mastermind
Studies of antineurogenic phenotypes induced by Notch proteins indicate that mastermind appears to function in an elaboration of a signal upstream of Notch to elaborate a signal that activates the receptor (Lieber, 1993). Suppressor of Hairless exhibits specific interactions with mastermind, indicating that the Notch pathway may regulate nuclear events by controlling mastermind transcription by means of Suppressor of Hairless (Fortini, 1994).
In embryos homozygotic for mutations in numb, the achaete-scute complex, daughterless and mastermind, the calmodulin transcription pattern is altered for each of these loci (Kovalick, 1992).
During signaling by the Notch receptor, Notch's intracellular domain is cleaved, moves to the nucleus and associates with a DNA-binding protein of the CSL class (CSL for CBF1, Suppressor of Hairless [Su(H)], LAG-1); as a result, target genes are transcriptionally activated. In C. elegans, a glutamine-rich protein called LAG-3 forms a ternary complex with the Notch intracellular domain and LAG-1 and appears to serve as a transcriptional activator that is critical for signaling. Although database searches have failed to identify a LAG-3-related protein, it has been surmised that Notch signaling in other organisms might involve an analogous activity.
To search for a LAG-3-like activity in mice, a modified yeast two-hybrid screen was used, similar to that used to identify LAG-3. Briefly, a complex bait was used to screen a library of mouse cDNAs fused to the Gal4 activation domain (Clontech). That bait included mouse CBF1 fused to the Gal4 DNA-binding domain (GD) as well as the intracellular domain of mouse Notch1. The bait proteins were co-expressed from a pBridge vector. Out of 6 million transformants, one positive with similarity to Drosophila Mastermind and human KIAA0200 was recovered. A focus was placed on this clone because Drosophila Mastermind is known to be critical for Notch signaling. The murine ortholog of Mastermind is called mMam1, and the human one hMam1. The mMam1 fragment recovered in the two-hybrid screen consists of 62 amino acids and included a conserved region present in both fly and human Mastermind proteins (Petcherski, 2000).
To explore the idea that Mastermind might have a role similar to LAG-3 in Notch signaling, a series of two-hybrid assays was conducted. mMam1 binds mCBF1-GD in the presence of either Notch1 or Notch3, but not in their absence. It was next asked whether Drosophila Mastermind might participate in a similar complex in flies. A fusion protein was used carrying the Gal4 activation domain and the amino-terminal 198 amino acids of fly Mastermind (dMam [1-198]; henceforth called dMam), which includes the conserved region of Mastermind that is critical for complex formation among mouse components. dMam was found to bind Su(H) strongly in the presence of the fly Notch intracellular domain, but not in its absence (Petcherski, 2000).
The interchangeability of proteins from different species was examined. Remarkably, the fly protein, dMam, interacts with murine Notch1 or Notch3 and murine CBF1, and mMam1 interacts with fly Notch and Su(H). In contrast, C. elegans LAG-3 does not form a complex with either murine or fly components, and mMam and dMam do not complex with worm components. It is concluded that both fly and murine Mastermind proteins form a ternary complex with either fly or murine receptors and CSL proteins. This interchangeability underscores the similarity between the fly and murine Notch pathways. Although murine Mastermind is not described, a full-length cDNA sequence for human Mastermind is available. Comparison of human and fly Mastermind sequences reveals only one short region of significant similarity that is limited to 60 amino acids at the amino terminus. Therefore, despite a low overall sequence similarity between mouse and Drosophila Mastermind proteins, the region crucial for complex formation is conserved (Petcherski, 2000).
The importance of Notch's ankyrin repeats for complex formation was examined. In C. elegans, formation of the ternary complex is dependent on the ankyrin repeats of the Notch-related receptor GLP-1. To ask whether the same situation holds for the murine complex, two missense mutants, M1 and M2, were used, each of which bears amino-acid substitutions in the fourth ankyrin repeat of mNotch1. Consistent with results in C. elegans, both M1 and M2 compromise interactions among Notch1, CBF1 and either mMam1 or dMam (Petcherski, 2000).
What is the role of Mastermind in Notch signaling? Previous studies have suggested a role in transcriptional control. In Drosophila, Mastermind is a nuclear protein and is bound to chromatin. Furthermore, in Drosophila, Mastermind acts downstream of Notch in signaling. The amino-acid sequences of both human and fly Mastermind proteins are rich in glutamine and proline, a common feature in transcriptional activators. In the work reported here, a physical link between Mastermind and the major CSL transcription factor of the Notch pathway is described. The interaction of both mMam and dMam with the Notch intracellular domain and CBF1 relies on the receptor's ankyrin repeats. These repeats are essential for Notch signaling and the transcriptional response. In C. elegans, point mutations in the ankyrin repeats severely compromise signaling by the Notch-related receptor GLP-1. In tissue culture cells, the M1 and M2 point mutations abolish receptor function and compromise the activation of transcription by Notch signaling. The simplest explanation for all these findings is that Mastermind functions as a transcriptional activator for Notch signaling (Petcherski, 2000).
Important parallels exist between LAG-3 in C. elegans and Mastermind in Drosophila and mammals. (1) All of these proteins form a ternary complex with an intracellular fragment of Notch and a CSL DNA-binding protein. (2) Mutations in the fourth ankyrin repeat of the receptor compromise ternary complex formation for C. elegans and mouse proteins, as is reported here. (3) All three proteins are rich in glutamine and proline: 27.6% in LAG-3, 29.4% in dMam and 22% in hMam1. (4) LAG-3 and Mastermind function downstream of Notch in C. elegans and Drosophila, respectively. It is proposed that LAG-3 and Mastermind perform analogous functions as activators for Notch (Petcherski, 2000).
What is the evolutionary relationship between LAG-3 and Mastermind? An intriguing idea is that LAG-3 and Mastermind share a common ancestor. The conservation in amino-acid sequence between Mastermind orthologs is much lower than is found for other components of the pathway: whereas hMam1 and dMam share similarity only in a stretch of 60 amino acids within a much larger protein, Notch and CSL proteins show high similarity (44.8% and 74.5% identity for hNotch1/dNotch and hCBF1/Su[H], respectively) over most of their length between these same species. It therefore seems plausible that the absence of similarity between LAG-3 and Mastermind may reflect a high rate of amino-acid substitution in these proteins rather than a distinct evolutionary origin (Petcherski, 2000).
