Mastermind-like transcriptional co-activators: emerging roles ... - Nature

Oncogene (2008) 27, 5138–5147

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REVIEW

Mastermind-like transcriptional co-activators: emerging roles in regulating cross talk among multiple signaling pathways AS McElhinny1, J-L Li2 and L Wu3 1 Ventana Medical Systems Inc., Tucson, AZ, USA; 2Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL, USA and 3Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL, USA

A family of Mastermind-like (MAML) genes encodes critical transcriptional co-activators for Notch signaling, an evolutionarily conserved pathway with numerous roles in both development and human diseases. Notch receptors are cleaved upon ligand engagement and the intracellular domain of Notch shuttles to the nucleus. MAMLs form a functional DNA-binding complex with the cleaved Notch receptor and the transcription factor CSL, thereby regulating transcriptional events that are specific to the Notch pathway. Here, we review recent studies that have utilized molecular, cellular and physiological model system strategies to reveal the pivotal roles of the MAML proteins in Notch signaling. Unexpectedly, however, emerging evidence implicate MAML proteins as exciting key transcriptional co-activators in other signal transduction pathways including: muscle differentiation and myopathies (MEF2C), tumor suppressor pathway (p53) and colon carcinoma survival (b-catenin). Thus, the MAML family appears to function in transcriptional coactivation in a multitude of cellular processes. It is hypothesized that MAML proteins mediate cross-talk among the various signaling pathways and the diverse activities of the MAML proteins converge to impact normal biological processes and human diseases, including cancers. Oncogene (2008) 27, 5138–5147; doi:10.1038/onc.2008.228 Keywords: Mastermind-like MEF2C; p53; Wnt/b-catenin

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ways critical for embryonic and postnatal development (including Notch, Hedgehog, Wnt and transforming growth factor-b, to name a few) enable cells to communicate with the external environment and neighboring cells (Pires-daSilva and Sommer, 2003). The exquisitely specific functional consequences of the responses generated from the pathways depend upon the induction and/or repression of downstream target gene expression. Therefore, a critical yet largely uncharacterized biological phenomenon is the conversion of cellular signaling inputs into the transcriptional regulation of target genes. Growing evidence indicates that transcription factors require other molecules (for example, co-activators and repressors) to regulate their activities (Hall and McDonnell, 2005; Li et al., 2007; Yu and Reddy, 2007). In this review, we focus on the Mastermind-like (MAML) family of transcriptional co-activators that are integral to the Notch signaling pathway. We also discuss unexpected roles for the MAML family in other signaling pathways that have been revealed recently. These findings highlight pivotal regulatory functions for MAML co-activators in multiple signaling pathways, and also implicate them as players in mediating crosstalks among pathways.

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Introduction Broadly speaking, cancer results from deregulation of the normal cellular processes that mediate proliferation, differentiation and cell death programs. Under physiological conditions, cells undergo these processes by responding to environmental stimuli through specific signaling transduction pathways. For instance, pathCorrespondence: Dr L Wu, Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, 1376 Mowry Road, Gainesville, FL 32610-3633, USA. E-mail: lzwu@ufl.edu

Transcriptional co-activators: molecules that regulate signaling-activated transcriptional events In the past, much attention has focused solely on the transcription factors that regulate gene expression in response to specific stimuli. However, a rapidly growing field offers a more complex view of gene transcription and emphasizes the importance of other molecules (including co-activators and co-repressors) in transcriptional regulation (Lai, 2002; Hall and McDonnell, 2005; Li et al., 2007; Yu and Reddy, 2007). In response to specific signals, the first step in gene expression is to assemble transcriptional complexes upstream of the core promoter (including the TATA box and transcriptional start site) by binding of the pathway-specific transcription factors. The basal transcriptional machinery is thus positioned at the core promoter, enabling transcription to proceed.

MAML co-activator in Notch and other pathways AS McElhinny et al

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Transcriptional co-activators are proteins that complex with transcription factors to regulate specific target gene expression. They promote transcriptional activation by various mechanisms including: assisting with the assembly of active transcriptional complexes, altering local chromatin structure for greater accessibility of the complex and communicating with the general transcription apparatus at target gene promoters. The coactivators are often required for maximal transcriptional activity, and are now recognized as important targets for developmental and physiological signals in many diverse biological processes. The MAML proteins, the focus of this review, are transcriptional co-activators that are essential for mediating cellular responses to the Notch signaling pathway. Recent research also implicates their coactivator functions in other signaling pathways, including those that involve MEF2C, p53 and Wnt/b-catenin. Thus, these recent data suggest broader roles for MAML proteins in regulating various biological processes.

