Mastermind-Like Transcriptional Co-Activators: Emerging Roles in Regulating Cross Talk Among Multiple Signaling Pathways

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Oncogene (2008) 27, 5138–5147 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $30.00 www.nature.com/onc REVIEW Mastermind-like transcriptional co-activators: emerging roles in regulating cross talk among multiple signaling pathways

AS McElhinny1, J-L Li2 and L Wu3

1Ventana 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 ways critical for embryonic and postnatal development critical transcriptional co-activators for Notch signaling, (including Notch, Hedgehog, Wnt and transforming an evolutionarily conserved pathway with numerous roles growth factor-b, to name a few) enable cells to in both development and human diseases. Notch receptors communicate with the external environment and neigh- are cleaved upon ligand engagement and the intracellular boring cells (Pires-daSilva and Sommer, 2003). The domain of Notch shuttles to the nucleus. MAMLs form a exquisitely specific functional consequences of the functional DNA-binding complex with the cleaved Notch responses generated from the pathways depend upon receptor and the transcription factor CSL, thereby the induction and/or repression of downstream target regulating transcriptional events that are specific to the gene expression. Therefore, a critical yet largely Notch pathway. Here, we review recent studies that have uncharacterized biological phenomenon is the conver- utilized molecular, cellular and physiological model sion of cellular signaling inputs into the transcriptional system strategies to reveal the pivotal roles of the MAML regulation of target genes. proteins in Notch signaling. Unexpectedly, however, Growing evidence indicates that transcription factors emerging evidence implicate MAML proteins as exciting require other molecules (for example, co-activators and key transcriptional co-activators in other signal transduc- repressors) to regulate their activities (Hall and tion pathways including: muscle differentiation and McDonnell, 2005; Li et al., 2007; Yu and Reddy, myopathies (MEF2C), tumor suppressor pathway (p53) 2007). In this review, we focus on the Mastermind-like and colon carcinoma survival (b-catenin). Thus, the (MAML) family of transcriptional co-activators that are MAML family appears to function in transcriptional co- integral to the Notch signaling pathway. We also discuss activation in a multitude of cellular processes. It is unexpected roles for the MAML family in other hypothesized that MAML proteins mediate cross-talk signaling pathways that have been revealed recently. among the various signaling pathways and the diverse These findings highlight pivotal regulatory functions for activities of the MAML proteins converge to impact MAML co-activators in multiple signaling pathways, normal biological processes and human diseases, including and also implicate them as players in mediating cross- cancers. talks among pathways. Oncogene (2008) 27, 5138–5147; doi:10.1038/onc.2008.228