The mastermind locus encodes a nuclear protein required in the Notch signaling pathway. In a screen for genes affecting wing pattern, an
EP element was identified that directs expression of an alternatively spliced form of the mastermind transcript that has been called mam[DN]. Unlike the conventional mam transcript, mam[DN] is spatially regulated in the developing embryonic nervous system and eye imaginal disc. mam[DN] corresponds to an endogenous transcript and encodes an alternate form of the Mam protein that dominantly interferes with activity of the conventional Mam protein.
Mam[DN] blocks Notch signaling downstream from the activated form of Notch but cannot interfere with an activated form of Su(H), suggesting that Mam[DN] may interfere with the activity of a ternary complex involving Mam, Notch and Su(H) (Giraldez, 2002).
The mam[DN] transcript shares one exon with the conventional mastermind transcript. mam[DN] has two short exons before the shared exon and one
short exon after it. The predicted open reading frame of mam[DN] begins in its second exon, which encodes only four amino acids, continues through
the common exon in the same reading frame as Mam and ends after an additional four amino acids encoded by its fourth exon. Thus, the alternate
transcript is predicted to produce a short form of the Mam protein that differs at both the N- and C- termini, but which is identical to the conventional
Mam protein through the shared exon. The alternate form of Mam protein encoded by GH07841 would lack the conserved N-terminal domain that is
needed for interaction with the Ankyrin repeats of Notch and Su(H) (Giraldez, 2002).
The conventional form of Mam is ubiquitously expressed in the embryo and in the imaginal discs. To analyse the pattern of expression of mam[DN] an exon-specific RNA probe was used. mam[DN] is first detected by in situ hybridization in two pairs of cells adjacent to the midline in each segment at stage 10-11. These cells are the first to be labelled with Mab22C10, suggesting that they are vMP2 and dMP2. Later, expression of mam[DN] concentrates in vMP2. Between stages 13 and 15 mam[DN] accumulates in the CNS and in the salivary glands. During larval stages, mam[DN] is not detectably expressed in the leg or wing imaginal discs. mam[DN] is expressed in developing photoreceptors behind the morphogenetic furrow in the eye disc. These observations suggest that spatial regulation of mam[DN] expression could be used to modulate Notch activity levels (Giraldez, 2002).
Cell-specific gene
regulation is often controlled by specific combinations of DNA binding sites in
target enhancers or promoters. A key question is whether these sites are
randomly arranged or if there is an organizational pattern or
'architecture' within such regulatory modules. During Notch signaling in
Drosophila proneural clusters, cell-specific activation of certain Notch
target genes is known to require transcriptional synergy between the Notch
intracellular domain (NICD) complexed with CSL proteins bound to 'S' DNA
sites and proneural bHLH activator proteins bound to nearby 'A' DNA
sites. Previous studies have implied that arbitrary combinations of S and A DNA
binding sites (an 'S+A' transcription code) can mediate the
Notch-proneural transcriptional synergy. By
contrast, this study shows that the Notch-proneural transcriptional synergy critically
requires a particular DNA site architecture ('SPS'), which consists of a
pair of specifically-oriented S binding sites. Native and synthetic promoter
analysis shows that the SPS architecture in combination with proneural A sites
creates a minimal DNA regulatory code, 'SPS+A', that is both sufficient
and critical for mediating the Notch-proneural synergy. Transgenic
Drosophila analysis confirms the SPS orientation requirement during Notch
signaling in proneural clusters. Evidence that CSL interacts
directly with the proneural Daughterless protein, thus providing a molecular
mechanism for this synergy. It is concluded that the SPS architecture
functions to mediate or enable the Notch-proneural transcriptional synergy which
drives Notch target gene activation in specific cells. Thus, SPS+A is an
architectural DNA transcription code that programs a cell-specific pattern of
gene expression (Cave, 2005).
The functional significance of the SPS element has not
been determined, but initially, it was proposed that the arrangement of the S
binding sites in the SPS may function to mediate cooperative DNA binding by CSL
proteins, or it may be necessary for the recruitment of other proteins to the
promoter. Subsequent
studies, though, showed that CSL, NICD, and Mam "ternary complexes" can
assemble on single S sites. To
date, no studies have experimentally addressed whether there are significant
functional differences between SPS elements and single S or other non-SPS
binding site configurations, and the mechanistic function of the SPS element is
not known (Cave, 2005).
In Drosophila, five of the seven bHLH repressor genes in the
E(spl)-Complex contain an SPS element in their promoter regions, and four
of these bHLH R genes contain both SPS and proneural bHLH A protein binding (A)
sites. These four bHLH R genes (the m7, m8, mγ, and
mδ genes, collectively referred to as the 'SPS+A bHLH
R' genes have been shown genetically to depend upon proneural bHLH A genes
for expression. In addition, transcription assays in Drosophila
cells with at least two of these four genes (m8 and mγ) have
shown that there is strong transcriptional synergy when NICD and proneural
proteins are expressed in combination. These SPS+A
bHLH R genes also have similar patterns of cell-specific expression within
proneural clusters. Following determination of the neural precursor cell from
within a proneural cluster of cells, Notch-mediated lateral inhibition is
initiated and these SPS+A bHLH R genes are specifically upregulated in all of
the nonprecursor cells but not in the precursor cell. The
absence of NICD, and the presence of specific repressor proteins such as
Senseless, prevent upregulation
of SPS+A bHLH R genes in the precursor cells (Cave, 2005).