(ICN) being released from the cell membrane and shuttling to the nucleus. This process activates the major downstream nuclear target for Notch, the CSL family of DNA-binding factors (CBF1/RBP-Jk in mammals, Su(H) in Drosophila and Lag-1 in Caenorhabditis elegans). The current prevailing model is that Notch activation results in the displacement of CSL family members’ repressors (including CIR, N-CoR/ SMRT, SPEN and KyoT2) and the recruitment of transcriptional co-activators, including MAMLs, p300 and GCN5/PCAF (Lai, 2002; Wu and Griffin, 2004; Kovall, 2007). The transcriptional complex with the core components of ICN, CSL and MAMLs then is able to activate target gene expression (including the basic helixloop-helix HES gene family), which in turn regulates the expression of tissue-specific transcription factors that control cell fates and other events. Currently, however, only a few Notch target genes have been identified and these few cannot fully explain the diverse responses of Notch receptor activation. It is also important to note that evidence indicates that Notch signaling can be mediated in a CSL-independent manner. However, the mechanisms responsible for this particular pathway are less characterized (Brennan and Gardner, 2002).

The MAML family: conserved transcriptional activators for the Notch pathway Overview of Notch signaling-mediated transcription Genetic and cellular evidence clearly demonstrate that the MAML proteins are integral to the Notch signaling pathway by regulating the transcriptional activation of Notch target gene expression. Notch signaling is a highly conserved pathway that modulates stem/precursor cells in their response to developmental cues and influences cellular decisions within multiple tissues (Artavanis-Tsakonas et al., 1999; Bray, 2006; Ilagan and Kopan, 2007). This pathway regulates diverse biological processes, including neurogenesis (Lasky and Wu, 2005), myogenesis (Luo et al., 2005), vasculogenesis (Anderson and Gibbons, 2007) and hematopoiesis (Aster et al., 2008), to name a few. Not surprisingly, aberrant Notch signaling (either deficient or abnormally increased) is linked to multiple developmental disorders and cancers (Roy et al., 2007). Exactly how one signaling pathway can exert such diverse effects on so many different cell types and cellular processes remains elusive. The multiplicity of Notch receptors and ligands expressed in different cell types, coupled with the combinations of tissue-specific transcription factors and transcriptional co-activators activated in response to specific stimuli, could at least partially explain this amazing diversity. Briefly, the Notch pathway involves the activation of Notch receptors (Notch1-4 in mammals and Notch in Drosophila) by their ligands that are expressed on neighboring cells (Jagged1, Jagged2, Delta-like1 (Dll1), Dll3 and Dll4 in mammals or Delta and Serrate in Drosophila) (Figure 1a). Upon ligand binding, the Notch receptors undergo proteolytic processing, which involves ADAM metalloproteases and the g-secretase complex, resulting in the intracellular domain of Notch

Identification of Maml proteins and investigations into their biochemical roles in Notch-mediated transcription Though we now realize the vital function of the MAML co-activators in the Notch pathway, the exact mechanisms and the roles of the MAML co-activators in signal-mediated transcription are still unclear. The identification of the MAML family members, their structural domains and known interactions are discussed below. The Mastermind homologues have been identified in Drosophila (Yedvobnick et al., 1988; Smoller et al., 1990), C. elegans (Petcherski and Kimble, 2000b), Xenopus (Katada and Kinoshita, 2003), mouse (Petcherski and Kimble, 2000b; Wu et al., 2004) and human (Wu et al., 2000, 2002; Kitagawa et al., 2001; Lin et al., 2002). The Mastermind (mam) gene first was identified in Drosophila as one of the original group of ‘neurogenic’ loci along with Notch, and loss of the function of Mam resulted in excessive neural cells at the expense of epithelial cells (Yedvobnick et al., 1988; Xu et al., 1990), indicating that the molecule regulated neuronal cell-fate decisions. The Mastermind gene has extensive genetic interactions with Notch components such as Su(H) and Deltex, and was repeatedly identified as a Notch modifier in genetic screens (Xu and Artavanis-Tsakonas, 1990; Xu et al., 1990; Fortini and Artavanis-Tsakonas, 1994; Go and Artavanis-Tsakonas, 1998). The gene was determined to encode a novel, glutamine-rich nuclear protein with novel protein sequence features suggestive of a role in transcriptional regulation (Smoller et al., 1990). Indeed, immunohistochemical studies revealed Mam binding on multiple sites of polytene chromosomes, likely co-localizing with RNA polymerase and the groucho co-repressor protein (Bettler et al., 1996). These studies identified Mastermind as an integral Oncogene