Keywords: Mastermind-like co-activator; Notch; MEF2C; p53; Wnt/b-catenin 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 Introduction response to specific stimuli. However, a rapidly growing field offers a more complex view of gene transcription Broadly speaking, cancer results from deregulation of and emphasizes the importance of other molecules the normal cellular processes that mediate proliferation, (including co-activators and co-repressors) in transcrip- differentiation and cell death programs. Under physio- tional regulation (Lai, 2002; Hall and McDonnell, 2005; logical conditions, cells undergo these processes by Li et al., 2007; Yu and Reddy, 2007). responding to environmental stimuli through specific In response to specific signals, the first step in gene signaling transduction pathways. For instance, path- expression is to assemble transcriptional complexes upstream of the core promoter (including the TATA box and transcriptional start site) by binding of Correspondence:Dr L Wu, Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, 1376 the pathway-specific transcription factors. The basal Mowry Road, Gainesville, FL 32610-3633, USA. transcriptional machinery is thus positioned at the E-mail:lzwu@ufl.edu core promoter, enabling transcription to proceed. MAML co-activator in Notch and other pathways AS McElhinny et al 5139 Transcriptional co-activators are proteins that complex (ICN) being released from the cell membrane and with transcription factors to regulate specific target gene shuttling to the nucleus. This process activates the expression. They promote transcriptional activation by major downstream nuclear target for Notch, the CSL various mechanisms including:assisting with the assem- family of DNA-binding factors (CBF1/RBP-Jk in bly of active transcriptional complexes, altering local mammals, Su(H) in Drosophila and Lag-1 in Caenor- chromatin structure for greater accessibility of the habditis elegans). The current prevailing model is that complex and communicating with the general transcrip- Notch activation results in the displacement of CSL tion apparatus at target gene promoters. The co- family members’ repressors (including CIR, N-CoR/ activators are often required for maximal transcriptional SMRT, SPEN and KyoT2) and the recruitment of activity, and are now recognized as important targets for transcriptional co-activators, including MAMLs, p300 developmental and physiological signals in many diverse and GCN5/PCAF (Lai, 2002; Wu and Griffin, 2004; biological processes. Kovall, 2007). The transcriptional complex with the core The MAML proteins, the focus of this review, are components of ICN, CSL and MAMLs then is able to transcriptional co-activators that are essential for activate target gene expression (including the basic helix- mediating cellular responses to the Notch signaling loop-helix HES gene family), which in turn regulates the pathway. Recent research also implicates their co- expression of tissue-specific transcription factors that activator functions in other signaling pathways, includ- control cell fates and other events. Currently, however, ing those that involve MEF2C, p53 and Wnt/b-catenin. only a few Notch target genes have been identified and Thus, these recent data suggest broader roles for these few cannot fully explain the diverse responses of MAML proteins in regulating various biological pro- Notch receptor activation. It is also important to note cesses. 