This study shows that there
are important functional differences between the SPS architecture and non-SPS
configurations of S binding sites. The SPS architecture is critical
for synergistic activation of the m8 SPS+A bHLH R gene by Notch
pathway and proneural proteins. Whereas previous studies have focused on which
regulatory genes and proteins function combinatorially to activate SPS+A bHLH R
gene expression, this study focuses on the underlying DNA transcription code that
programs the Notch-proneural transcriptional synergy that drives cell-specific
gene transcription. The results of previous studies have implied that an
apparently arbitrary combination of S and A binding sites (S+A transcription
code) is sufficient for transcriptional activation of SPS+A bHLH R genes. By
contrast, this study shows that a minimal transcription code, SPS+A, is sufficient and
critical for mediating Notch-proneural synergistic activation of these
genes. The SPS+A code is composed of the specific SPS binding site architecture
in combination with proneural A binding sites. Furthermore,
evidence is presented that direct physical interactions between the Drosophila Su(H)
and Daughterless protein mediate the transcriptional synergy, thus providing a
molecular mechanism for the Notch-proneural synergy. Together, these studies
show that the SPS architecture functions to mediate or enable the
transcriptional synergy between Notch pathway and proneural proteins and that
SPS+A is an architectural transcription code sufficient for cell-specific target
gene activation during Notch signaling (Cave, 2005).
To test whether the SPS binding site architecture is important for Notch-proneural
synergy, the ability of Drosophila NICD (dNICD) and proneural
bHLH A proteins, such as Achaete and Daughterless (Ac/Da) to synergistically
activate the wild-type native m8 promoter and SPS architecture variants was examined.
Whereas the native m8 promoter carries the
wild-type SPS architecture of S binding sites,
the m8 promoter variants contain either a
disrupted S site, leaving a single functional S site (SF-X or
X-SR), or orientation variants in which the orientation of one or
both S sites have been reversed (SR-SF, SF-
SF, and SR-SR) (Cave, 2005).
The native m8 promoter is synergistically activated in transcription assays by
coexpression of dNICD and Ac/Da, but it is only weakly activated by expression
of dNICD or proneural Ac/Da proteins alone. However, neither promoter with a
single S binding site (SF-X or X-SR) can mediate
synergistic interactions between dNICD and proneural proteins. In fact, both single
S site promoters are only
weakly activated when proneural and dNICD proteins are expressed individually
or together. Thus, single S sites are not sufficient to mediate Notch-proneural
synergy in these contexts, even though they are in the same position as the SPS
in the wild-type m8 promoter (Cave, 2005).
When the number of S binding sites are
maintained, but the orientation of these sites within the SPS is varied
(SR-SF, SF-SF, and
SR-SR), only the wild-type (SF-SR)
SPS orientation is synergistically activated by coexpression of dNICD and
proneural Ac/Da proteins. Thus, the wild-type
SPS architecture of S binding sites is clearly necessary for the m8
promoter to mediate transcriptional synergy between NICD and the proneural
protein complexes assembled on the SPS and A sites, respectively (Cave, 2005).
The transcriptional synergy between NICD and proneural proteins
mediated by the SPS element is crucial for the coactivation by the Mastermind
(Mam) protein. Coexpression of Mam with both dNICD and proneural proteins provides a
strong coactivation of transcription of the wild-type m8 promoter.
However, this strong coactivation is not observed with any of the non-wild-type
m8 SPS variants, which also cannot mediate
Notch-proneural synergy. Thus, coactivation by both the NICD and Mam cofactors
is strongly dependent on synergistic interactions with proneural combinatorial
cofactors, and the specific SPS architecture is critical for mediating this
synergy (Cave, 2005).
The native m8 promoter studies tested
whether the organization of the S binding sites in the SPS are
necessary to mediate the Notch-proneural synergy. In order to test which of
these architectural features are sufficient to mediate that synergy,
a set of synthetic promoters was created carrying the same SPS variants mentioned above in
combination with A sites (SPS-4A reporter). These
synthetic promoters thus contain the sites predicted to mediate the synergy but
lack the other sites present in the native m8 promoter, which might also
be necessary. This reductionist approach allows for the identification of a
minimal promoter that contains only those sites that are necessary and
sufficient to mediate the Notch-proneural synergy. All of these synthetic reporters are
modestly activated by expression of proneural proteins alone, but expression of
dNICD alone gives no activation. By contrast, only the SPS-4A reporter containing
the wild-type SPS (SF-SR) mediates clear synergistic
activation when dNICD and proneural proteins are coexpressed, and none of the
SPS variants do so (Cave, 2005).
Given that functional CSL/NICD/Mam ternary complexes
have been shown to assemble on single S sites and activate transcription,
it was expected that promoters with single S sites could be
activated at low levels by expression of dNICD in the absence of the proneural
proteins and that promoters with two S sites might have more activity than
single S sites. However, it was surprising to observe that all of the m8
and synthetic promoters, even with the wild-type SPS element, have very low or
no activity when dNICD is expressed alone. Thus, the SPS binding site
architecture does not appear to facilitate recruitment of functional NICD
coactivator. This argues against previous proposals that suggested that the SPS
architecture might function to recruit other proteins to the promoter.
Thus, given that the
wild-type SPS architecture is necessary and sufficient for Notch-proneural
synergy, these results indicate that the function of the SPS element is to enable
synergistic interactions with proneural proteins (Cave, 2005).
The synthetic promoters do
not carry bHLH R sites, which are present in all E(spl)-C gene promoters.
Thus, these sites clearly are not necessary for
Notch-proneural synergy, although they may modulate it in vivo. It has been
proposed that other repressor proteins bind the mγ and
mδ SPS+A bHLH R gene promoters to restrict their expression to a
subset of proneural clusters. Although these
hypothetical repressor binding sites may be necessary to program the full
mγ and mδ gene expression pattern, the current results
indicate that they are not necessary for the Notch-proneural synergy that drives
nonprecursor cell-specific upregulation (Cave, 2005).
Both the m8 and SPS-4A
synthetic reporter contain a hexamer sequence that has been coconserved with the
SPS element. Elimination of that hexamer site in a synthetic
promoter does not disrupt Notch-proneural, suggesting that Notch-proneural synergy
in vivo is not dependent on the hexamer site (Cave, 2005).