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Figure 1 Mastermind-like transcriptional co-activator (MAML) co-activates the Notch signaling pathway. (a) The Notch signaling pathway. Ligand binding between neighboring cells induces proteolytic cleavage of Notch receptors, producing the free intracellular domain of Notch (ICN). ICN translocates to the nucleus and binds to a transcription factor, CSL. An active Notch transcriptional complex, consisting of the ICN/MAML/CSL core components, is formed through displacement of the transcriptional co-repressor complex (Co-R), and likely, recruitment of additional, unidentified transcriptional co-activators (Co-A). The transcription of Notch target genes is then activated. (b) Structure and interacting domains of MAML co-activators. The human MAML family (MAML1, 2 and 3) is structurally conserved with each other and with their Drosophila Mastermind orthologue (D. Mam). They contain an Nterminal basic domain (BD) and two acidic domains. The N-terminal domain is responsible for interactions with the ankyrin domain of the Notch receptors. The sequences after the BD have transcriptional activity and are denoted as transcriptional activation domains (TADs). In MAML1, there are two TADs: TAD1, containing the CBP/p300-binding site and TAD2, whose activities are required for Notch signaling in vivo. The diagram is not drawn to scale. (c) Inhibition of Notch-mediated transcription by expression of a dominantnegative MAML1 mutant, dnMAML1. dnMAML1 contains the BD of MAML1 but lacks the entire TADs; therefore, it retains the ability to form a complex with Notch and CSL, but fails to activate transcription. It therefore interferes with endogenous Notch signaling. Oncogene


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component of the Notch signaling pathway in Drosophila and pointed toward its involvement in transcriptional regulation. Moreover, the C. elegans orthologue, LAG-3, was confirmed to have Mastermind-like activity although it exhibits limited sequence homology to Drosophila Mastermind and human MAML1 (Petcherski and Kimble, 2000a, b). LAG-3 formed a ternary complex with the LAG-1 DNA-binding protein and the intracellular domain of the receptor (GLP-1) in the nucleus and exhibited potent transcriptional activity; therefore, it was proposed to function as transcriptional co-activator. The Xenopus Mastermind 1 (XMam1) orthologue has high homology with human MAML1 and is required for primary neurogenesis by Notch signaling (Katada and Kinoshita, 2003). Taken together, these studies laid the foundation for investigating the involvement of Mastermind and its orthologues in transcriptional regulation of the Notch signaling pathway. Our group and others previously identified a family of three mammalian MAML co-activators as essential regulators of Notch-induced transcriptional events (Wu et al., 2000, 2002; Petcherski and Kimble, 2000a; Fryer et al., 2002; Lin et al., 2002; Wu and Griffin, 2004). The three human MAML genes encode nuclear proteins and have limited sequence homology with their othologues, yet are highly conserved in their structure (Figure 1b). The MAML co-activators contain a highly conserved N-terminal basic domain (BD) and two acidic domains in their middle region and C terminus. Using the functional clues obtained from Drosophila studies discussed above, the hypothesis that human MAML proteins are involved in the transcriptional regulation of Notch target gene expression was investigated. Indeed, the human MAML proteins are transcriptional coactivators because although they do not bind to DNA directly, they potentiate transcriptional activity when localized to promoters by fusion with the Gal4 DNAbinding domain. Further studies indicate that MAML proteins contain a BD that is responsible for Notch ICN binding, and transcriptional domains required for the transcriptional activation of Notch target genes (Figure 1b). Biochemically, all MAML proteins appear to form stable DNA-binding complexes with ICNs and the transcription factor CSL upon Notch activation (Lin et al., 2002; Wu et al., 2002) and potentiate transcription of Notch target genes. These data led to a model in which MAML proteins are recruited to Notch/CSL on the target gene promoters when Notch receptors are activated, contributing to transcriptional activation. In support of this model, MAML1 mutants that are either deficient in transcriptional activities or incapable of binding ICN1 behave as dominant negatives and interfere with the ability of MAML1 to activate Notch signaling (Wu et al., 2000). The isolation of a large, stable multiprotein complex containing the endogenous MAML1, ICN and CSL, is also consistent with MAML proteins being core components of a Notch transcriptional complex (Jeffries et al., 2002). Finally, crystal structure analyses of the Notch core complex confirmed that the interaction of ICN and CSL creates the