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 Identification of Maml proteins and investigations into Overview of Notch signaling-mediated transcription their biochemical roles in Notch-mediated transcription Genetic and cellular evidence clearly demonstrate that Though we now realize the vital function of the MAML the MAML proteins are integral to the Notch signaling co-activators in the Notch pathway, the exact mechan- pathway by regulating the transcriptional activation of isms and the roles of the MAML co-activators in Notch target gene expression. Notch signaling is a signal-mediated transcription are still unclear. The highly conserved pathway that modulates stem/precur- identification of the MAML family members, their sor cells in their response to developmental cues and structural domains and known interactions are dis- influences cellular decisions within multiple tissues cussed below. (Artavanis-Tsakonas et al., 1999; Bray, 2006; Ilagan The Mastermind homologues have been identified in and Kopan, 2007). This pathway regulates diverse Drosophila (Yedvobnick et al., 1988; Smoller et al., biological processes, including neurogenesis (Lasky 1990), C. elegans (Petcherski and Kimble, 2000b), and Wu, 2005), myogenesis (Luo et al., 2005), vasculo- Xenopus (Katada and Kinoshita, 2003), mouse (Petch- genesis (Anderson and Gibbons, 2007) and hemato- erski and Kimble, 2000b; Wu et al., 2004) and human poiesis (Aster et al., 2008), to name a few. Not (Wu et al., 2000, 2002; Kitagawa et al., 2001; Lin et al., surprisingly, aberrant Notch signaling (either deficient 2002). The Mastermind (mam) gene first was identified or abnormally increased) is linked to multiple develop- in Drosophila as one of the original group of ‘neuro- mental disorders and cancers (Roy et al., 2007). Exactly genic’ loci along with Notch, and loss of the function of how one signaling pathway can exert such diverse effects Mam resulted in excessive neural cells at the expense of on so many different cell types and cellular processes epithelial cells (Yedvobnick et al., 1988; Xu et al., 1990), remains elusive. The multiplicity of Notch receptors and indicating that the molecule regulated neuronal cell-fate ligands expressed in different cell types, coupled with the decisions. The Mastermind gene has extensive genetic combinations of tissue-specific transcription factors and interactions with Notch components such as Su(H) and transcriptional co-activators activated in response to Deltex, and was repeatedly identified as a Notch specific stimuli, could at least partially explain this modifier in genetic screens (Xu and Artavanis-Tsakonas, amazing diversity. 1990; Xu et al., 1990; Fortini and Artavanis-Tsakonas, Briefly, the Notch pathway involves the activation of 1994; Go and Artavanis-Tsakonas, 1998). The gene was Notch receptors (Notch1-4 in mammals and Notch in determined to encode a novel, glutamine-rich nuclear Drosophila) by their ligands that are expressed on protein with novel protein sequence features suggestive neighboring cells (Jagged1, Jagged2, Delta-like1 (Dll1), of a role in transcriptional regulation (Smoller et al., Dll3 and Dll4 in mammals or Delta and Serrate in 1990). Indeed, immunohistochemical studies revealed Drosophila) (Figure 1a). Upon ligand binding, the Mam binding on multiple sites of polytene chromo- Notch receptors undergo proteolytic processing, which somes, likely co-localizing with RNA polymerase and involves ADAM metalloproteases and the g-secretase the groucho co-repressor protein (Bettler et al., 1996). complex, resulting in the intracellular domain of Notch These studies identified Mastermind as an integral