Together, the synthetic and
m8 promoter results indicate that SPS+A is a minimal transcription code
that is both necessary and sufficient for Notch-proneural synergy in
Drosophila. The results with the promoters that were tested show that
Notch-proneural transcriptional synergy requires the specific organization or
architecture of the SPS element, in addition to its combination with proneural A
binding sites. All of the promoters with SPS variants failed to mediate this
synergy. This clearly indicates that arbitrary combinations of S and A binding
sites are not sufficient to mediate Notch-proneural synergy (Cave, 2005).
An important question is whether there are other DNA binding
transcription factors that can combinatorially synergize with CSL/NICD
transcription complexes. Previous studies have shown that Notch pathway
factors can synergize with a nonproneural transcription factor,
Grainyhead, suggesting
that synergy with the CSL/NICD transcription complexes could be very general or
nonspecific. To test whether a general coactivator, the VP16 transcription
activation domain, can synergistically interact with dNICD, an
essentially identical wild-type SPS-containing synthetic promoter was created in which the A
sites were replaced by UAS binding sites for the yeast Gal4 transcription
factor (SPS-5U). Expression of a fusion protein
containing the Gal4 DNA binding domain and the constitutively active VP16
activation domain can activate the synthetic SPS-5U promoter.
However, the Gal4-VP16 fusion protein does not
synergize with NICD. Thus, CSL/NICD complexes do not synergize with every nearby
DNA bound transcription factor, and there is at least some specificity to the
synergy with bHLH A proteins. This interaction specificity could contribute
significantly to selective activation of Notch target genes. Further studies
will be required to determine whether other DNA binding transcription factors
can combinatorially synergize with Notch signaling and whether such factors fall
into distinct classes (Cave, 2005).
Given that Notch signaling and neural
bHLH A proteins have been conserved between Drosophila and mammals, it was
next asked whether the transcriptional synergy between these proteins is also
conserved in mammalian cells. Using the same set of synthetic promoters as
mentioned above, activation following expression of the mammalian
NICD and neural bHLH A protein homologs (Notch-1 ICD [mNICD] and MASH1/E47,
respectively) was tested in murine NIH 3T3 cells. As in the Drosophila system,
expression of MASH1/E47 proteins alone produces modest activation of the
wild-type (SF-SR) SPS-4A promoter, and mNICD alone does not
produce any significant activation of the promoter.
However, clear transcriptional synergy is observed with the wild-type
SPS promoter when both mNICD and neural bHLH A proteins are coexpressed.
Moreover, SPS-mediated synergy requires nearly the same organizational features
of S binding sites as observed in Drosophila. Neither of the single S
site promoters can mediate that synergy, nor
can most of the orientation variants. Although
the SR-SR promoter is activated following coexpression of
both the mNICD and bHLH A proteins, it is not activated by mNICD alone (Cave, 2005).
These results indicate that the potential for
transcriptional synergy between NICD and neural bHLH A proteins has been
conserved in a mammalian cell system and that the SPS+A code is sufficient and
critical for mediating that transcriptional synergy. This raises the possibility
that there may be mammalian genes that are regulated by neural bHLH A proteins
and Notch signaling via this code. Although there is an SPS element
conserved in the HES-1 promoter, HES-1 does not have an A site in
its proximal promoter region, and HES-1 is not activated by expression of
bHLH A genes. Thus, HES-1 appears
to be similar to the Drosophila E(spl)-C m3 bHLH R gene, which also has
an SPS but no obvious nearby A site. Whole-genome
searches are being performed for genes in mammalian systems that may be regulated by the SPS+A
code (Cave, 2005).
It has been proposed that the architecture of the
SPS element may mediate cooperative binding of a second CSL protein once an
initial CSL protein binds the DNA. Using electromobility gel shift assays to test for
cooperative binding, the ability was compared of bacterially expressed and
partially purified Drosophila Su(H) protein to bind DNA probes containing
either the wild-type m8 SPS or an m8 SPS with one S site mutated.
If there is cooperativity, one would expect to observe the band corresponding to
two DNA bound CSL proteins to be as strong or stronger than the band
corresponding to a single CSL protein bound to DNA. The single S site probe
serves as a control because it cannot be cooperatively bound by two Su(H)
proteins, and it also serves to identify the band corresponding to a single
Su(H) protein bound to the wild-type SPS probe.
Similar amounts of Su(H) protein bind strongly to
the wild-type probe and to the single-site probe. In particular, because single
protein binding to the wild-type DNA probe did not
facilitate or stabilize simultaneous binding of two S proteins,
Su(H) does not appear to bind cooperatively to the two S sites in the
wild-type probe. These results suggest that CSL proteins do not bind
cooperatively to the SPS in vivo, although posttranslational modifications in
vivo could affect these binding properties Cave, 2005).
In addition, the protein binding affinity for the SF-SR and
SR-SF probes appears to be comparable,
although the reversed orientation of the two S
sites would have likely disrupted cooperative binding if it were present. This
result strongly suggests that the complete lack of activation by
SR-SF sites in all of the promoters tested is not due
simply to decreased ability of Su(H) protein to bind to the
SR-SF orientation variant Cave, 2005).
To test the in vivo relevance of the conserved S binding site orientation in SPS
elements, transgenic flies were created carrying β-galactosidase reporter
genes driven by native m8 promoters containing either the wild-type
(SF-SR) or SR-SF variant SPS
elements. Wing and eye imaginal discs containing m8 promoters with the
wild-type SPS element produced strong expression in proneural cluster regions,
similar to the pattern
described for endogenous m8. By contrast,
comparably stained wing and eye discs carrying the m8 promoter reporters
with the SR-SF SPS variant showed no expression or very
low levels of expression, respectively.
Extended staining of discs containing the SR-SF element
revealed clear but weak expression in a pattern of single cells that resembles
the distribution of neural precursors in the wing discs and eye discs.