recruitment site for MAML proteins (Nam et al., 2006; Wilson and Kovall, 2006). Mechanistically, MAML1 is required for chromatin-dependent transactivation by the reconstituted Notch ICN-CSL enhancer complex in vitro (Fryer et al., 2002; Wallberg et al., 2002). Currently, the underlying mechanisms responsible for MAML co-activation functions remain unclear and identification of interacting molecules is required. Though more detailed mapping studies are needed, two transcriptional activation domains (TAD) of the MAML1 protein have been proposed (Figure 1b). (1) TAD1 is located in the region of aa 75–300 of MAML1, and contains a p300/CBP-binding site. Thus, MAML1 recruits the chromatin modification enzyme p300/CBP and leads to nucleosome acetylation at Notch enhancers to activate transcription in vitro (Fryer et al., 2002). However, the recruitment of p300/CBP is not sufficient to activate the transcription of Notch target genes in vivo, because an MAML1 mutant, MAML1 (1-302) containing TAD1 and retaining the p300 binding activity failed to activate Notch target genes and actually interfered with Notch signaling in vivo (Wu et al., 2000). Moreover, the MAML1 and p300 interaction leads to mutual modifications: MAML1 causes p300 phosphorylation, whereas p300 mediates MAML1 acetylation (Fryer et al., 2002; Saint Just Ribeiro et al., 2007). The exact functional significance of these post-translational modifications in Notch signalbased transcription remains unknown. (2) TAD2 extends from the center of MAML1 to its C-terminal region (aa 303–1016), which contains glutamine-rich sequences. TAD2 is required for transcription in vivo. Therefore, unknown proteins that interact with TAD2 likely are required for Notch target gene activation. A protein that interacts directly with MAML1 is CDK8 that can cause ICN1 phosphorylation and degradation (Fryer et al., 2004). The exact binding site was not mapped, but neither MAML1 mutant with deletion aa 75–300 nor 1–301 binds to CDK8. This suggests an additional regulatory role for MAML1 to recruit CDK8 to target gene promoters to degrade the Notch enhancer complex, thereby terminating Notch signaling. Although more studies have investigated MAML1, it has been determined that both MAML2 and MAML3 interact with Notch receptors and also interact with p300 (Lin et al., 2002; Wu et al., 2002; L Wu et al., unpublished). Additional interacting proteins have not yet been identified. It is intriguing to speculate that there are binding proteins specific for each MAML member, contributing to their differential activation and activities. Therefore, much work is required to dissect the structural domains of the MAML family and to identify their specific interacting partners before their functional roles and mechanisms of actions can be elucidated. Investigations into MAML co-activators that regulate Notch signaling in various biological processes The three Maml members exhibit distinct expression patterns during embryonic development (Wu et al., Oncogene


