Myc - What We have Learned from Flies

590 Current Drug Targets, 2009, 10, 590-6011389-4501/09 $55.00+.00© 2009 Bentham Science Publishers Ltd.Myc - What We have Learned from FliesN.A. Siddall 1,2, J.I. Lin 3,4, G.R. Hime 1,2 and L.M. Quinn 1,*1Department of Anatomy and Cell Biology, University of Melbourne, Parkville 3010, Melbourne, Australia; 2Australian Research Coun cil Cen tre of Excellen ce in Biotechn ology an d Developmen t, 3Peter MacCallum Can cer Cen tre, St An drews Place, East Melbourn e 3002, Melbourn e, Australia; 4Departmen t of Biochemistry an d Molecular Biology, University of Melbourne, Parkville 3010, Melbourne, AustraliaAbstract :The Myc family proteins are key regulators of animal growth and development. dMyc, the only Drosophila member of the Myc gene family, is orthologous to the mammalian c-Myc oncoprotein. Extensive studies have revealed much about both upstream regulators and downstream target genes in the sphere of Myc regulation. Here, we review some of the critical discoveries made using the Drosophila model, in particular those studies that have explored the essential role of the Myc family in growth and cell cycle progression and identified many of the upstream signals and downstream targets common to both c-Myc and dMyc.Keywords : Myc, Drosophila , growth, cell cycle, ribosome biogenesis, stem cells, cell competition, cancer. INTRODUCTIONRegulation of cell growth and cell cycle progression is critical for normal animal development. Mis-regulation will lead to significant developmental defects; too little growth and/or cell cycle progression leads to small organs and small body size whilst ectopic proliferation can lead to tissue overgrowth, genomic instability [1] and cancer [2]. Since the identification of the oncogenic potential of c-myc in the early 1980s [3], the mammalian Myc family, which is comprised of c-Myc, N-Myc, and L-Myc, has been the focus of extensive investigation [4-6]. dMyc, which is encoded by the diminutive (dm ) locus, is the sole Drosophila Myc member and is orthologous to c-Myc [7, 8]. Myc proteins contain an N-terminal domain for regulating transcription and a C-terminal basic-helix-loop-helix-zipper (bHLHZ) that mediates interaction with a second bHLHZ protein, Max. Myc proteins can heterodimerize with Max and bind E-box sequences in the promoters of target genes to directly control expression of genes involved in DNA synthesis, RNA metabolism and cell cycle progression [9-11]. Alternatively, Max can heterodimerize with Mxd (formerly Mad)/Mnt proteins, which also contain a bHLHZ motif, but binding of the Max-Mxd/Mnt complex results in repression of Myc-Max targets. The heterodimerization and DNA binding behaviour of the Myc-Max-Mxd/Mnt network is conserved in Drosophila , but with each gene represented by a single ortholog [7, 11-14].In studies using mouse models, c-myc knockout mice die at embryonic day 10.5 [15], whilst mice haplo-insufficient for c-myc are viable but grow more slowly and are smaller than wildtype [16]. c-Myc is therefore essential for the accumulation of cellular mass or cell growth in mammals [17, 18], whilst upregulation of c-myc can lead to the*Address correspondence to this author at the Department of Anatomy and Cell Biology, University of Melbourne, Parkville 3010, Melbourne, Australia; E-mail: l.quinn@.au uncontrolled growth associated with cancer [1, 19-21]. As in mice, dm haplo-insufficiency results in reduced cell growth, which leads to small adult body size [8]. Functional conservation with c-Myc has been demonstrated by the ability of dMyc to transform primary mammalian cells and rescue proliferation defects in c-myc null fibroblasts [22]. Conversely the human c-Myc protein can rescue lethal mutations of dMyc, demonstrating the biological relevance of this model [23].Downstream - Myc Hits Multiple TargetsA collection of genetic experiments, microarray profiling analyses and genome binding studies in mammals [24, 25] and Drosophila [8, 10, 14] have led to an understanding of the incredibly wide-ranging function of Myc (reviewed in [6]). Myc’s chief role in cell behaviour is highlighted by the finding that Myc proteins can bind to the promoters and control the transcription of 10-15% of all genes [5, 10]. Thus the regulatory targets of c-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 [6, 26, 27]). Small changes in c-Myc protein levels, either up or down, will modify these critical cellular processes, emphasizing the requirement for extremely tight control of c-myc expression [16, 28].T he Myc Family Regulates Growth and S Phase ProgressionMyc is Critical for GrowthAlthough Myc proteins affect multiple targets, this review will focus on the ability of Myc to drive cell growth and cell cycle progression, processes critical