This is likely due to activation via the A
site by proneural proteins because proneural levels are highest in the precursor
cells. However, there was no expression in the surrounding nonprecursor cells
within the proneural clusters even though Notch signaling is activated in
these cells. Similar neural precursor-specific m8 reporter expression
patterns have been observed when the S binding sites are eliminated,
indicating that reversal of
the S binding site orientations is functionally equivalent to eliminating them
for this aspect of Notch target gene expression. These in vivo results
confirm that the conserved orientation of the S binding sites in the wild-type
SPS element is essential for nonprecursor cell specific upregulation of the
SPS+A bHLH R m8 genes in response to Notch signaling in proneural clusters (Cave, 2005).
To gain an insight into the
molecular mechanism underlying the strong transcriptional synergy between
Notch signaling and bHLH A proteins on the m8 and SPS-4A
promoters, whether this synergy involves a direct physical interaction
was tested by using yeast two-hybrid assays with the Drosophila proteins.
These experiments revealed that the Daughterless N-terminal domain directly
and specifically interacts with the Su(H) protein in the absence of the bHLH
domain and C terminus (Cave, 2005).
Using transcription assays in Drosophila cells,
whether the Da N terminus (DaN construct), which contains a
transcription activation domain,
can synergistically activate the m8 promoter was tested in the absence of both its
bHLH DNA binding domain and a heterodimerization partner, like Ac.
The Da N-terminal protein synergistically
activates the m8 promoter when dNICD is coexpressed, apparently by
direct binding of the DaN protein to endogenous CSL bound to the SPS element.
These results indicate that the Notch-proneural transcriptional
synergy is not mediated by cooperative DNA binding interactions between the
Su(H) and proneural proteins, although such cooperative binding may mediate
transcriptional synergy between some combinatorial cofactors.
These results suggest that a direct interaction between
Su(H) and the Da N-terminal fragment, which can occur independent of NICD,
facilitates the formation of an active transcription complex when NICD is also
present during Notch signaling (Cave, 2005).
These results suggest
that the SPS architecture functions to enable a direct physical interaction
between Su(H) and Da proteins, thus providing a molecular mechanism for the
observed Notch-proneural synergy that is mediated by the SPS element. This
interaction could stabilize the recruitment or functional activity of NICD,
which then recruits Mam, and could explain the strong dependence of both NICD
and Mam coactivation functions on the presence of proneural proteins (Cave, 2005).
In
previous studies, it has been proposed that neither the synergistic activation
nor the transcriptional repression mediated by CSL protein complexes imply
direct interactions between CSL and DNA bound combinatorial cofactors; rather,
it is likely that CSL proteins exert their effects through the recruitment of
non-DNA binding cofactors, such as chromatin modifying enzymes.
While this might be the case for some Notch target
gene promoters, in the case of m8, the results indicate that the
mechanism underlying the synergistic interactions between CSL/NICD and bHLH A
proteins does involve direct physical interactions (Cave, 2005).
A mechanistic model is proposed for programming Notch-proneural synergy with the SPS+A
transcription code. These studies demonstrate that there are important
functional differences between SPS and non-SPS organizations of S binding sites.
The critical role of the SPS binding site architecture is not
predicted or explained by the previous models for Notch target gene
transcription. Previous models suggest that
transcription is promoted by the binding of NICD to CSL, which displaces CSL
bound corepressors, thus allowing transcriptional synergy with other DNA bound
combinatorial cofactors. These models have not distinguished between
Notch target genes with regulatory modules that contain SPS or non-SPS
configurations of S binding sites, nor do they explain or predict the critical
function of the SPS binding site architecture in mediating Notch-proneural
transcriptional synergy (Cave, 2005).
A revised model is proposed that
incorporates the essential requirement for the specific SPS binding site
architecture in combination with the proneural A binding sites for
transcriptional activation of m8 and the other SPS+A bHLH R genes. These
genes each contain an SPS+A module and exhibit similar cell-specific
upregulation in nonprecursor cells in proneural clusters.
In this new model, the specific architecture of the S sites in the SPS
element directs the oriented binding of Su(H) so that it is in the proper
orientation and/or conformation to enable a direct interaction with Da. This
interaction is an essential prerequisite for subsequent recruitment and/or
functional coactivation by NICD during Notch signaling. This
Notch-proneural complex is then further activated by subsequent recruitment of
Mam (Cave, 2005).
It is interesting to note that the mammalian homologs of each
of the Su(H), NICD, and Da proteins have been shown to interact with the p300
coactivator; thus, when complexed together, these proteins could
potentially function combinatorially to recruit p300 or a related coactivator (Cave, 2005).
In Drosophila and mammals, Notch signaling is used
throughout development to activate many different target genes, and in multiple
developmental pathways. Thus, it is of paramount importance that the proper
target genes are selectively activated in the proper cell-specific patterns. It
is known that Notch signaling can activate genes through non-SPS
configurations of S sites in certain other target genes. For example, expression
of the Drosophila genes single minded, Su(H), and vestigal
have all been shown to be regulated by Notch
signaling, and all have single S sites or multiple unpaired S sites but no SPS
elements in their promoter and/or enhancer regions (Cave, 2005).
The results show that for
essentially every promoter tested, NICD cannot activate in the absence of neural
bHLH A combinatorial cofactors, suggesting that NICD may always require a
combinatorial cofactor to activate target genes. If so, the non-SPS Notch
target genes are likely also to have specific combinatorial cofactors. The
results also clearly show that the Notch-proneural combinatorial synergy
requires a specific configuration of S sites, the SPS. There may be other
specific configurations of S binding sites that mediate synergy for different
classes of combinatorial cofactors for Notch signaling (Cave, 2005).
Together, these
observations suggest that specific, but unknown, non-SPS configurations of sites
may program the interactions between Notch complexes and the proper
combinatorial cofactors. It is speculated that these non-SPS configurations might be
unique to each target gene, or it is possible that there are specific patterns
or classes of S binding site configurations -- an 'S binding site
subcode' -- that determine cofactor specificity. Thus, the results
suggest that selective Notch target gene activation may be programmed by
distinct Notch transcription codes in which specific configurations of S
binding sites mediate selective interactions with specific combinatorial
cofactors (Cave, 2005).