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2002, 2004), supporting the hypothesis that they play specific roles in different tissues. On the basis of reporter assays in cultured cells, the three human MAML proteins have overlapping and differential activities in terms of their abilities to activate Notch receptormediated transcription: MAML1 and MAML2 coactivate all four Notch receptors, whereas MAML3 seems only to effectively co-activate Notch4. Thus, MAMLs have the potential to cooperate differentially with various Notch receptors in the activation of target genes, which could contribute to the wide range of biological effects upon activation of the Notch pathway. Studies have begun to investigate the functional roles of MAML family members in regulating Notch-based transcriptional events in multiple cellular processes. One valuable tool is a dominant-negative MAML1 mutant, DNMAML1 (Figure 1c). This mutant consists of a 62 amino-acid peptide of the N-terminal BD of MAML1 that structurally was shown to interact with the Notch– CSL transcriptional complex (Nam et al., 2006). DNMAML1 is a Pan-Notch inhibitor: it interferes with the endogenous function of MAML proteins and inhibits transcriptional activation from all four Notch receptors (Weng et al., 2003). Other strategies that have been successful in determining Notch roles in cellular processes is by the inhibition of Notch receptor processing by inactivating the g-secretase complex with specific inhibitors (GSI), and development of mouse models that are deficient of various Notch signaling components. These studies have shown, for example, that Notch signaling has well-established roles in lymphoid development, although the exact functions of the four Notch receptors are not equal. Loss of the Notch1 receptor perturbs T-cell development, yet the B-cell compartment appears unaffected (Radtke et al., 1999). Conversely, the targeted inactivation of the Notch2 receptor or Dll1 ligand results in a loss of marginal zone B cells (MZB cells), but T-cell development appears normal (Saito et al., 2003; Hozumi et al., 2004). Importantly, these effects are consistent with phenotypes caused by the inactivation of canonical Notch signaling in these compartments, because loss of the transcription factor CSL blocks both T-cell development and MZB cell generation (Han et al., 2002; Tanigaki et al., 2002). Not unexpectedly, then, the expression of DNMAML1 in bone marrow cells abrogated canonical Notch signaling, inhibiting both T-cell development and MZB cell generation (Maillard et al., 2004). Taken together, these data revealed that the MAML genes collectively are essential in mediating physiological Notch functions. Importantly, DNMAML1 knock-in mouse models recently were generated to investigate the in vivo functional consequences of inhibiting the endogenous MAML functions in Notch signaling. Defects were readily determined in several systems, including: Thelper cell development (Tu et al., 2005), skin (Proweller et al., 2006), vascular smooth muscle (Proweller et al., 2007) and cardiovascular development (High et al., 2007). These studies revealed that MAML proteins indeed are critical to mediate Notch signaling in Oncogene

multiple tissues in vivo. It should be noted that DNMAML1 seems to be specific for Notch signaling. However, the possibility that DNMAML1 might interfere with other Notch-independent functions cannot be ruled out. More studies are required to characterize the effects of DNMAML1 in light of newly discovered Notch-independent MAML functions (discussed below). Current studies have begun to dissect the roles of individual MAML co-activators in vivo. A mouse model with a targeted disruption of the Maml1 gene was recently reported (Shen et al., 2006; Oyama et al., 2007; Wu et al., 2007). The Maml1-knockout mice exhibited several defects (including muscle defects which is discussed later). They remained small in size and died within the perinatal period. The effect of Maml1 knockout on lymphoid development was characterized to evaluate its contribution in mediating Notch functions. Intriguingly, the Maml1 deficiency had no obvious effect on T-cell development, but did result in the absence of MZB cells, a phenotype similar to the phenotype of Notch2 or Dll1 ligand deficiencies described above. Moreover, after analyses of the Maml1 heterozygote mice, there appeared to be a dosage effect for Maml1 levels in MZB cell formation. These studies revealed an unexpected, in vivo role for Maml1 in mediating the signaling of a specific Notch receptor, Notch2. The specific requirement for Maml1 in the Bcell compartment could not be explained simply by expression patterns of Mamls, because at least two Maml members, Maml1 and 2 are expressed in splenic B cells. Rather, it is hypothesized that Maml1 provides specific molecular features to the Notch2 transcriptional activation complex that are not compensated by other Maml members. These data favor the model that individual Maml co-activators contribute to the molecular specificity of Notch receptor functions in vivo. Currently, the roles for Maml1 in mediating Notch signaling in other tissues have not been carefully examined. However, one additional interesting finding is that there appear to be defects in a subpopulation of muscle precursors (satellite cells) that are responsible for muscle regeneration in Maml1-deficient mice (H Shen et al., unpublished). Therefore, we speculate that Maml1 loss inhibits the Notch response that is known to be essential for the activation and expansion of satellite cells, the early steps of myogenic process. These ideas warrant further investigation and are being tested. In summary, the precise roles of individual MAML co-activators in vivo, and whether they contribute to the diverse biological effects of Notch signaling, remain to be addressed. Fortunately, detailed structural information is now available regarding the trimolecular complex between CSL, Notch1 and the N-terminal Notchbinding domain of MAML1 (Nam et al., 2006; Wilson and Kovall, 2006). Therefore, investigating the structural differences among various combinations of transcriptional complexes containing distinct Notch and Maml family members will provide valuable information about their in vivo specificity. Finally, complete expression profiles of the three MAMLs in


