissue, which is essential for understanding tumorigenesis. For gene knockdown, GAL4 drivers can be used for expression of a UAS-transgene for an inverted repeat targeting the gene of interest [84]. Extensive UAS-RNAi collections have been developed and made publicly available and, as these cover most (97%) of the Drosophila genome (e.g. Vienna Drosophila resource centre [85]), they have provided an invaluable resource for conducting non-biased genetic screens to elucidate novel signaling mechanisms and gene function (Figure 3C,D). For example, “flip-out” clones generated using a hairpin targeting Hfp, were used to demonstrate that loss of Hfp results is increased growth compared with the surrounding control cells [40]. Moreover, using the dmyc-lacZ enhancer trap described below, we have demonstrated that Hfp is required for repressing dmyc promoter activity (Figure 4, [40]).

In mammalian systems, in vitro transcription assays (e.g., luciferase assays) are a standard method for monitoring effects of a particular factor on promoter/enhancer activity. However, if we are to draw connections between the signaling environment and changes to the expression of cell cycle genes, it is essential to investigate transcription from endogenous promoters in vivo. In Drosophila, promoter and enhancer trap activity can be measured using reporter elements inserted into endogenous promoters in order to determine factors capable of modulating transcription of a given gene. Furthermore, random P-element mutagenesis screens generated extensive enhancer trap collections, where each line contains the insertion of a visible reporter (e.g., LacZ or GFP) for monitoring gene activity within a particular region of the genome [86,87]. As these enhancer traps often land in regions of active chromatin, such as gene promoters, they can often be used to measure endogenous promoter or enhancer activity, as an indicator of transcriptional activation of the gene located downstream of the insertion [88].

For example, a random P-lacZ insertional mutagenesis screen for the X-chromosome resulted in many hits in the dMyc promoter [89], providing potential enhancer traps to identify changes in dMyc promoter activity. Analysis of these lacZ insertions revealed that insert [P{lacW}l(1)G0354 [89]], located just prior to the 5'UTR in the dMyc promoter, in a region that is responsive to wing enhancers of dMyc expression (Figure 4A). By using the ®-gal antibody, this dMyc-lacZ line can, therefore, be used to monitor changes to dMyc promoter activity in wing imaginal discs [40,42]. In particular, UAS-hfp RNAi “flip-out” clones in the dMyc-lacZ enhancer trap background, revealed that Hfp is required for repression of dMyc promoter activity (Figure 4B and [40]). The enhancer trap appears to reflect changes in dMyc transcription, as mRNA abundance is significantly increased in Hfp loss of function imaginal tissues [40] and can provide an in vivo read out for potential enhancers and suppressors of dMyc transcription.

As detailed above, Myc is a potent mitogen, but despite the large number of transcriptional targets and the associated oncogenic potential of Myc dysregulation, how Myc is regulated at the transcriptional level is largely unknown. In Drosophila using the genetic systems described above, we have demonstrated that repression of dMyc promoter activity and cell growth requires Hfp function (Figure 4B and [40]). Furthermore, the increased dMyc-lacZ reporter activity, compared to surrounding wild type cells (Figure 4B) was associated with significantly increased dMyc mRNA levels [40]. Thus like its mammalian counterpart FIR, Hfp behaves as a tumor suppressor to repress dMyc, which suggests that the mechanism proposed for transcriptional repression of c-Myc by FIR is conserved in Drosophila. These data suggest that the loss-of-function FIR mutants described in colorectal cancer may be sufficient to increase Myc expression, which would be predicted to lead to cancer initiation and progression.