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EVOLUTIONARY HOMOLOGS

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CBP interaction with bHLH transcription factors

NeuroD1/BETA2 is a key regulator of pancreatic islet morphogenesis and insulin hormone gene transcription in islet beta cells. This factor also appears to be involved in neurogenic differentiation, because NeuroD1/BETA2 is able to induce premature differentiation of neuronal precursors and convert ectoderm into fully differentiated neurons upon ectopic expression in Xenopus embryos. Amino acid sequences in mammalian and Xenopus NeuroD1/BETA2 have been identified that are necessary for insulin gene expression and ectopic neurogenesis. Evolutionarily conserved sequences spanning the basic helix-loop-helix (amino acids [aa] 100 to 155) and C-terminal (aa 156 to 355) regions are important for both of these processes. The transactivation domains (AD1, aa 189 to 299; AD2, aa 300 to 355) are within the carboxy-terminal region, as analyzed by using GAL4:NeuroD1/BETA2 chimeras. Selective activation of mammalian insulin gene enhancer-driven expression and ectopic neurogenesis in Xenopus embryos is regulated by two independent and separable domains of NeuroD1/BETA2, located between aa 156 to 251 and aa 252 to 355. GAL4:NeuroD1/BETA2 constructs spanning these sequences demonstrate that only aa 252 to 355 contain activation domain function, although both aa 156 to 251 and 300 to 355 are found to interact with the p300/CREB binding protein (CBP) coactivator. These results implicate p300/CBP in NeuroD1/BETA2 function and further suggest that comparable mechanisms are utilized to direct target gene transcription during differentiation and in adult islet beta cells (Sharma, 1999).

PCAF is a histone acetyltransferase that associates with p300/CBP and competes with E1A for access to them. While exogenous expression of PCAF potentiates both MyoD-directed transcription and myogenic differentiation, PCAF inactivation by anti-PCAF antibody microinjection prevents differentiation. MyoD interacts directly with both p300/CBP and PCAF, forming a multimeric protein complex on the promoter elements. Viral transforming factors that interfere with muscle differentiation disrupt this complex without affecting the MyoD-DNA interaction, indicating functional significance of the complex formation. Exogenous expression of PCAF or p300 promotes p21 expression and terminal cell-cycle arrest. Both of these activities are dependent on the histone acetyltransferase activity of PCAF, but not on that of p300. These results indicate that recruitment of histone acetyltransferase activity of PCAF by MyoD, through p300/CBP, is crucial for activation of the myogenic program (Puri, 1997).

In response to decreased cellular oxygen concentrations, the basic helix-loop-helix (bHLH)/PAS (Per, Arnt, Sim) hypoxia-inducible transcription factor, HIF-1alpha, mediates the activation of networks of target genes involved in angiogenesis, erythropoiesis and glycolysis. The mechanism for activation of HIF-1alpha has been demonstrated to be a multi-step process that includes hypoxia-dependent nuclear import and activation (derepression) of the transactivation domain, resulting in recruitment of the CREB-binding protein (CBP)/p300 coactivator. Inducible nuclear accumulation is dependent on a nuclear localization signal (NLS) within the C-terminal end of HIF-1alpha which also harbors the hypoxia-inducible transactivation domain. Nuclear import of HIF-1alpha is inhibited by either deletion or a single amino acid substitution within the NLS sequence motif and, within the context of the full-length protein, these mutations also resulted in inhibition of the transactivation activity of HIF-1alpha and recruitment of CBP. However, nuclear localization per se is not sufficient for transcriptional activation, since fusion of HIF-1alpha to the heterologous GAL4 DNA-binding domain generates a protein that shows constitutive nuclear localization but requires hypoxic stimuli to function as a CBP-dependent transcription factor. Thus, hypoxia-inducible nuclear import and transactivation by recruitment of CBP can be functionally separated from one another and both can play critical roles in signal transduction by HIF-1alpha (Kallio, 1998).