Oncogene MAML co-activator in Notch and other pathways AS McElhinny et al 5140

Signaling cell

Delta Jagged Notch1-4

ICN

Signal-receiving cell

Transcription Activation Domains (TADs)

Basic Acidic Acidic 1016 1 MAML1 BD

TAD1 TAD2 Notch p300/CBP 1 1153 MAML2

1 1133 MAML3

1 1596 D. Mam

Co-A ICN DNMAML1

ICN MAML DNMAML1 CSL (BD only) STOP CSL

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 N- terminal 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 dominant- negative 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 MAML co-activator in Notch and other pathways AS McElhinny et al 5141 component of the Notch signaling pathway in Droso- recruitment site for MAML proteins (Nam et al., phila and pointed toward its involvement in transcrip- 2006; Wilson and Kovall, 2006). Mechanistically, tional regulation. Moreover, the C. elegans orthologue, MAML1 is required for chromatin-dependent transac- LAG-3, was confirmed to have Mastermind-like activity tivation by the reconstituted Notch ICN-CSL enhancer although it exhibits limited sequence homology to complex in vitro (Fryer et al., 2002; Wallberg et al., Drosophila Mastermind and human MAML1 (Petch- 2002). erski and Kimble, 2000a, b). LAG-3 formed a ternary Currently, the underlying mechanisms responsible for complex with the LAG-1 DNA-binding protein and the MAML co-activation functions remain unclear and intracellular domain of the receptor (GLP-1) in the identification of interacting molecules is required. nucleus and exhibited potent transcriptional activity; Though more detailed mapping studies are needed, therefore, it was proposed to function as transcriptional two transcriptional activation domains (TAD) of the co-activator. The Xenopus Mastermind 1 (XMam1) MAML1 protein have been proposed (Figure 1b). (1) orthologue has high homology with human MAML1 TAD1 is located in the region of aa 75–300 of MAML1, and is required for primary neurogenesis by Notch and contains a p300/CBP-binding site. Thus, MAML1 signaling (Katada and Kinoshita, 2003). Taken together, recruits the chromatin modification enzyme p300/CBP these studies laid the foundation for investigating the and leads to nucleosome acetylation at Notch enhancers involvement of Mastermind and its orthologues in to activate transcription in vitro (Fryer et al., 2002). transcriptional regulation of the Notch signaling pathway. However, the recruitment of p300/CBP is not sufficient Our group and others previously identified a family of to activate the transcription of Notch target genes in three mammalian MAML co-activators as essential vivo, because an MAML1 mutant, MAML1 (1-302) regulators of Notch-induced transcriptional events containing TAD1 and retaining the p300 binding (Wu et al., 2000, 2002; Petcherski and Kimble, 2000a; activity failed to activate Notch target genes and Fryer et al., 2002; Lin et al., 2002; Wu and Griffin, actually interfered with Notch signaling in vivo (Wu 2004). The three human MAML genes encode nuclear et al., 2000). Moreover, the MAML1 and p300 proteins and have limited sequence homology with their interaction leads to mutual modifications:MAML1 othologues, yet are highly conserved in their structure causes p300 phosphorylation, whereas p300 mediates (Figure 1b). The MAML co-activators contain a highly MAML1 acetylation (Fryer et al., 2002; Saint Just conserved N-terminal basic domain (BD) and two acidic Ribeiro et al., 2007). The exact functional significance of domains in their middle region and C terminus. Using these post-translational modifications in Notch signal- the functional clues obtained from Drosophila studies based transcription remains unknown. (2) TAD2 discussed above, the hypothesis that human MAML extends from the center of MAML1 to its C-terminal proteins are involved in the transcriptional regulation of region (aa 303–1016), which contains glutamine-rich Notch target gene expression was investigated. Indeed, sequences. TAD2 is required for transcription in vivo. the human MAML proteins are transcriptional co- Therefore, unknown proteins that interact with TAD2 activators because although they do not bind to DNA likely are required for Notch target gene activation. A directly, they potentiate transcriptional activity when protein that interacts directly with MAML1 is CDK8 localized to promoters by fusion with the Gal4 DNA- that can cause ICN1 phosphorylation and degradation binding domain. Further studies indicate that MAML (Fryer et al., 2004). The exact binding site was not proteins contain a BD that is responsible for Notch ICN mapped, but neither MAML1 mutant with deletion aa binding, and transcriptional domains required for the 75–300 nor 1–301 binds to CDK8. This suggests an transcriptional activation of Notch target genes additional regulatory role for MAML1 to recruit CDK8 (Figure 1b). to target gene promoters to degrade the Notch enhancer Biochemically, all MAML proteins appear to form complex, thereby terminating Notch signaling. stable DNA-binding complexes with ICNs and the Although more studies have investigated MAML1, it transcription factor CSL upon Notch activation (Lin has been determined that both MAML2 and MAML3 et al., 2002; Wu et al., 2002) and potentiate transcription interact with Notch receptors and also interact with of Notch target genes. These data led to a model in p300 (Lin et al., 2002; Wu et al., 2002; L Wu et al., which MAML proteins are recruited to Notch/CSL on unpublished). Additional interacting proteins have not the target gene promoters when Notch receptors are yet been identified. It is intriguing to speculate that there activated, contributing to transcriptional activation. In are binding proteins specific for each MAML member, support of this model, MAML1 mutants that are either contributing to their differential activation and activ- deficient in transcriptional activities or incapable of ities. Therefore, much work is required to dissect the binding ICN1 behave as dominant negatives and structural domains of the MAML family and to identify interfere with the ability of MAML1 to activate Notch their specific interacting partners before their functional signaling (Wu et al., 2000). The isolation of a large, roles and mechanisms of actions can be elucidated. stable multiprotein complex containing the endogenous MAML1, ICN and CSL, is also consistent with MAML proteins being core components of a Notch transcrip- Investigations into MAML co-activators that regulate tional complex (Jeffries et al., 2002). Finally, crystal Notch signaling in various biological processes structure analyses of the Notch core complex confirmed The three Maml members exhibit distinct expression that the interaction of ICN and CSL creates the patterns during embryonic development (Wu et al.,