Elucidating the various transcription codes controlling target gene
activation during Notch signaling will be an important goal for future
studies. The results have clearly shown that the architecture of transcription
factor binding sites can be crucial for control of cell-specific Notch
target gene activation. The studies presented here give a glimpse into the
molecular mechanisms by which a one dimensional pattern of DNA binding sites can
program cell-specific patterns of gene expression (Cave, 2005).
The Notch receptor controls development by activating transcription of specific target genes in response to extracellular signals. The factors that control assembly of the Notch activator complex on target genes and its ability to activate transcription are not fully known. This study shows, through genetic and molecular analysis, that the Drosophila Nipped-A protein is required for activity of Notch and its coactivator protein, Mastermind, during wing development. Nipped-A and Mastermind also colocalize extensively on salivary gland polytene chromosomes, and reducing Nipped-A activity decreases mastermind binding. Nipped-A is the fly homologue of the yeast Tra1 and human TRRAP proteins and is a key component of both the SAGA and Tip60 (NuA4) chromatin-modifying complexes. Like Nipped-A, the Ada2b component of SAGA and the Domino subunit of Tip60 are also required for Mastermind function during wing development. Based on these results, it is proposed that Nipped-A, through the action of the SAGA and Tip60 complexes, facilitates assembly of the Notch activator complex and target gene transcription (Gause, 2006).
Nipped-A mutations were isolated in a genetic screen for factors that regulate activation of cut by the wing margin enhancer, and it was found that they reduce Notch activity both at the wing margin and in the developing wing veins. Heterozygous Nipped-A mutations increase the severity of the mutant wing margin and blade reduction phenotype caused by the weak loss-of-function Notch (Nnd-1) mutation and decrease the severity of the vein-shortening phenotype caused by a gain-of-function Notch mutation (NAx-E2)(Gause, 2006).
Other genetic data also indicate that Nipped-A is important for Notch signaling. Mastermind is a coactivator protein required for transcriptional activation by Notch, and heterozygous Nipped-A mutations dramatically increase the weak wing-nicking phenotype caused by heterozygous mastermind mutations. The vestigial gene is directly activated by Notch, and flies heterozygous for both Nipped-A and vestigial mutations display wing margin defects. The Notch intracellular fragment binds to the Suppressor of Hairless [Su(H)] protein on target genes, and a Nipped-A Su(H) double mutant displays a dominant wing-nicking phenotype. Together, the effects that the Nipped-A dosage has on the mutant phenotypes displayed by Notch, mastermind, and vestigial mutants indicate that Nipped-A encodes a factor critical for Notch activity in the developing wing (Gause, 2006).
Two Nipped-A mutants have point mutations in the gene encoding the Drosophila homologue of the yeast Tra1 and mammalian TRRAP proteins. Tra1/TRRAP is a key component of the SAGA and Tip60 (NuA4) chromatin-remodeling complexes in yeast, flies, and humans (Gause, 2006).
Tra1/TRRAP is a direct target of transcriptional activators and helps them recruit the SAGA and Tip60 chromatin modification complexes to aid in gene activation. Mammalian Tra1/TRRAP was first identified as a coactivator that interacts directly with the Myc and E2F activators. Tra1/TRRAP is also a target of several other activators in yeast and mammalian cells, including Gal4, E1A, VP16, nuclear receptors, and p53. Tra1/TRRAP contains an ATM-phosphatidylinositol-3 (PI-3) kinase-like domain near the C terminus that is important for recruitment of histone acetyltransferase (HAT) activity in mammalian cells. The C terminus is also critical for interaction of yeast Tra1 with acidic activators (Gause, 2006 and references therein).
There is evidence that SAGA, which contains Tra1/TRRAP and the Gcn5/PCAF HAT, may be involved in transcriptional activation by the Notch complex. Several components of the Notch activator complex are known and functionally identical in worms, flies, and mammals. Upon binding of ligands such as Serrate or Delta to the extracellular EGF repeats of Notch, an intracellular fragment of Notch (NICD) is proteolytically released, allowing it to enter the nucleus, where it interacts with a DNA-bound CSL [CBF1/Su(H)/Lag-1] protein. NICD helps recruit the Mastermind coactivator. An N-terminal region of Mastermind interacts with both the CSL protein and an ankyrin repeat domain of NICD. The p300/CBP (CREB-binding protein) HAT coactivator is recruited by interactions with both the NICD ankyrin repeats and a specific region in the N-terminal half of Mastermind. The Gcn5/PCAF HAT is also recruited by the Notch activator complex in cultured mouse cells; this requires the ankyrin repeat region of NICD. The NICD ankyrin repeats bind other proteins, such as Mastermind and CBP, and thus it is possible that these proteins are also required to recruit Gcn5/PCAF. Because Tra1/TRRAP is the SAGA subunit targeted by several transcriptional activators, it is a distinct possibility that it is required for recruitment of Gcn5/PCAF by the Notch activator complex (Gause, 2006).
This study presents a molecular genetic analysis of several Nipped-A mutations that provides new insights into the roles of the Tra1/TRRAP protein and its complexes in Notch signaling. Reducing the Nipped-A gene dosage by half reduces both Mastermind and Notch activities during wing development and, surprisingly, certain mutant alleles can replace one copy of wild-type Nipped-A. These data also show that other subunits of the SAGA and Tip60 complexes that contain Nipped-A are required for Mastermind and Notch function in wing development and that Nipped-A is required for binding of Mastermind to chromosomes. Taken together, the results indicate that Nipped-A plays multiple roles in Notch signaling (Gause, 2006).
The evidence provided here, combined with the finding that two Nipped-A mutants have point mutations in the Tra1/TRRAP gene, demonstrates conclusively that Nipped-A encodes Tra1/TRRAP. All EMS-induced Nipped-A alleles sequenced to date have point mutations in the Tra1/TRRAP gene that affect the protein coding sequence or, in one case, the 3' UTR. A seventh allele generated by gamma rays, Nipped-A323, does not produce Tra1/TRRAP mRNA. Additional Nipped-A mutant alleles have been sequenced, and all contain point mutations that alter the protein coding sequence (Gause, 2006).