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different tissues and at different development stages, as well as characterization of mouse models lacking each co-activator individually and in combination, will provide a better understanding of the functions of Maml co-activators in Notch signaling-based responses. Emerging Notch-independent functions of the MAML proteins and their involvement with other signaling pathways MAML1 co-activates MEF2C muscle-based transcriptional events and is required for myogenesis The first indication that MAML functioned independently of the Notch pathway came from investigations using an Maml1 knockout mouse model (Shen et al., 2006). These mice display a severe perturbation of overall skeletal muscle structure consistent with a muscular dystrophy-like phenotype, including degeneration of myofibers and muscle necrosis. Maml1-null muscle embryonic fibroblasts (MEFs) failed to initiate myogenic differentiation after MyoD transduction, suggesting an intrinsic requirement for Maml1 in myogenesis. Indeed, Maml1 is required for muscle differentiation from myoblast to myotube formation, because Maml1 RNAi-mediated knockdown blocked the differentiation of C2C12 cells (a myoblast cell line). Maml1 also promotes myogenic differentiation, as C2C12 cells that overexpress MAML1 exhibited dramatically enhanced levels of muscle myosin and formed strikingly large myotubes. These data indicated that MAML1 expression is essential for muscle differentiation events, and for mediating muscle gene expression. The pro-myogenic activities of MAML1 were surprising and unexpected, as previous studies established that activation of Notch signaling actually inhibited myogenic differentiation in C2C12 cells (Kopan et al., 1994; Lindsell et al., 1995). In fact, Notch signaling is inhibited by the Notch antagonist, Numb, during the later step of myogenesis, myogenic differentiation (Luo et al., 2005). Therefore, we hypothesized that Maml1 possesses Notch-independent functions that might account for its pro-myogenic activities. This led us to investigate the functional interaction of Maml1 and transcription factors important for myogenic differentiation, resulting in the finding that Maml1 interacts with and co-activates MEF2C, a critical muscle-specific transcription factor. Furthermore, our preliminary studies indicate that myogenin, also a key muscle transcription factor, is a direct target of Maml1, and its expression is regulated by the Maml1/MEF2C interaction. Consistent with these data, activation of the Notch pathway (by ligand stimulation or the ectopic expression of the constitutively activated form of ICN) inhibited MAML1enhanced myogenesis in C2C12 cells (Shen et al., 2006). Biochemically, the blockage of MAML1’s promyogenic effects by Notch activation was associated with the recruitment of MAML1 away from MEF2C to the Notch transcriptional complex.

These studies revealed novel and unique roles for MAML1 that are independent of the Notch signaling pathway. A model was proposed where the Notch pathway was active during myoblast proliferation, and then silenced upon activation of myoblasts to differentiate into mature muscle. MAML1 then would ‘switch’ to function as a potent transcriptional coactivator of MEF2C for muscle differentiation transcriptional events to occur. Importantly, these studies suggested the exciting possibility that MAML family members may serve as co-activators for other pathways than Notch, and perhaps mediate cross talks between various pathways and Notch (Shen et al., 2006). Other studies described below support this intriguing hypothesis. MAML1 co-activates p53, which is required for p53-mediated germ-cell apoptotic response The p53 tumor suppressor mediates cell-cycle arrest and apoptosis in response to cellular stresses, including DNA damage, hypoxia and oncogene activation, and is important in both normal developmental processes and cancer (Vousden and Lane, 2007). Another exciting, recent finding is that MAML1 exhibits Notch-independent functions in co-activating p53-dependent gene expression, thereby potentially regulating p53-mediated responses (Zhao et al., 2007). Chromatin immunoprecipitation assays indicate that MAML1 is a component of the activator complex that binds to native p53-response elements within the promoters of p53 target genes. The MAML1/p53 interaction involves the N-terminal region of MAML1 and the DNA-binding domain of p53. Overexpression of wild-type MAML1 enhanced the specific induction of p53-dependent genes in mammalian cells. Conversely, MAML1 knockdown reduced p53dependent gene expression. Thus, these studies indicate that MAML1 is essential for p53-dependent gene expression. Interestingly, MAML1 also increases the half-life of the p53 protein and enhances its phosphorylation/ acetylation upon DNA damage in cells. In C. elegans, knockdown of the MAML homologue Lag-3 abrogated the p53-mediated germ-cell apoptotic response that typically occurs in response to DNA damage. Furthermore, the knock down of Lag-3 reduced the normal expression patterns of downstream pro-apoptotic targets of the C. elegans p53 homologue, Cep-1, including Ced-13 and Egl-1. These data clearly demonstrate a role for Lag-3 in p53-mediated apoptosis in C. elegans, although more work is required to decipher the mechanisms involved. However, these studies have set the stage for investigating the intriguing regulation of p53-mediated gene expression by MAML family members. MAML1 co-activates b-catenin-based transcriptional events and is essential for colon carcinoma cell survival Misregulation of the Wnt signaling pathway, a pathway critical for cellular proliferation and differentiation, is associated with various disorders including colon Oncogene


