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Gene name - mastermind Synonyms - Cytological map position - 50C20-23 Function - presumptive transcription factor Keywords - neurogenic |
Symbol - mam FlyBase ID:FBgn0002643 Genetic map position - 2-70.3 Classification - novel Cellular location - nuclear |
mastermind is a neurogenic gene. Neurogenic gene mutants produce neural hyperplasia (too many nerves). The distribution of mastermind protein (Mam) has been examined. Mam is expressed through all germlayers during early embryogenesis, including ectodermal precursors to both neuroblasts and epidermoblasts. In early stage 6 embryos, the presumptive mesoderm expresses Mam at lower levels than other blastoderm cells. At late stage 7 and early stage 8 there is a higher level of staining in the nuclei of cells along the ventral midline and cephalic furrow. The mesoderm expresses Mam at lower levels than does the ectoderm. The delaminating neuroblasts stain similarly to the ectodermal layer, however, it is apparent that some neuroblasts do not stain as intensely as the ectoderm. Mam is subsequently down-regulated within the nervous system and then reexpressed. Mam is expressed in the segmental cells of the midline; there is overlap between Prospero and Mam at regular intervals at the midline. Neuroblasts express Mam after delamination and apparently after division. Numerous Prospero-expressing cells do not contain significant levels of Mam. These small cells are presumably GMCs, immature neurons and/or glial cells that have down-regulated Mam. Cells that express Mam strongly may derive from a late wave of neuroblast delamination. Mam persists in the nervous system through late embryogenesis and postembryonically. The mesoderm, anterior midgut, and posterior midgut express Mam at low levels, whereas the proventriculus, pharynx, Malpighian tubules and hindgut maintain high levels of Mam. Expression in the nervous system continues postembryonically in second and third instar larvae. Mam is detected throughout the brain and is expressed strongly within the optic lobe and within thoracic regions of the CNS known to contain clusters of neuroblasts and their progeny. Mam is ubiquitously expressed in wing and leg imaginal discs and is not down-regulated in sensory organ precursor cells of the wing margin or notum. In the eye disc, Mam shows most prominent expression posterior to the morphogenetic furrow. Expression of the protein during oogenesis appears limited to follicle cells. Immunohistochemical detection of Mam on polytene chromosomes reveals binding at >100 sites. Chromosome colocalization studies with RNA polymerase and the Groucho corepressor protein implicate Mam in transcriptional regulation (Bettler, 1996).
If Mam functions late in the neurogenic pathway as a nuclear regulatory protein, there are two principal roles to consider: activation of products of the E(spl) complex and/or repression of the proneural loci. The genetic interaction between mam and Suppressor of Hairless points to the former possibility. Based on its similarity to CBF1, it has been suggested that Su(H) protein may need to recruit a coactivator for E(spl) induction; it is conceivable that Mam performs this function (Bettler, 1996). Mam has been shown to be an evolutionarily conserved protein, with identifiable homologs in C. elegans and humans. Mam has been shown to interact with Su(H) in the presence of the fly Notch intracellular domain, but not in its absence (Petcherski, 2000).
Mastermind acts downstream of Notch to determine alternate cell fates in neural lineages. Neural precursors (or neuroblasts) divide in a stem cell lineage to generate a series of ganglion mother cells, each of which divides once to produce a pair of postmitotic neurons or glial cells. An exception to this rule is the MP2 neuroblast, which divides only once to generate two neurons. A screen was carried out for genes expressed in the MP2 neuroblast and its progeny as a means of identifying the factors that specify cell fate in the MP2 lineage. A P-element insertion line was identified that expresses the reporter gene tau-beta-galactosidase in the MP2 precursor and its progeny, the vMP2 and dMP2 neurons. The transposon disrupts the neurogenic gene mastermind, but does not lead to neural hyperplasia. However, the vMP2 neuron is transformed into its sibling cell, dMP2. By contrast, expression of a dominant activated form of the Notch receptor in the MP2 lineage transforms dMP2 to vMP2. Notch signaling requires Mastermind, suggesting that Mastermind acts downstream of Notch to determine the vMP2 cell fate. Mastermind plays a similar role in the neurons derived from ganglion mother cells 1-1a and 4-2a, where it specifies the pCC and RP2sib fates, respectively. This suggests that Notch signaling through Mastermind plays a wider role in specifying neuronal identity in the Drosophila central nervous system. Notch is expressed in both MP2 progeny. Notch signaling is blocked by Numb, which segregates exclusively to dMP2 when the MP2 precursor divides. Numb interacts directly with the intracellular domain of Notch. By antagonizing Notch, Numb promotes the dMP2 cell fate. Thus it is likely that Numb antagonism of Notch signaling in dMP2 confines Mastermind function, acting downstream of Notch, to the vMP2 neuron (Schuldt, 1998).
Studies of mammalian homolog of Mastermind provide insight into the molecular interactions of Mastermind as a co-activator in the Notch pathway. Signaling through the Notch pathway activates the proteolytic release of the Notch intracellular domain (ICD), a dedicated transcriptional coactivator of CSL (CBF-1, Suppressor of Hairless, and Lag-1) 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 (Drosophila homolog Nejire) 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).
Genomic DNA length - 67 kb
cDNA clone length - 6.3 kb. There are four zygotic transcripts, differing in their times of expression, and one maternal transcript. There are two sites at which transcription can start, and multiple lengths of the 3' UTR (Smoller, 1990).
Bases in 5' UTR - 754
Exons - seven
Bases in 3' UTR - 1791 and longer
date revised: 30 May 2006
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