Recruitment of p300/CBP by the hypoxia-inducible factor, HIF-1, is essential for the transcriptional response to hypoxia and requires an interaction between the p300/CBP CH1 region and HIF-1alpha. A new p300-CH1 interacting protein, p35srj, has been identified and cloned. p35srj is an alternatively spliced isoform of MRG1, a human protein of unknown function. Virtually all endogenous p35srj is bound to p300/CBP in vivo, and it inhibits HIF-1 transactivation by blocking the HIF-1alpha/p300 CH1 interaction. p35srj did not affect transactivation by transcription factors that bind p300/CBP outside the CH1 region. Endogenous p35srj is up-regulated markedly by the HIF-1 activators hypoxia or deferoxamine, suggesting that it could operate in a negative-feedback loop. In keeping with this notion, a p300 CH1 mutant domain, defective in HIF-1 but not p35srj binding, enhances endogenous HIF-1 function. In hypoxic cells, p35srj may regulate HIF-1 transactivation by controlling access of HIF-1alpha to p300/CBP, and may keep a significant portion of p300/CBP available for interaction with other transcription factors by partially sequestering and functionally compartmentalizing cellular p300/CBP (Bhattacharya, 1999).

Histone acetyltransferases (HATs) play a critical role in transcriptional control by relieving the repressive effects of chromatin, and yet how HATs themselves are regulated remains largely unknown. Here, it is shown that Twist directly binds two independent HAT domains of acetyltransferases, p300 and p300/CBP-associated factor (PCAF), and directly regulates their HAT activities. Twist strongly binds the C-terminal fragment (amino acids 1257-2414) of p300 spanning the HAT domain as well as the CH3 domain. Further deletion reveals that this interaction requires the CH3 domain (compare 1572-2414 and 1869-2414), which is known to interact with other proteins. Of particular interest, Twist retains an interaction with a HAT domain even in the absence of the CH3 domain (1257-1572). Twist also binds the N terminus of p300 (1-566 and 1-744), although these interactions are 5- to 10-fold weaker than those with the CH3 and HAT domains. Twist shows strong binding to PCAF. Intriguingly, experiments using a series of PCAF internal deletion mutants reveal that this interaction required the presence of the intact HAT domain and bromodomain. Thus, Twist interacts independently with the HAT domains of two different proteins, p300 and PCAF, suggesting that Twist may recognize common motifs present in these HAT domains. The N terminus of Twist is a primary domain interacting with both acetyltransferases, and the same domain is required for the inhibition of p300-dependent transcription by Twist. Taken together, these findings support the view that Twist suppresses the coactivator functions of p300 and PCAF through physical interactions mediated by the N terminus of Twist. Adenovirus E1A protein mimics the effects of Twist by inhibiting the HAT activities of p300 and PCAF. These findings establish a cogent argument for considering the HAT domains as a direct target for acetyltransferase regulation by both a cellular transcription factor and a viral oncoprotein (Hamamori, 1999).

E1A has been shown to bind the CH3 domain of p300/CBP and to displace PCAF from this domain. The effect of E1A has been interpreted as a simple competition between E1A and PCAF for the CH3 domain. The present study adds a further level of complexity by demonstrating that E1A and Twist may exert their inhibition not only by physically disrupting the p300-PCAF complex formation but also through suppression of their enzymatic activities. The interaction of Twist at the CH3 domain raises the intriguing possibility that Twist might also prevent PCAF association with p300/CBP by competing with PCAF for the common CH3 domain. These two mechanisms may not necessarily work simultaneously, and cells would have exquisite control mechanisms that determine how these two mechanisms of p300 and PCAF regulation may be differentially utilized in a given situation. Individual histone acetyltransferases have distinct roles. For instance, myogenic transcription and differentiation are dependent on the HAT activity of PCAF but not on that of p300/CBP. Similar observations are made in other systems, indicating that the transcriptional activities of the HAT domains of p300 and PCAF are highly promoter dependent. The dual inhibitory mechanisms involving the HAT inhibition as well as the competitive displacement of cofactors would allow E1A and possibly Twist to regulate a broad range of transcriptional activators that are differentially dependent on p300 and PCAF and their HAT activities (Hamamori, 1999 and references).

The mechanisms by which neural stem cells give rise to neurons, astrocytes, or oligodendrocytes are beginning to be elucidated. However, it is not known how the specification of one cell lineage results in the suppression of alternative fates. In addition to inducing neurogenesis, the bHLH transcription factor neurogenin (Ngn1) inhibits the differentiation of neural stem cells into astrocytes. While Ngn1 promotes neurogenesis by functioning as a transcriptional activator, Ngn1 inhibits astrocyte differentiation by sequestering the CBP-Smad1 transcription complex away from astrocyte differentiation genes, and by inhibiting the activation of STAT transcription factors that are necessary for gliogenesis. Thus, two distinct mechanisms are involved in the activation and suppression of gene expression during cell-fate specification by neurogenin (Sun, 2001).