to the oncogenic properties of c-Myc. Extensive studies have shown that like mammalian Myc proteins, dMyc is essential for the accumulation of cellular mass or cell growth [8, 10, 29]. In Drosophila , dMyc regulates cell and organismal size:Myc - What We have Learned from Flies Current Drug Targets, 2009, Vol. 10, No. 7 591hypomorphic mutants of dm are small due to reduced cell growth [8] whilst null mutations of dm lead to larval lethality due to a growth arrest [29]. Conversely, overexpression of dMyc produces larger flies [8, 29]. A pertinent question then remains is how does dMyc achieve this effect on growth?Mammalian studies suggest that Myc’s ability to regulate cell growth requires transcriptional control through the three RNA polymerases. c-Myc regulates a large number of RNA polymerase II-transcribed genes, many of which encode ribosomal proteins, translation factors and other components of the biosynthetic apparatus [25]. c-Myc is also particularly efficient at activating transcription via RNA polymerase I [5] and III [30]. The capacity to drive production of rRNA is central to c-Myc's powerful cell growth effects, as ribosome biogenesis is well known to be rate-limiting for cell growth.InDrosophila, dMyc can also activate RNA polymerase I (Pol I) transcription and ribosome biogenesis. Expression-profiling experiments on dMyc overexpression or knockdown, in both S2 cells and whole larvae, have highlighted dMyc’s role in ribosome biogenesis [10, 11, 14]. Genetic experiments also confirmed that dMyc can stimulate expression of a Pol I factor TIF-IA. DNA microrarrays on cells or tissue overexpressing dmyc revealed upregulation of Pol I transcripts, highlighting the ability of dMyc to regulate Pol I transcription [10].Furthermore, recent work by Steiger et al . has shown that dMyc can also stimulate RNA polymerase III (Pol III) transcribed genes. Reducing dMyc levels in S2 cells resulted in reduced expression of Pol III targets, whilst overexpression of dMyc increased the levels of these same targets [31]. Like c-Myc [30], dMyc most likely regulates Pol III targets via interaction with the Pol III factor TFIIIB-component, Brf. This study demonstrated that the Pol III functions of dMyc are independent of dMax, suggesting that dMyc can activate Pol III genes via direct interaction with Brf [31]. In line with this, c-myc mutants lacking DNA binding function, which are unable to regulate direct target genes, can rescue much of the growth defect in c-myc (-/-) primary fibroblasts, suggesting the possibility of Max-independent functions for c-Myc [32].In mammals, c-Myc exerts transcriptional control over the mitochondrial metabolic network, thus providing a direct link between c-Myc and energy metabolism [33, 34]. While this has not been reported for Drosophila, recent work revealed that dMyc is an important mediator of PI3K/TOR-dependent regulation of ribosome biogenesis [35]. Using a functional genomics approach, the PI3K/TOR target FOXO was shown to downregulate dMyc to reduce translation when muscle tissues were nutrient deprived. The expression profiling revealed that the interactions between FOXO and dMyc were however tissue-specific, as FOXO was unable to downregulate dMyc in adipose tissue under similar conditions. Not only did FOXO regulate dm mRNA levels, but direct regulation was suggested by identification of FOXO binding sites in the dm promoter [35].Examination of the other arm of the PI3K/TOR pathway also showed that dMyc can regulate the transcription of TORC1 targets. Chromatin immunoprecipitation (ChIP) experiments confirmed dMyc protein enrichment in the promoter regions of TORC1 target genes [35]. The amount of dMyc protein binding could be increased or decreased with the addition of insulin or rapamycin, respectively, suggesting TORC1 controls dMyc activity by managing the amount of dMyc protein bound to the promoters of TORC1 targets. Thus, Teleman’s work neatly ties dMyc, which can also control expression of ribosome synthesis genes, into the PI3K/TOR pathway.Myc Couples Growth with S Phase ProgressionLike c-Myc, dMyc regulates the activation of genes required for G1-S phase progression [8, 10, 14, 29, 36-40]. dMyc drives G1 to S phase progression via upregulation of the G1/S phase cyclins (Cyclin E and Cyclin D), which lead to phosphorylation and inactivation of the key inhibitor of G1-S phase progression, Rbf, a member of the Drosophila Retinoblastoma family [39]. The inhibition of Rbf results in release of E2F1 from the inhibitory complex with Rbf, which permits upregulation of E2F1 dependent S phase genes [41]. Like c-Myc, dMyc therefore regulates cell growth and the activation of genes required for DNA replication and progression through S phase.In Drosophila, dMyc can drive growth and