Oncogene MAML co-activator in Notch and other pathways AS McElhinny et al 5142 2002, 2004), supporting the hypothesis that they play multiple tissues in vivo. It should be noted that specific roles in different tissues. On the basis of reporter DNMAML1 seems to be specific for Notch signaling. assays in cultured cells, the three human MAML However, the possibility that DNMAML1 might inter- proteins have overlapping and differential activities in fere with other Notch-independent functions cannot be terms of their abilities to activate Notch receptor- ruled out. More studies are required to characterize the mediated transcription:MAML1 and MAML2 co- effects of DNMAML1 in light of newly discovered activate all four Notch receptors, whereas MAML3 Notch-independent MAML functions (discussed be- seems only to effectively co-activate Notch4. Thus, low). MAMLs have the potential to cooperate differentially Current studies have begun to dissect the roles of with various Notch receptors in the activation of target individual MAML co-activators in vivo. A mouse model genes, which could contribute to the wide range of with a targeted disruption of the Maml1 gene was biological effects upon activation of the Notch pathway. recently reported (Shen et al., 2006; Oyama et al., 2007; Studies have begun to investigate the functional roles Wu et al., 2007). The Maml1-knockout mice exhibited of MAML family members in regulating Notch-based several defects (including muscle defects which is transcriptional events in multiple cellular processes. One discussed later). They remained small in size and died valuable tool is a dominant-negative MAML1 mutant, within the perinatal period. The effect of Maml1 DNMAML1 (Figure 1c). This mutant consists of a 62 knockout on lymphoid development was characterized amino-acid peptide of the N-terminal BD of MAML1 to evaluate its contribution in mediating Notch func- that structurally was shown to interact with the Notch– tions. Intriguingly, the Maml1 deficiency had no CSL transcriptional complex (Nam et al., 2006). obvious effect on T-cell development, but did result in DNMAML1 is a Pan-Notch inhibitor:it interferes with the absence of MZB cells, a phenotype similar to the the endogenous function of MAML proteins and phenotype of Notch2 or Dll1 ligand deficiencies inhibits transcriptional activation from all four Notch described above. Moreover, after analyses of the Maml1 receptors (Weng et al., 2003). Other strategies that have heterozygote mice, there appeared to be a dosage effect been successful in determining Notch roles in cellular for Maml1 levels in MZB cell formation. These studies processes is by the inhibition of Notch receptor revealed an unexpected, in vivo role for Maml1 in processing by inactivating the g-secretase complex with mediating the signaling of a specific Notch receptor, specific inhibitors (GSI), and development of mouse Notch2. The specific requirement for Maml1 in the B- models that are deficient of various Notch signaling cell compartment could not be explained simply by components. expression patterns of Mamls, because at least two These studies have shown, for example, that Notch Maml members, Maml1 and 2 are expressed in splenic B signaling has well-established roles in lymphoid devel- cells. Rather, it is hypothesized that Maml1 provides opment, although the exact functions of the four Notch specific molecular features to the Notch2 transcriptional receptors are not equal. Loss of the Notch1 receptor activation complex that are not compensated by other perturbs T-cell development, yet the B-cell compartment Maml members. These data favor the model that appears unaffected (Radtke et al., 1999). Conversely, the individual Maml co-activators contribute to the mole- targeted inactivation of the Notch2 receptor or Dll1 cular specificity of Notch receptor functions in vivo. ligand results in a loss of marginal zone B cells (MZB Currently, the roles for Maml1 in mediating Notch cells), but T-cell development appears normal (Saito signaling in other tissues have not been carefully et al., 2003; Hozumi et al., 2004). Importantly, these examined. However, one additional interesting finding effects are consistent with phenotypes caused by the is that there appear to be defects in a subpopulation of inactivation of canonical Notch signaling in these muscle precursors (satellite cells) that are responsible for compartments, because loss of the transcription factor muscle regeneration in Maml1-deficient mice (H Shen CSL blocks both T-cell development and MZB cell et al., unpublished). Therefore, we speculate that Maml1 generation (Han et al., 2002; Tanigaki et al., 2002). Not loss inhibits the Notch response that is known to be unexpectedly, then, the expression of DNMAML1 in essential for the activation and expansion of satellite bone marrow cells abrogated canonical Notch signaling, cells, the early steps of myogenic process. These ideas inhibiting both T-cell development and MZB cell warrant further investigation and are being tested. generation (Maillard et al., 2004). Taken together, these In summary, the precise roles of individual MAML data revealed that the MAML genes collectively are co-activators in vivo, and whether they contribute to the essential in mediating physiological Notch functions. diverse biological effects of Notch signaling, remain to Importantly, DNMAML1 knock-in mouse models be addressed. Fortunately, detailed structural informa- recently were generated to investigate the in vivo tion is now available regarding the trimolecular complex functional consequences of inhibiting the endogenous between CSL, Notch1 and the N-terminal Notch- MAML functions in Notch signaling. Defects were binding domain of MAML1 (Nam et al., 2006; Wilson readily determined in several systems, including:T- and Kovall, 2006). Therefore, investigating the struct- helper cell development (Tu et al., 2005), skin (Proweller ural differences among various combinations of et al., 2006), vascular smooth muscle (Proweller et al., transcriptional complexes containing distinct Notch 2007) and cardiovascular development (High et al., and Maml family members will provide valuable 2007). These studies revealed that MAML proteins information about their in vivo specificity. Finally, indeed are critical to mediate Notch signaling in complete expression profiles of the three MAMLs in