The results show that the major Nipped-A transcript differs from a previously reported splicing pattern, which appears to be a rare variant. Antibodies against a polypeptide encoded largely by the rare exons detect a weak Tra1/TRRAP signal in Western blot assays of concentrated nuclear extracts or purified complexes, confirming that the variant produces Tra1/TRRAP protein in vivo. The rare transcript does not, however, support at least one essential function of Nipped-A and Notch signaling in the wing margin because mutation of a splice site in Nipped-ANC106 for an exon that is not included in the rare variant is lethal and causes defects in Notch signaling. Nipped-ANC106, however, had little effect on the NAx-E2 wing vein phenotype, raising the possibility that the alternatively spliced product can support Notch function in developing wing veins (Gause, 2006).
An unexpected finding is that the Nipped-ANC105 allele, which encodes the N-terminal 2,048 residues of Tra1/TRRAP, suffices to replace one wild-type copy of Nipped-A to support Notch and Mastermind function in vivo. This was unexpected because the protein encoded by Nipped-ANC105 lacks the ATM-PI3 kinase motif which, in mammalian cell culture experiments, is required for Tra1/TRRAP to associate with Gcn5 and Tip60. One possible explanation is that the C terminus of the Nipped-A protein is not required for Notch and Mastermind function and that the truncated protein can replace the full-length protein. Because the effects of the Nipped-A mutations on Notch functions in wing development could only be studied in the presence of a wild-type allele, it is also possible that a truncated protein somehow increases the activity of the remaining full-length Nipped-A protein. The truncated protein could not be detected in Western blot assays of extracts or by immunostaining, suggesting that if this is the case, only a small amount of the mutant protein is sufficient. It is considered improbable that linked second-site mutations are masking effects of Nipped-ANC105 on both Notch mutant phenotypes and the mastermind phenotype. Many mutations have effects similar to Nipped-A, and few have opposing effects, and it would likely require multiple mutations to counteract the effects of Nipped-ANC105 on all three phenotypes. It is also unlikely that there is a linked second-site mutation that counteracts the effects of Nipped-ANC105 by increasing the expression of wild-type Nipped-A, because mutant embryos and larvae show the expected decrease in full-length Nipped-A protein (Gause, 2006).
The Nipped-ANC194 allele, which encodes residues 1 to 1500, had a significant effect on both of the Notch mutant phenotypes but did not increase the severity of the wing-nicking phenotype displayed by mamg2. Again, this differs from null alleles of Nipped-A, which affect all three phenotypes, suggesting that Nipped-ANC194 retains sufficient activity to replace one copy of the wild type in support of Mastermind activity. Again, one possible explanation is that Nipped-A residues 1 to 1500 are sufficient to support Mastermind function, although it is conceivable that the truncated protein somehow increases the activity of the remaining wild-type Nipped-A protein. It was not possible to detect this truncated protein, suggesting that if a truncated protein is responsible, only low levels are required. Despite extensive screens with a deficiency collection and candidate genes, no mutations that suppress mastermind mutant phenotypes have been mapped to chromosome 2. Thus, it is unlikely that a linked second-site mutation masks an effect of Nipped-ANC194 on the mastermind phenotype. Similar to Nipped-ANC105, heterozygous Nipped-ANC194 mutants display the expected reduced levels of full-length protein, although the possibility cannot be excluded of a subtle increase in the expression of the wild-type Nipped-A allele that is sufficient to rescue the mastermind phenotype but not the Notch mutant phenotypes (Gause, 2006).
Isolation and analysis of additional Nipped-A truncation alleles and development of more sensitive biochemical assays will lead to a fuller understanding of how Nipped-A alleles encoding truncated proteins support Notch signaling (Gause, 2006).
The experiments presented in this study indicate that the roles of Nipped-A in supporting Mastermind function likely involve both the SAGA and Tip60 complexes. The Ada2b protein is specific to SAGA, and Ada2b mutations affect the mastermind phenotype but not the two Notch mutant phenotypes. It is thought unlikely that the effect of the Ada2b mutations is more specific than Nipped-A mutations because the mastermind phenotype is more sensitive. As shown by the Nipped-ANC96 hypomorph, the Nnd-1 phenotype is more sensitive to the Nipped-A dosage than is mastermind. Moreover, the Nipped-ANC194 allele has a specificity opposite that of the Ada2b mutations and affects the Notch mutant phenotypes but not the mastermind phenotype. Combined, the contrasts in the effects of Ada2b and various Nipped-A mutations show that Nipped-A and its complexes play multiple roles in Notch signaling. They suggest that the SAGA complex, or at least the Ada2b subunit, is more specific for Mastermind function and that Nipped-A has additional functions (Gause, 2006).
Another possibility raised by the specificity of the effects of Ada2b mutations for effects on Mastermind activity in wing margin development is that Mastermind may have functions in margin development independent of Notch. For example, Mastermind could conceivably function as a coactivator for other activator proteins in addition to Notch. This possibility is consistent with the binding of Mastermind to several sites in polytene chromosomes, including the ecdysone-dependent puffs (Gause, 2006).
The Domino protein, a putative ATPase remodeling enzyme, is a subunit of the Tip60 complex. The Nnd-1 and NAx-E2 phenotypes and the Mastermind phenotype are modified by domino mutations, although the effect on NAx-E2 is modest. These effects are similar to those of the Nipped-ANC106 allele and thus suggest that the Tip60 complex also supports Mastermind function and Notch signaling during wing development. It is possible, however, that Domino functions independently of Tip60 and Nipped-A because the human Domino homologue SRCAP interacts directly with the CBP HAT enzyme that interacts with Mastermind. Nevertheless, the likely involvement of the Tip60 complex raises the possibility that histone exchange could facilitate transcriptional activation by Notch because, in addition to acetylating histone H4, Tip60 exchanges histone H2 variants during DNA repair (Gause, 2006).