S phase progression of both mitotic and endoreplicating cells [29, 40]. In the Drosophila larvae and adult ovary many cells undergo endoreplication, where cell growth and DNA replication occur in the absence of cell division. In the adult ovary, dMyc is required for growth and endoreplication of both germline and somatic cells [40]. Analysis of dm mutant clones in the ovary revealed that although dMyc was essential for growth and endoreplication, mitotic divisions occurred normally in dm loss-of-function germline and somatic cells. Although there was a clear requirement for dMyc in the genomic endoreplication cycles, the later follicle cell endocycles necessary for chorion gene amplification occurred normally in dm mutant cells. Loss of dMyc function using the same null dmyc alleles used in the ovary study results in developmental arrest at the second larval instar. The larvae are smaller than normal as endoreplicating tissues such as the salivary glands and fat body fail to grow and replicate their DNA. Conversely, overexpression of dMyc in endoreplicating cells results in dramatic increases in cell growth (nucleolar size) and nuclear DNA content (DNA replication). Thus, dMyc is both necessary and sufficient for endoreplication and larval growth [29].Analysis of endoreplicating tissues in Drosophila has highlighted the antagonistic behaviour of Mxd/Mnt towards to dMyc function. Mxd/Mnt proteins repress transcription by interacting with the mSin3 co-repressor complex, which contains histone deacetylase (HDAC) activity (reviewed in [42, 43]). Drosophila contains a single ortholog of the mammalian Mxd/Mnt transcriptional repressor protein (dMnt). Loss of dMnt function flies are overgrown, whilst dMnt overexpression inhibits growth and proliferation [13]. To demonstrate antagonism between dMyc and dMnt in vivo, the effect of dmnt loss of function was examined in the dm null background. Larvae homozygously mutant for both dMyc and dMnt outgrow dMyc single mutants. This increased body size correlates with increased endoreplication and growth of larval tissues in the double mutant. Thus the loss of dMnt allows sufficient expression of dMyc targets required for growth and endoreplication [44]. These findings592 Current Drug Targets, 2009, Vol. 10, No. 7 Siddall et al.suggest that dMyc must normally overcome the antagonistic effects of dMnt repression in order to upregulate the genes required for larval growth and endoreplication. Expression analysis revealed that the E-box sequence was not required for restoration of dMyc target expression in the Mnt/Myc background. It is possible that like c-Myc, dMyc might play a more direct role in DNA replication. Studies in vertebrates (human, mouse and Xenopus) have shown that Myc proteins can control the initiation of DNA replication by directly interacting with the pre-replication complex [45]. If dMyc is involved in DNA replication in Drosophila then the ability of dMyc to drive endocycles might not be a consequence of a tight coupling of growth and endoreplication as once predicted [29].Upstream Regulation of MycMyc proteins respond to multiple developmental and environmental signals to direct cell growth and proliferation. Due to the fast turnover and low abundance of both c-myc mRNA and protein in most normal tissues, multiple mechanisms must operate to constrain c-Myc levels [16, 38, 46-49]. The importance of maintaining a tight rein on Myc is reflected by the multiple tiers of its regulation, including RNA synthesis, translation and protein degradation (reviewed in [6]). As mis-regulation of c-myc is associated with the majority of human malignancies [21], the upstream signalling pathways, transcriptional, translational and proteolytic mechanisms that regulate Myc are therefore of critical importance.Many key signalling pathways have been linked with the regulation of Myc in both Drosophila and mammals. In mammals, c-myc is upregulated in response to the growth factors in serum, but no single signal can account for full activation of c-myc. In Drosophila the regulation of Myc by developmental morphogens has been particularly well studied in the larval wing disc [8, 39, 50-53], where dMyc is a key mediator of growth and S phase progression [8]. Coordination of proliferation and patterning in the wing imaginal disc depends on key signalling pathways, such as Wingless (Wg), Decapentaplegic (Dpp) and Notch [54], which are conserved in vertebrates where they are critical mediators of development and disease [55].Regulation of dmyc by Wg/Wnt Signalling Depends on Developmental ContextThe Wingless (Wg) protein is the founding member of the Wnt family of secreted morphogens. Wg is secreted in a band across the dorsal-ventral (D/V) boundary in the wing pouch [56] and is essential