Oncogene MAML co-activator in Notch and other pathways AS McElhinny et al 5143 different tissues and at different development stages, These studies revealed novel and unique roles for as well as characterization of mouse models lacking MAML1 that are independent of the Notch signaling each co-activator individually and in combination, pathway. A model was proposed where the Notch will provide a better understanding of the functions pathway was active during myoblast proliferation, and of Maml co-activators in Notch signaling-based re- then silenced upon activation of myoblasts to differ- sponses. entiate into mature muscle. MAML1 then would ‘switch’ to function as a potent transcriptional co- activator of MEF2C for muscle differentiation tran- Emerging Notch-independent functions of the MAML scriptional events to occur. Importantly, these studies proteins and their involvement with other signaling suggested the exciting possibility that MAML family pathways members may serve as co-activators for other pathways than Notch, and perhaps mediate cross talks between MAML1 co-activates MEF2C muscle-based various pathways and Notch (Shen et al., 2006). transcriptional events and is required for myogenesis Other studies described below support this intriguing The first indication that MAML functioned indepen- hypothesis. dently of the Notch pathway came from investigations using an Maml1 knockout mouse model (Shen et al., MAML1 co-activates p53, which is required for 2006). These mice display a severe perturbation of p53-mediated germ-cell apoptotic response overall skeletal muscle structure consistent with a The p53 tumor suppressor mediates cell-cycle arrest and muscular dystrophy-like phenotype, including degenera- apoptosis in response to cellular stresses, including tion of myofibers and muscle necrosis. Maml1-null DNA damage, hypoxia and oncogene activation, and is muscle embryonic fibroblasts (MEFs) failed to important in both normal developmental processes and initiate myogenic differentiation after MyoD transduc- cancer (Vousden and Lane, 2007). Another exciting, tion, suggesting an intrinsic requirement for Maml1 recent finding is that MAML1 exhibits Notch-indepen- in myogenesis. Indeed, Maml1 is required for dent functions in co-activating p53-dependent gene muscle differentiation from myoblast to myotube expression, thereby potentially regulating p53-mediated formation, because Maml1 RNAi-mediated knockdown responses (Zhao et al., 2007). Chromatin immunopreci- blocked the differentiation of C2C12 cells (a myoblast pitation assays indicate that MAML1 is a component of cell line). Maml1 also promotes myogenic differentia- the activator complex that binds to native p53-response tion, as C2C12 cells that overexpress MAML1 exhibited elements within the promoters of p53 target genes. The dramatically enhanced levels of muscle myosin and MAML1/p53 interaction involves the N-terminal region formed strikingly large myotubes. These data indicated of MAML1 and the DNA-binding domain of p53. that MAML1 expression is essential for muscle Overexpression of wild-type MAML1 enhanced the differentiation events, and for mediating muscle gene specific induction of p53-dependent genes in mammalian expression. cells. Conversely, MAML1 knockdown reduced p53- The pro-myogenic activities of MAML1 were surpris- dependent gene expression. Thus, these studies indicate ing and unexpected, as previous studies established that that MAML1 is essential for p53-dependent gene activation of Notch signaling actually inhibited myo- expression. genic differentiation in C2C12 cells (Kopan et al., 1994; Interestingly, MAML1 also increases the half-life of Lindsell et al., 1995). In fact, Notch signaling is inhibited the p53 protein and enhances its phosphorylation/ by the Notch antagonist, Numb, during the later step of acetylation upon DNA damage in cells. In C. elegans, myogenesis, myogenic differentiation (Luo et al., 2005). knockdown of the MAML homologue Lag-3 abrogated Therefore, we hypothesized that Maml1 possesses the p53-mediated germ-cell apoptotic response that Notch-independent functions that might account for typically occurs in response to DNA damage. Further- its pro-myogenic activities. This led us to investigate the more, the knock down of Lag-3 reduced the normal functional interaction of Maml1 and transcription expression patterns of downstream pro-apoptotic tar- factors important for myogenic differentiation, resulting gets of the C. elegans p53 homologue, Cep-1, including in the finding that Maml1 interacts with and co-activates Ced-13 and Egl-1. These data clearly demonstrate a role MEF2C, a critical muscle-specific transcription factor. for Lag-3 in p53-mediated apoptosis in C. elegans, Furthermore, our preliminary studies indicate that although more work is required to decipher the myogenin, also a key muscle transcription factor, is a mechanisms involved. However, these studies have direct target of Maml1, and its expression is regulated set the stage for investigating the intriguing regulation by the Maml1/MEF2C interaction. Consistent with of p53-mediated gene expression by MAML family these data, activation of the Notch pathway (by ligand members. stimulation or the ectopic expression of the constitutively activated form of ICN) inhibited MAML1- enhanced myogenesis in C2C12 cells (Shen et al., MAML1 co-activates b-catenin-based transcriptional 2006). Biochemically, the blockage of MAML1’s pro- events and is essential for colon carcinoma cell survival myogenic effects by Notch activation was associated Misregulation of the Wnt signaling pathway, a pathway with the recruitment of MAML1 away from MEF2C to critical for cellular proliferation and differentiation, is the Notch transcriptional complex. associated with various disorders including colon