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carcinoma (Segditsas and Tomlinson, 2006). The tight control of nuclear levels of b-catenin is a key regulatory event for Wnt/b-catenin signaling. In the absence of Wnt signaling, b-catenin remains sequestered in the cytosol in a phosphorylated state, which leads to its degradation by a proteosome. The binding of the Wnt ligand to Frizzled and LDL receptors on the cell surface initiates multiple events, including the recruitment of Dishevelled and inactivation of glycogen synthase kinase 3b (GSK-3b). Inhibiting the phosphorylation of b-catenin by GSK-3b leads to the translocation of b-catenin to the nucleus, where it interacts with the T-cell factor (TCF) family of transcription factors and activates target gene expression. Aberrant Wnt signaling can lead to constitutive transcriptional activation of b-catenin/TCF target genes, resulting in enhanced cellular proliferation in the absence of appropriate cues. MAML1 recently was determined to be a co-activator for b-catenin-mediated transcription (Alves-Guerra et al., 2007). In these studies, MAML1 interacted with b-catenin in vitro and in vivo and dramatically increased the transcriptional activity of b-catenin on promoters containing TCF-binding sites. The other two MAML family members have similar co-activating functions. Mechanistically, it appears that MAML1 is recruited by b-catenin on Wnt target gene promoters (for example, cyclin D1 and c-Myc) even in the presence of the Notch signaling inhibitor, GSI. These data indicate that MAML1 functions in the Wnt/bcatenin pathway independently of Notch signaling. Strikingly, the knockdown of MAML proteins in colonic carcinoma cells, SW480, resulted in cell death, which correlated with decreased b-catenin-induced expression of cyclin D1 and c-Myc. Thus, MAML proteins appear to be essential for SW480 colonic cell survival by transcriptional co-activation of b-cateninmediated transcription. Other potential Notch-independent functions from Xenopus and Drosophila systems Evidence that MAML proteins have Notch-independent activities also has been established from studies using Xenopus and Drosophila systems. In Xenopus embryos, overexpression of XMam1 caused the formation of a pigmented cell mass on their surface and induced expression of RNA-binding protein nrp-1. However, this effect was not observed when Notch signaling was activated ectopically through constitutive activation of the Notch receptor (Katada et al., 2006). This indicates that Xmam1 affects a neurogenic lineage in a Notchindependent pathway. Recently, a genetic screen was performed using the Exelixis collection of insertional mutations with a goal to identify Mastermind modifiers (Kankel et al., 2007). This screen took advantage of a Drosophila mutant (C96-MamN) that displays a fully penetrant wingmargin phenotype associated with the expression of the N-terminal Mastermind protein, MamN (Helms et al., 1999). The screen searched for mutations in the Oncogene

Exelixis collection that dominantly modified the wing-margin phenotype of C96-MamN. The genes identified with enhancer or suppressor functions were subsequently subjected to a second screening to determine Notch pathway-dependent or -independent functions. In the end, 175 known and 160 novel genes were found to interact with Notch components and designated as Notch interactors, whereas 79 failed to interact with the Notch pathway thus were defined as Mastermind-specific interactors (MSI). The MSI genes were classified into several distinct functional categories, including: negative transcriptional regulators, factors with RNA polymerase II transcriptional activities, factors with small GTPase regulator activities and factors that negatively regulate metabolism. It is possible that these MSIs potentially interact with other Notch pathway components that were not included in this screen. However, this study verified that the interaction of one MSI gene, the Drosophila b-catenin homologue, armadillo, interacts with Mam independently of Notch in both the wing and eye. These data confirm the finding that MAML proteins function independently of Notch as a co-activator for b-catenin signaling (as discussed above). Therefore, multiple MSIs from this screen indicate a broad and prominent role for Mam in various developmental processes.