for downregulation of dMyc and cell cycle arrest in a region of the wing disc called the "Zone of Non-Proliferating Cells", or ZNC, at the end of larval development (Fig. 1A-C) [8, 39, 50, 51]. In mammals c-mycis also regulated by the Wnt pathway [57]. Whilst evidence for binding by the Wnt responsive transcription factor TCF has not been reported for Drosophila, binding sites for TCF4 have been identified in the c-myc promoter [58].dMyc is required for driving growth and S phase progression in the wing imaginal disc, but developmental signals are required to ensure that dm expression is reduced in the ZNC. First, mRNA in situ analysis has shown that dm transcription is high in the cycling cells of the pouch, but decreased in the ZNC. In addition, ectopic expression of dMyc in the ZNC has been shown to prevent cell cycle exit [8]. As mentioned above, the Wg morphogen regulates cell cycle exit at the D-V boundary to form the ZNC late in the third larval instar, which is essential for differentiation of the adult wing (Fig. 1A-C )[50]. The Wg pathway is required for downregulation of dm transcription in the ZNC and this repression of dMyc by the Wg signal is essential, since inhibition of the Wg pathway within the ZNC prevents downregulation of dMyc [8] and its G1 targets (Cyclin E and E2F1) [50].The effect of Wg to drive downregulation of dmyc, cell cycle exit and differentiation in the wing pouch contrasts with much of the mammalian Wnt literature, which documents the function of Wnt signalling in driving increased cell proliferation in human oncogenesis [59]. In mammals, c-myc is a target of Wnt/Wg signalling in both normal intestine and in colorectal carcinomas [57, 60]. Genetic mouse experiments have revealed Wnt signalling is required for c-myc expression and proliferation in intestinal crypt stem cells [61, 62].In vitro, c-Myc can prevent the cell cycle arrest normally induced by blocking the Wnt pathway [58], suggesting c-Myc acts downstream of Wnt signals and can drive proliferation in the absence of Wnt activity. In addition, genetic mouse studies have shown that c-Myc is required for the majority of the tumourigenic effects that result from deregulated Wnt target gene activation [63].However, accumulated evidence has demonstrated that the effects of the Wnt pathway on c-Myc, growth and proliferation are far more complex. Numerous studies have documented that Wnt proteins show tumour suppressor activity and the ability to inhibit cell transformation through growth inhibition and promotion of differentiation [64-68]. These seemingly opposite effects reflect the ability of the Wg/Wnt signalling pathway to elicit different biological responses, which depend upon cell and tissue context. Thus the effect of the Wg/Wnt signal on Myc expression depends on developmental context.The action of Wg in the Drosophila larval wing imaginal disc reflects the complexity of the pathway. Wg behaves in either a tumour suppressor or oncogenic manner depending on the developmental context. Wg acts like a tumour suppressor in the pouch of the larval wing disc, where it is essential for cell cycle exit prior to differentiation (as described above [50]). In contrast, Wg behaves in an oncogenic manner in the neighbouring hinge compartment of the same imaginal tissue, where it promotes proliferation [51]. Similar differential effects have been reported in the intestinal crypt, where Wnt proteins have been explored extensively due to the link between deregulated Wnt signalling and colorectal carcinoma. Wnt signals near the bottom of crypts are crucial for the proliferation and maintenance of the undifferentiated progenitors, whilst Paneth cells require Wnt signals to achieve terminal differentiation. These observations imply that Wnt signals in the crypt can separately drive stem-cell proliferation and Paneth cell differentiation [69]. The fact that both gene programmes are deregulated simultaneously in invasive colorectal cancer reinforces the importance of understanding the diverse biological effects of the Wnt pathway on Myc regulation [70].Myc - What We have Learned from Flies Current Drug Targets, 2009, Vol. 10, No. 7 593Fig. (1). (A-C) - Wg protein, dmyc expression and cell cycle patterning in the Drosophila wing pouch.(A) Wg protein (red) is strongly expressed along the dorsal-ventral boundary of the wing pouch.(B) -gal antibody staining (blue) of dmyc-lacZ(w67c23 P{lacW}l(1)G0354G0354;(161)) discs shows a pattern consistent with dmyc transcription throughout the cycling cells of the pouch and downregulation of dmyc within the G1 arrested cells of the ZNC.(C) The zone of non-proliferating cells (ZNC) can be seen by the reduced BrdU staining (green) for S phase along the dorsal-ventral boundary. Wing imaginal discs are aligned dorsal (D) to the top of the image, ventral (V) the bottom.