Neuronal differentiation is promoted by both platelet-derived growth factor (PDGF) and by neurotrophin-3 (NT3). The cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are potent inducers of astrocyte production, and thyroid hormone induces oligodendrocyte differentiation. LIF and CNTF exert their effects primarily via the JaK/STAT signaling pathway. LIF and CNTF bind to related receptors, which activate a receptor-associated tyrosine kinase, the Janus kinase (JaK1). Activated JaK1 phosphorylates two cytoplasmic proteins, the signal transducers and activators of transcription 1 and 3 (STAT1 and STAT3). This leads to STAT dimerization and translocation to the nucleus where the STATs activate cell type and stimulus-specific programs of gene expression (Sun, 2001 and references therein).

Other factors, such as bone morphogenetic protein (BMP), can enhance both neuronal and astrocyte differentiation, depending on the age of the stimulated cortical progenitors. BMP-induced astrocyte differentiation appears to be mediated by the downstream Smad signaling proteins. BMPs bind a multimeric receptor, which in turn results in the direct phosphorylation of Smad1. This permits Smad1 to dimerize with Smad4 and to translocate to the nucleus, where these factors cooperate with STATs to activate glial-specific programs of gene expression (Sun, 2001 and references therein).

The cooperation between Smads and STATs on glial promoters such as the glial fibrillary acidic protein (GFAP) promoter appears to be facilitated by a family of coactivator proteins termed p300/CBP. CBP (CREB binding protein) and p300 are ubiquitously expressed and are involved in the transcriptional coactivation of many different transcription factors. STATs and Smads bind to different domains of CBP/p300, and the STAT/p300/Smad complex, acting at the STAT binding element in the astrocyte-specific GFAP promoter, is particularly effective at inducing astrocyte differentiation in neural stem cells (Sun, 2001 and references therein).

Many transcription factors require CBP/p300 in order to activate transcription, and there is evidence that the levels of CBP/p300 are limiting, i.e., that there is competition among the various families of transcription factors for CBP/p300 binding. For example, nuclear steroid receptors indirectly inhibit AP-1-dependent transcription by sequestering CBP/p300 away from AP-1 and onto sites where the nuclear receptors are bound. Similarly, the anti-adenoviral actions of interferon are attributed to interferon's ability to activate STATs, which then sequester CBP/p300 away from the adenoviral transcription factor E1A. During early cortical development, endogenous Ngn1 associates with both CBP and Smad1, and the presence of neurogenin blocks STAT binding to CBP. Xenopus neurogenin has been shown to recruits CBP/p300 to the NeuroD promoter to activate transcription and induce neurogenesis. The characterization of the domains of CBP that interact with neurogenin reveal that both an N- and a C-terminal domain are involved. Interestingly, the neurogenin binding domains of CBP overlap with the STAT binding sites on CBP (but not with the Smad binding sites). This is consistent with the finding that neurogenin competes with STAT proteins for binding to CBP. By sequestering CBP, neurogenin may not only inhibit STAT-mediated transcription, but may also inhibit the function of other CBP-dependent transcription factors. Ngn1 also inhibits AP-1-dependent transcription. This may be relevant to Ngn's ability to inhibit astrocyte differentiation since the analysis of the GFAP promoter identifies multiple sites, including an AP-1 site, that contribute to neurogenin's inhibition of the GFAP promoter. Taken together, these findings suggest that CBP/p300 may orchestrate broad programs of gene expression that are relevant to cell fate determination. The effect of CBP/p300 on cell fate may then be determined by the relative binding affinity and abundance of different transcription factors that either compete or cooperate with one another for binding to CBP/p300 (Sun, 2001 and references therein).

In addition to sequestering the CBP-Smad1 complex, neurogenin also inhibits the activation of astrocyte-specific genes by blocking STAT activation. The mechanism by which Ngn1 reduces the level of phospho-STAT1 and -STAT3 is unknown. The Ngn1 deficient in binding DNS can also inhibit STAT phosphorylation, though not to the extent seen with wild-type Ngn1. This suggests that Ngn1 inhibits STAT phosphorylation only in part by a mechanism that is independent of Ngn1 binding to DNA (Sun, 2001).