Oncogene MAML co-activator in Notch and other pathways AS McElhinny et al 5144 carcinoma (Segditsas and Tomlinson, 2006). The Exelixis collection that dominantly modified the tight control of nuclear levels of b-catenin is a key wing-margin phenotype of C96-MamN. The genes regulatory event for Wnt/b-catenin signaling. In the identified with enhancer or suppressor functions were absence of Wnt signaling, b-catenin remains sequestered subsequently subjected to a second screening to deter- in the cytosol in a phosphorylated state, which leads to mine Notch pathway-dependent or -independent func- its degradation by a proteosome. The binding of the tions. In the end, 175 known and 160 novel genes Wnt ligand to Frizzled and LDL receptors on the cell were found to interact with Notch components and surface initiates multiple events, including the recruit- designated as Notch interactors, whereas 79 failed to ment of Dishevelled and inactivation of glycogen interact with the Notch pathway thus were defined synthase kinase 3b (GSK-3b). Inhibiting the phospho- as Mastermind-specific interactors (MSI). The MSI rylation of b-catenin by GSK-3b leads to the transloca- genes were classified into several distinct functional tion of b-catenin to the nucleus, where it interacts categories, including:negative transcriptional regula- with the T-cell factor (TCF) family of transcription tors, factors with RNA polymerase II transcriptional factors and activates target gene expression. Aberrant activities, factors with small GTPase regulator activities Wnt signaling can lead to constitutive transcriptional and factors that negatively regulate metabolism. activation of b-catenin/TCF target genes, resulting in It is possible that these MSIs potentially interact with enhanced cellular proliferation in the absence of other Notch pathway components that were not appropriate cues. included in this screen. However, this study verified MAML1 recently was determined to be a co-activator that the interaction of one MSI gene, the Drosophila for b-catenin-mediated transcription (Alves-Guerra b-catenin homologue, armadillo, interacts with Mam et al., 2007). In these studies, MAML1 interacted independently of Notch in both the wing and eye. These with b-catenin in vitro and in vivo and dramatically data confirm the finding that MAML proteins increased the transcriptional activity of b-catenin on function independently of Notch as a co-activator for promoters containing TCF-binding sites. The other b-catenin signaling (as discussed above). Therefore, two MAML family members have similar co-activating multiple MSIs from this screen indicate a broad and functions. Mechanistically, it appears that MAML1 is prominent role for Mam in various developmental recruited by b-catenin on Wnt target gene promoters processes. (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/b- catenin pathway independently of Notch signaling. Speculations on functional contributions: cross talk Strikingly, the knockdown of MAML proteins in regulation by the MAML co-activators colonic carcinoma cells, SW480, resulted in cell death, which correlated with decreased b-catenin-induced Figure 2 summarizes the findings that we discussed expression of cyclin D1 and c-Myc. Thus, MAML above, showing that MAML proteins participate in proteins appear to be essential for SW480 colonic cell multiple signaling pathways:Notch, MEF2C, p53, b- survival by transcriptional co-activation of b-catenin- catenin and potentially others. These findings reveal mediated transcription. pivotal regulatory functions for MAML co-activators in diverse biological processes including cell proliferation, differentiation and survival, and highlight the central Other potential Notch-independent functions roles for MAML co-activators in signaling cross talk. from Xenopus and Drosophila systems Many questions remain, however, including whether the Evidence that MAML proteins have Notch-independent MAML co-activators participate in multiple signaling activities also has been established from studies using pathways simultaneously or within certain signaling Xenopus and Drosophila systems. In Xenopus embryos, contexts, and how these activities contribute to the overexpression of XMam1 caused the formation of a functional biological outcomes. pigmented cell mass on their surface and induced From the biochemical aspects, some clues can be expression of RNA-binding protein nrp-1. However, inferred. On the basis of immunoprecipitation studies, this effect was not observed when Notch signaling was the MAML interactions with Notch/CSL appear to be activated ectopically through constitutive activation of more stable compared to MAML interactions with the Notch receptor (Katada et al., 2006). This indicates MEF2C, p53 and b-catenin (L Wu et al., unpublished). that Xmam1 affects a neurogenic lineage in a Notch- Although preliminary, these data lead to the hypothesis independent pathway. that MAML proteins mainly function as co-activators Recently, a genetic screen was performed using the for Notch/CSL when Notch receptors are activated, but Exelixis collection of insertional mutations with a goal are co-activators for other signaling pathways when to identify Mastermind modifiers (Kankel et al., 2007). Notch is inactive. This hypothesis was supported by our This screen took advantage of a Drosophila mutant previous findings that the Notch effects are dominant (C96-MamN) that displays a fully penetrant wing- over MAML1’s myogenic activities with MEF2C margin phenotype associated with the expression of (Shen et al., 2006). However, it is anticipated that there the N-terminal Mastermind protein, MamN (Helms may be more complicated scenarios than just simple et al., 1999). The screen searched for mutations in the functional switches. Future investigations into relative