(D) Schematic of the egg producing tissue of the Drosophila ovary, the germarium. The germarium contains three main stem cell populations; germline stem cells (GSC), somatic stem cells (SSC) and Escort stem cells (ESC) (162, 163).Differentiation of the GSC gives rise to the cystoblast daughter cell, which undergoes 4 rounds of mitotic division to produce mature 16 cell cysts. The SSCs produce the follicle cells, which form an epithelium surrounding the 16-cell cyst, to produce an egg chamber. The terminal filament (TF), cap cells (CC) and inner-germarium sheath cells (IGS) are somatically derived and make up the stem cell niche. The germline and somatic stem cells together are responsible for generation and maintenance of the ovary.(E) Schematic of the proliferating region of the Drosophila testis. In the apex of the testis, typically 7-9 GSCs abut a group of somatic hub cells. Each GSC is surrounded by a pair of SSCs, called cyst progenitor cells (CPCs). GSCs asymmetrically divide to produce a daughter gonialblast, which subsequently undergoes four rounds of mitosis, thereby creating cysts of 2, 4, 8, and 16 secondary spermatogonia, with each germline cyst always surrounded by two differentiated cyst cells, the daughters of the CPCs. The spermatogonia will differentiate into mature spermatocytes, undergo meiosis and finally produce mature sperm (137).(F- I) Myc expression in the Drosophila male and female gonads.(F) Analysis of germaria with the dMyc monoclonal antibody (red) (a gift from B. Eisenman) shows that dMyc protein is normally present at low levels in the GSCs, cystoblast daughter cells, and depleted further in developing germline cysts. dMyc expression is upregulated in mature germ line cysts (GLC), and high levels of dMyc can be observed in the mature nurse cells of the stage 1 ovary. dMyc is also present in SSCs and in somatic follicle cells, but not in the SCs that comprise the stem cell niche (i.e the TF, CC or IGS cells).(G) Depletion of dMyc (red) can be observed in germ cells of the germarium via expression of a UAS-dmyc RNAi specifically in the germline. Somatic cells, including SSCs maintain dMyc expression. Specific depletion of dmyc from the GCs using RNAi shows specificity of the antibody for dMyc protein.(H) Using a rabbit polyclonal antibody (a gift from D. Stein), we observed dMyc expression in both somatic (green arrowhead) and germline (yellow arrowhead) stem cell populations in the developing larval testis. dMyc was also expressed in proliferating gonial cells of the testis, however, expression was not observed in the somatic niche cells (hub, *).(I) Knockdown of dMyc in the early germ cells using the UAS-dmyc RNAi resulted in a loss of GSCs abutting the hubs. In this single confocal planar image, vasa staining (red) shows only 2 GSCs (one is marked with a yellow arrowhead) adjacent to the hub, with large, dMyc-expressing somatic cells (green arrowhead) taking up the positions where the GSCs should lie.594 Current Drug Targets, 2009, Vol. 10, No. 7 Siddall et al.Wg, Dpp and Notch Coordinate Patterning and Growth Cross-talk between the Wg pathway and the Dpp and Notch pathways is required to coordinate proliferation and patterning of the wing imaginal disc. Drosophila Dpp, a member of the mammalian transforming growth factor-beta (TGF-beta) family of secreted proteins, is required to restrict Wg expression during wing development [71]. In mammals, TGF-beta can behave as a tumour-suppressor or oncogene depending on the tissue microenvironment, thus pathway inhibition or activation can result in cancer progression [72-78]. Dpp is expressed in a band of cells in the anterior compartment along the anterior-posterior boundary of the larval wing disc [79, 80] and is required for cell cycle progression and tissue growth [81]. Yet although ectopic expression of Dpp or the activated Dpp receptor results in overgrowth [81, 82], the Dpp targets required to drive this growth are yet to be reported.Like Wg, the Notch pathway also plays a role in cell cycle arrest during wing development [50, 52]. Notch is activated in cells along the dorso-ventral (D/V) boundary (ZNC) of the wing disc and is required for induction of Wg expression [83]. The activation of Wg target genes achaete (ac) and scute(sc) specifically within the anterior compartment of the cells flanking the D/V boundary results in downregulation of the mitotic inducer, Cdc25c/Stg, to arrest these cells in G2 [50]. The expression of Notch specifically within the D/V boundary prevents the G2 arrest, allowing Wg to mediate G1 arrest within the anterior cells comprising the D/V boundary and all cells comprising the posterior compartment ZNC[8, 50]. The interplay of these signalling pathways