An intricate array of heterogeneous transcription factors participate in programming tissue-specific gene expression through combinatorial interactions that are unique to a given cell-type. The zinc finger-containing transcription factor GATA4, which is widely expressed in mesodermal and endodermal derived tissues, is thought to regulate cardiac myocyte-specific gene expression through combinatorial interactions with other semi-restricted transcription factors such as myocyte enhancer factor 2, nuclear factor of activated T-cells, serum response factor, and Nkx2.5. GATA4 also interacts with the cardiac-expressed basic helix-loop-helix transcription factor dHAND (also known as HAND2). GATA4 and dHAND synergistically activate expression of cardiac-specific promoters from the atrial natriuretic factor gene, the b-type natriuretic peptide gene, and the alpha-myosin heavy chain gene. Using artificial reporter constructs this functional synergy was shown to be GATA site-dependent, but E-box site-independent. A mechanism for the transcriptional synergy is suggested by the observation that the bHLH domain of dHAND physically interacted with the C-terminal zinc finger domain of GATA4 forming a higher order complex. This transcriptional synergy observed between GATA4 and dHAND is associated with p300 recruitment, but not with alterations in DNA binding activity of either factor. Moreover, the bHLH domain of dHAND directly interacts with the CH3 domain of p300 suggesting the existence of a higher order complex between GATA4, dHAND, and p300. These results suggest the existence of an enhanceosome complex comprised of p300 and multiple semi-restricted transcription factors that together specify tissue-specific gene expression in the heart (Dai, 2002).

CBP interaction with NFkappaB

The nuclear factor kappaB (NF-kappaB) transcription factor is responsive to specific cytokines and stress and is often activated in association with cell damage and growth arrest in eukaryotes. NF-kappaB is a heterodimeric protein, typically composed of 50- and 65-kilodalton subunits of the Rel family, of which RelA(p65) stimulates transcription of diverse genes. Specific cyclin-dependent kinases (CDKs) were found to regulate transcriptional activation by NF-kappaB through interactions with the coactivator p300. The transcriptional activation domain of RelA(p65) interacts with an amino-terminal region of p300 distinct from a carboxyl-terminal region of p300 required for binding to the cyclin E-Cdk2 complex. The CDK inhibitor p21 or a dominant negative Cdk2, which inhibits p300-associated cyclin E-Cdk2 activity, stimulates kappaB-dependent gene expression, which is also enhanced by expression of p300 in the presence of p21. The interaction of NF-kappaB and CDKs through the p300 and CBP coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progression (Perkins, 1996).

The transcriptional activity of NF-kappa B is stimulated upon phosphorylation of its p65 subunit on serine 276 by protein kinase A (PKA). The transcriptional coactivator CPB/p300 associates with NF-kappa B p65 through two sites, an N-terminal domain that interacts with the C-terminal region of unphosphorylated p65, and a second domain that only interacts with p65 phosphorylated on serine 276. Accessibility to both sites is blocked in unphosphorylated p65 through an intramolecular masking of the N terminus by the C-terminal region of p65. Phosphorylation by PKA both weakens the interaction between the N- and C-terminal regions of p65 and creates an additional site for interaction with CBP/p300. Therefore, PKA regulates the transcriptional activity of NF-kappa B by modulating its interaction with CBP/p300 (Zhong, 1998).

Homodimers of the NF-kappaB p50 subunit are transcriptionally repressive in cells, whereas they can promote transcription in vitro, suggesting that their endogenous effects are mediated by association with other factors. Transcriptionally inactive nuclear NF-kappaB in resting cells consists of homodimers of either p65 or p50 complexed with the histone deacetylase HDAC-1. Only the p50-HDAC-1 complexes bind to DNA and suppress NF-kappaB-dependent gene expression in unstimulated cells. Appropriate stimulation causes nuclear localization of NF-kappaB complexes containing phosphorylated p65 that associates with CBP and displaces the p50-HDAC-1 complexes. Phosphorylation of p65 determines whether it associates with either CBP or HDAC-1, ensuring that only p65 entering the nucleus from cytoplasmic NF-kappaB:IkappaB complexes can activate transcription (Zhong, 2002).

The effects of HDAC-1 and CBP/p300 underscore the importance of acetylation in regulating NF-kappaB activity, although the identity of CBP/p300 targets remains to be fully determined. CBP/p300 can acetylate the four core histones, loosening chromatin and facilitating transcription. Histones associated with NF-kappaB-dependent genes are acetylated following stimulation. Alternative targets include p53, where acetylation is important for CBP-mediated p53-dependent transcription, although unlike p53 and despite many attempts, p65 acetylation by CBP/p300 has not been detected. Thus, it appears that the major effect of CBP/p300 on NF-kappaB-dependent transcription is via acetylation of histones or other proteins in the chromatin remodeling and transcriptional apparatus (Zhong, 2002).