Speculations on functional contributions: cross talk regulation by the MAML co-activators Figure 2 summarizes the findings that we discussed above, showing that MAML proteins participate in multiple signaling pathways: Notch, MEF2C, p53, bcatenin and potentially others. These findings reveal pivotal regulatory functions for MAML co-activators in diverse biological processes including cell proliferation, differentiation and survival, and highlight the central roles for MAML co-activators in signaling cross talk. Many questions remain, however, including whether the MAML co-activators participate in multiple signaling pathways simultaneously or within certain signaling contexts, and how these activities contribute to the functional biological outcomes. From the biochemical aspects, some clues can be inferred. On the basis of immunoprecipitation studies, the MAML interactions with Notch/CSL appear to be more stable compared to MAML interactions with MEF2C, p53 and b-catenin (L Wu et al., unpublished). Although preliminary, these data lead to the hypothesis that MAML proteins mainly function as co-activators for Notch/CSL when Notch receptors are activated, but are co-activators for other signaling pathways when Notch is inactive. This hypothesis was supported by our previous findings that the Notch effects are dominant over MAML1’s myogenic activities with MEF2C (Shen et al., 2006). However, it is anticipated that there may be more complicated scenarios than just simple functional switches. Future investigations into relative


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MAML co-activators: links to human cancers Though it is not a major focus for this review, it is worthwhile to point out that growing evidence implicates MAML co-activators in cancers, including mucoepidermoid carcinoma, leukemia and potentially cervical cancer (Wu and Griffin, 2004). This is expected considering the essential regulatory roles for MAML proteins in Notch signaling as well as other signaling pathways, as discussed in this review. Importantly, the MAML co-activators may be excellent candidates as targets to modulate the growing number of signaling pathways in which they participate. Currently, we know more about the functions of the MAML proteins in the regulation of Notch signaling than other signaling pathways. Inhibition of Notch signaling has been achieved by interference with the recruitment of the MAML proteins to Notch/CSL complex (using either DNMAML1 or small molecular compounds). Thus, more work is needed to support preliminary studies that show aberrant Notch signaling can be controlled to inhibit cell growth and survival in cancer cells with overactive Notch signaling (Weng et al., 2003; Liu et al., 2006). In addition, investigations into MAML functions in other pathways, including Wnt, p53 and MEF2C-based signaling, may be of therapeutic importance in the future.

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β-catenin TCF

Figure 2 Summary of the co-activator functions for Mastermindlike transcriptional co-activator (MAML) in both Notch-dependent and Notch-independent pathways. (a) MAMLs co-activate Notch-mediated transcription, as explained in Figure 1. (b) MAMLs also co-activate MEF2C-mediated transcription and are required for muscle development. MAML/MEF2C interaction may be direct or indirect. (c) MAMLs also co-activate p53mediated transcription in a Notch-independent manner. (d) MAMLs also were recently determined to co-activate b-cateninmediated transcription in a Notch-independent manner. It is possible that unidentified other co-activators (CoA, yellow) are required for these events to occur.

contributions of pathway-specific effects mediated by the MAML co-activators and the potential interactions of these pathways in various biological systems will shed light on their functions in developmental and oncogenic contexts.

Concluding remarks The family of MAML genes encodes transcriptional co-activators required for Notch signaling. Recent discoveries of MAML involvement in the regulation of other signaling pathways have indicated much broader roles for the MAML co-activators in regulating biological functions, and also point toward a central role for MAML co-activators in mediating signaling cross talks. However, many questions remain to be addressed regarding the mechanisms underlying the coactivator functions of MAML proteins in Notch and other relevant pathways. These issues include investigations into the interactions of MAML-regulated signaling pathways and the individual and collective contributions of the MAML proteins in distinct cell types and diseased contexts. Understanding these questions will provide guidance for manipulating signaling pathways critical for cancer development and therapeutic treatments. Acknowledgements This work was supported in part by NIH (R01 CA097148) and Muscular Dystrophy Association (MDA).

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