therefore achieves the pattern of cell cycles within the wing pouch required for wing disc differentiation. Recently a more linear pathway has been proposed for controlling G1-S progression by Wg and Notch at the D-V boundary of the wing pouch ZNC [84]. This work suggests that Wg and Notch act in a linear pathway to control expression of dMyc and the bantam micro-RNA, which regulate the activity of the S phase transcription factor E2F1 [52].Steroid Hormones in Myc RegulationA vast body of literature has provided strong connections between mammalian steroid hormone pathways and regulation of cell cycle genes [85]. For example, in murine embryonic stem (ES) cells, the estrogen steroid hormone has been shown to promote proliferation associated with increased expression of c-myc, which leads to upregulation of the cyclins and their kinase partners [86]. In the larval wing disc, the receptor for the ecdysone steroid hormone (EcR) is required to regulate Wg, cell cycle progression [87] and dmyc transcription [88]. Ectopic wg expression occurs when the EcR pathway is blocked using dominant negative EcR transgenes, suggesting that suppression of wg transcription in the wing pouch is dependent on the EcR pathway. As Wg causes downregulation of dmyc in the pouch [8, 50], this finding is consistent with the reduced dm expression and cell cycle progression observed in EcR loss-of-function clones [87]. Thus proper signalling through the EcR pathway is required for endogenous dMyc expression in the Drosophila wing imaginal disc. Regulation of Myc at the Level of Transcription As c-Myc is a potent mitogen, the level of c-myc transcription must be tightly regulated. Myc transcription responds to developmental signalling molecules [38, 39, 50], which are likely to modulate the complement of a wide variety of transcription factors at the myc promoter [89, 90]. Hfp/FIR - a Clamp on Myc TranscriptionOne level of c-myc promoter regulation involves the presence of a paused, but transcriptionally engaged, RNA polymerase II (Pol II) at the start site, which must escape the promoter to allow elongation [91-96]. The function of the paused polymerase is potentially twofold: 1) to allow rapid response to developmental/mitogenic signals and 2) to protect the c-myc promoter from unwanted activation, since the polymerase will act to block the start site and prevent pre-initiation complex formation.Activation of transcription from the blocked c-myc promoter is dependent on the general transcription factor IIH (TFIIH), the basal transcription initiation factor for RNA polymerase II transcribed genes [97]. TFIIH is required for basal transcription and DNA repair [98-102], but also has a more specialised role in regulating c-myc transcription [89, 103, 104]. The TFIIH complex is a multi-complex protein, but the key subunit for Pol II escape and the transcriptional control of c-myc is the DNA helicase, XPB [89, 103, 104].Pol II complex movement within the promoter of the c-myc gene is controlled by interactions between TFIIH/XPB and two DNA structure-sensitive regulatory proteins called FBP and FIR [103-107]. This system centres around a regulatory sequence 1.2 kB upstream of the major P2 promoter of the c-myc transcription start site known as the Far Upstream Sequence Element (FUSE) [105, 108]. Interactions occur between the FUSE, the Fuse Binding Protein (FBP), the Fuse Interacting Repressor (FIR) and the XPB helicase to regulate Pol II movement [89, 106, 107]. In this system, FBP and FIR act as dominant regulators of Myc expression: FBP is potent activator of c-myc, while FIR returns FBP stimulated c-myc to basal levels. The antagonistic interactions between FBP and FIR are critical for tight regulation of c-myc transcription from the FUSE and the action of both FBP and FIR is channeled through XPB [103-107].FIR is therefore the key negative regulator within this system, which is required to neutralise FBP’s stimulation of XPB and return c-myc transcription to basal levels [89, 103]. FIR can independently bind all the key components of this system, including the FUSE [109, 110], XPB and FBP [103, 104]. In particular the co-ordinate binding of FIR to the FUSE sequence and the XPB helicase is thought to create a loop upstream of the c-myc promoter to 1) tether the TFIIH complex [89, 103, 105] and 2) disrupt upstream effector elements and transcription factor binding to further repress c-myc transcription [108].Studies of the temporal association of activators and repressors on the c-myc promoter revealed that binding of FIR is one of the key events at the c-myc promoter in the early response to mitogens, which is required for returning transcription to basal levels. The dynamics of c-myc promoter binding by FIR is reciprocal to c-myc expression,。