In summary, it has been shown that p65 phosphorylation determines whether nuclear NF-kappaB associates with HDAC-1 (inactive) or CBP/p300 (active) and that p50-HDAC-1 represses NF-kappaB-dependent gene expression in resting cells. Such a regulatory mechanism ensures that only stimulus-induced NF-kappaB activates transcription, and NF-kappaB in the nucleus for any other reason is transcriptionally silent. This mechanism is unique among the inducible transcription factors, since it imposes an additional layer of control on NF-kappaB that most likely reflects the necessity of maintaining it as a true inducible transcription factor (Zhong, 2002).

The nuclear function of the heterodimeric NF-kappaB transcription factor is regulated in part through reversible acetylation of its RelA subunit. The p300 and CBP acetyltransferases play a major role in the in vivo acetylation of RelA, principally targeting lysines 218, 221 and 310 for modification. Analysis of the functional properties of hypoacetylated RelA mutants containing lysine-to-arginine substitutions at these sites and of wild-type RelA co-expressed in the presence of a dominantly interfering mutant of p300 reveals that acetylation at lysine 221 in RelA enhances DNA binding and impairs assembly with IkappaBa. Conversely, acetylation of lysine 310 is required for full transcriptional activity of RelA in the absence of effects on DNA binding and IkappaBa assembly. Together, these findings highlight how site-specific acetylation of RelA differentially regulates distinct biological activities of the NF-kappaB transcription factor complex (Chen, 2002).

In summary, these studies demonstrate that acetylation of RelA at distinct sites differentially regulates various biological functions of NF-kappaB. Acetylation of lysine 310 of RelA is required for full transactivation by the NF-kappaB complex, most likely by recruiting an unidentified cofactor. Acetylation of lysine 221 enhances RelA binding to the kappaB enhancer,while acetylation of lysine 221 alone or in combination with lysine 218 impairs the assembly of RelA with IkappaBa. Lysines 218 and 221 are highly conserved within all Rel family members, including Dorsal from Drosophila. The possibility that these evolutionarily conserved lysine residues are targets for reversible acetylation and contribute to the regulation of the biological functions of other Rel factors remains an intriguing possibility (Chen, 2002).

CBP and the Notch pathway

Signaling through the Notch pathway activates the proteolytic release of the Notch intracellular domain (ICD), a dedicated transcriptional coactivator of CSL enhancer-binding proteins. Chromatin-dependent transactivation by the recombinant Notch ICD-CBF1 enhancer complex in vitro requires an additional coactivator, Mastermind (MAM). MAM provides two activation domains necessary for Notch signaling in mammalian cells and in Xenopus embryos. The central MAM activation domain (TAD1) recruits CBP/p300 to promote nucleosome acetylation at Notch enhancers and activate transcription in vitro. MAM expression induces phosphorylation and relocalization of endogenous CBP/p300 proteins to nuclear foci in vivo. Moreover, coexpression with MAM and CBF1 strongly enhances phosphorylation and proteolytic turnover of the Notch ICD in vivo. Enhanced phosphorylation of the ICD and p300 requires a glutamine-rich region of MAM (TAD2) that is essential for Notch transcription in vivo. Thus MAM may function as a timer to couple transcription activation with disassembly of the Notch enhancer complex on chromatin (Fryer, 2002).

Unexpectedly, expression of MAM induces endogenous CBP/p300 proteins to accumulate in multiple nuclear foci in vivo. These structures do not form upon expression of a mutant MAM protein lacking the C-terminal TAD2 region (1-301MM). Thus, binding of MAM to CBP/p300, which is mediated through TAD1, is not sufficient to cause CBP/p300 to accumulate in these structures. Expression of other Notch components (ICD, CBF1) did not affect the subnuclear localization of CBP/p300, indicating that these foci are not a consequence of high levels of Notch signaling in the nucleus. One possibility is that MAM may regulate the expression or modification of CBP/p300 independently of Notch signaling. Indeed, the MAM-induced foci are accompanied by increased phosphorylation of CBP, and this phosphorylation requires the C-terminal TAD2 domain of MAM. Consequently, overexpression of MAM in the nucleus may promote widespread phosphorylation of CBP, which may cause the CBP/p300 proteins to concentrate in these structures. Changes in CBP/p300 phosphorylation have been shown to alter its activity and differentially affect its interactions with other transcription factors. It will therefore be important to assess whether MAM promotes CBP/p300 phosphorylation within the Notch enhancer complex, and whether phosphorylation of CBP/p300 is important for transcriptional activation by Notch (Fryer, 2002).