Oncogene MAML co-activator in Notch and other pathways AS McElhinny et al 5145 Notch-dependent MAML functions MAML co-activators: links to human cancers

Co-A 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 muco- ICN MAML epidermoid carcinoma, leukemia and potentially cervi- cal cancer (Wu and Grifﬁn, 2004). This is expected CSL considering the essential regulatory roles for MAML proteins in Notch signaling as well as other signaling pathways, as discussed in this review. Notch-independent MAML functions Importantly, the MAML co-activators may be excellent candidates as targets to modulate the growing MAML 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 MEF2C 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 MAML Co-A can be controlled to inhibit cell growth and survival in cancer cells with overactive Notch signaling (Weng p53 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.

MAML Co-A Concluding remarks β-catenin The family of MAML genes encodes transcriptional TCF co-activators required for Notch signaling. Recent discoveries of MAML involvement in the regulation of other signaling pathways have indicated much broader Figure 2 Summary of the co-activator functions for Mastermind- roles for the MAML co-activators in regulating like transcriptional co-activator (MAML) in both Notch-dependent and Notch-independent pathways. (a) MAMLs co-activate biological functions, and also point toward a central Notch-mediated transcription, as explained in Figure 1. (b) role for MAML co-activators in mediating signaling MAMLs also co-activate MEF2C-mediated transcription and are cross talks. However, many questions remain to be required for muscle development. MAML/MEF2C interaction addressed regarding the mechanisms underlying the co- may be direct or indirect. (c) MAMLs also co-activate p53- mediated transcription in a Notch-independent manner. (d) activator functions of MAML proteins in Notch and MAMLs also were recently determined to co-activate b-catenin- other relevant pathways. These issues include investiga- mediated transcription in a Notch-independent manner. It is tions into the interactions of MAML-regulated signaling possible that unidentiﬁed other co-activators (CoA, yellow) are pathways and the individual and collective contributions required for these events to occur. 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. contributions of pathway-speciﬁc effects mediated by the MAML co-activators and the potential interactions Acknowledgements of these pathways in various biological systems will shed light on their functions in developmental and oncogenic This work was supported in part by NIH (R01 CA097148) and contexts. Muscular Dystrophy Association (MDA).

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