The timing of Notch signaling is tightly controlled in developmental processes such as somite formation, during which Notch target genes such as cHairy1 and mHES1 undergo periodic cycles of expression at the direction of a molecular oscillator, or vertebrate segmentation clock. This clock may be established through the intrinsic timing of Notch signaling as well as the half-life of Notch-induced transcriptional repressors. The Notch ICD is subject to proteolytic degradation in the nucleus through the action of the ubiquitin ligases such as Sel-10. Rapid turnover of the ICD may be required to allow genes to respond rapidly to subsequent cycles of Notch signaling. Coexpression with MAM and CBF1 promotes the phosphorylation and proteolytic turnover of the ICD in vivo, indicating that MAM couples transcription activation with degradation of the ICD. In this respect, MAM may act as a timer to control the length of time that the Notch complex remains associated with the enhancer. By extension, MAM might contribute to the periodic expression of Notch target genes during somitogenesis through its potential effects on the disassembly of the Notch enhancer complex (Fryer, 2002).

The data indicate that CBF1 acts in concert with MAM to control the proteolytic turnover of the ICD in vivo. Importantly, both MAM and CBF1 appear to be stable upon coexpression with the ICD, and thus it appears that the ICD can be destabilized independently of its interacting partners. The requirement for CBF1 may reflect its ability to enhance binding of MAM to the ICD, or alternatively CBF1 might be needed to target the Notch enhancer complex to DNA. Stability of a mutant ICD protein lacking the PEST domain is unaffected by coexpression with MAM and CBF1, and turnover is accompanied by increased phosphorylation of the ICD. Importantly, the MAM TAD2 domain is necessary for both enhanced phosphorylation and turnover of the ICD. Because p300 has been shown to be critical for the regulated turnover of the p53 transactivator by MDM2, it will be important to assess whether recruitment of p300 by MAM may similarly be required for proteolytic degradation of the ICD. Nevertheless, it is clear that recruitment of CBP/p300 through the MAM TAD1 region is not sufficient to couple activation with turnover of the Notch ICD under the conditions examined in this study (Fryer, 2002).

Thus the TAD2 region is required for MAM to promote the phosphorylation of its two associated factors, CBP/p300 and the Notch ICD. Because MAM does not possess intrinsic ICD protein kinase activity, it is attractive to consider that the Notch ICD and CBP/p300 may instead be targeted for phosphorylation by cyclin-dependent kinases that associate with the transcription complex and are recruited to the promoter by MAM. Phosphorylation events mediated by CDK7 and Srb10 (the CDK8 homolog in yeast) have been implicated in the proteolytic destruction of other enhancer factors. The CDK9 subunit of the positive transcription elongation factor, P-TEFb, also associates with RNAPII, whereas CDK8 interacts with RNAPII as a component of human and yeast mediator complexes that have been variously implicated in activation and repression of transcription. Another possibility is that the ICD is phosphorylated by a protein kinase that associates with MAM directly. It remains to be determined whether the MAM-induced phosphorylation is accompanied by increased ubiquitination of the ICD, and whether the degradation of the ICD observed is caused by ubiquitin-dependent proteolysis such as that described for the nuclear Sel-10 ubiquitin ligase. It will also be important to learn whether modification of the ICD regulates its transcriptional activity, as has been observed for other transcription factors, and whether these steps may ultimately be coupled to disassembly of the Notch enhancer complex and turnover of the Notch ICD (Fryer, 2002).

In summary, MAM is an essential component of the Notch enhancer complex in vitro as well as in vivo. The human MAM protein recruits p300/CBP to the Notch enhancer complex and controls the stability of the Notch ICD through the action of its unique C-terminal activation domain. Further studies will be needed to evaluate whether these properties are shared among the various MAM proteins in different species, and to learn how MAM-induced phosphorylation of the ICD and CBP/p300 proteins is coordinated with the regulation of Notch transcription (Fryer, 2002).

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