As revealed by immunostaining of salivary gland polytene chromosomes, at least one function of Nipped-A is to regulate the binding of Mastermind to chromosomes. The reduction in binding of Mastermind to polytene chromosomes caused by the hypomorphic Nipped-ANC96 and Nipped-ANC186 alleles is dramatic. Supporting the idea that Nipped-A directly regulates Mastermind binding, virtually all sites on polytene chromosomes that bind Mastermind also bind Nipped-A. A few possible explanations for these results are envisioned. The SAGA and Tip60 complexes that contain Nipped-A could acetylate Mastermind, proteins in the Notch activator complex, and/or possibly histones to facilitate binding of the Notch activator complex to chromatin. These modifications could be made by Gcn5 and/or Tip60, which acetylate histones H3 and H4, respectively. Alternatively, Nipped-A or its complexes could bind to chromosomes cooperatively with Mastermind. This would be consistent with the published observation that the ankyrin repeats of the NICD fragment of Notch, which help recruit Mastermind to the Notch activator complex, are also required to recruit Gcn5/PCAF SAGA subunit in transfected mouse cells. Both the Ada2b component of SAGA and the Domino subunit of Tip60 affect Mastermind function, so it is likely that Nipped-A supports Mastermind function in more than one way (Gause, 2006).
Because the evidence suggests that Nipped-A supports Mastermind function through both the SAGA and Tip60 chromatin-modifying complexes, it is theorized that, in addition to controlling the binding of Mastermind to chromosomes, Nipped-A could also cooperate with Mastermind to recruit these complexes to facilitate transcriptional activation through chromatin modification (Gause, 2006).
The data indicate that the SAGA complex, or at least its Ada2b subunit, is not required for some functions of Nipped-A in Notch signaling. Unlike Nipped-A and domino mutations, Ada2b mutations did not affect Notch mutant phenotypes, while they did enhance the phenotype caused by a mastermind mutation. It is postulated, therefore, that the Tip60 complex is also required for functions of Nipped-A beyond controlling the binding of Mastermind to chromosomes. The Tip60 complex could affect the expression of Notch activator complex components, or it could modify proteins in the Notch activator complex. It is also possible that Tip60 modifies chromatin to either aid binding of the Su(H) protein to the Notch target genes or, as mentioned above, to aid transcriptional activation by the Notch activator complex. In any case, the evidence indicates that two subunits of Tip60, Nipped-A and Domino, play more than one role in Notch signaling during wing development (Gause, 2006).
See the embryonic expression pattern of mam at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
mastermind and neuralized, another neurogenic locus, are expressed early in the ventral mesoderm anlage, prior to gastrulation (Bettler, 1991). The boundaries of its dorsal expression are probably involved in structuring the mesectoderm, as is the case with neuralized and Notch (Martin-Bermudo, 1995). mam expression is seen on either side of the cephalic furrow. During early embryogenesis, mam is expressed ubiquitously; during gastrulation, the predominant domain of MAM RNA and protein accumulation is found along the ventral longitutinal surface, including cells of the mesoderm, endoderm, mesectoderm and neuroectoderm. Thus regions of elevated MAM accumulation appears to be under control of dorsoventral patterning genes (Bettler, 1991).
mastermind mutants are lethal to the embryo. A neural hyperplasia is observed, caused by failure of the most ventral ectodermal cells to differentiate as epidermal cells rather than neuroblasts. Neural hyperplasia can be suppressed by mutation in proneural genes (Brand, 1988).
mastermind has a similar profile of embryonic defects as those produced by other neurogenic mutations, including Notch and Delta (Hartenstein, 1992), except that defects do not include the endoderm. A full list of tissues affected by neurogenic mutations is found at the Notch pathway.
The phenotypes and genetic interactions associated with mutations in the Drosophila mastermind(mam) gene have implicated mam as a component of the Notch
signaling pathway. However, its function and site of action within many tissues requiring Notch signaling have not been thoroughly investigated. To address these
questions, truncated versions of the Mam protein have been constructed that elicit dominant phenotypes when expressed in imaginal tissues under GAL4-UAS
regulation. By several criteria, these effects appear to phenocopy loss of function for the Notch pathway. When expressed in the notum, truncated Mam results in
failure of lateral inhibition within proneural clusters and perturbations in cell fate specification within the sensory organ precursor cell lineage. Expression in the wing is
associated with vein thickening and margin defects, including nicking and bristle loss. The truncation-associated wing margin phenotypes are modified by mutations in
Notch and Wg pathway genes and are correlated with depressed expression of wg, cut, and vg. These data support the idea that Mam truncations have lost key
effector domains and therefore behave as dominant-negative proteins. Coexpression of Delta or an activated form of Notch suppresses the effects of the Mam
truncation, suggesting that Mam can function upstream of ligand-receptor interaction in the Notch pathway. This system should prove useful for the investigation of
the role of Mam within the Notch pathway (Helms, 1999).
Chip may encode an enhancer-facilitator, acting to facilitate the activity of distal enhancers. The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are
unknown but are blocked by the Drosophila suppressor of Hairy-wing protein [su(Hw)] that binds to
gypsy retrovirus insertions between enhancers and promoters. su(Hw) bound to a gypsy insertion in the
cut gene also appears to act interchromosomally to antagonize enhancer-promoter interactions on the
homologous chromosome when activity of the Chip gene is reduced. Chip is needed for the wing margin enhancer of cut. The Chip mutation dominantly enhances the mutant phenotypes displayed by partially suppressed gypsy insertions in both cut and Ultrabithorax and is a homozygous larval lethal, indicating that Chip regulates multiple genes. Chip is normally required for wing margin enhancer function of cut because Chip mutations also enhance the cut wing phenotype of a cut mutation and heterozygotes for Chip display cut wing phenotypes when either scalloped or mastermind (mam) are also heterozygous mutant. Both Sc and Mam are known to regulate the cut distal enhancer, but in contrast to sd and mam mutants, Chip mutants display stronger genetic interactions with gypsy insertions than with wing margin enhancer deletions. Thus, in a heterozygous Chip mutant, a heterozygous gypsy insertion in cut displays a cut wing phenotype, whereas a heterozygous enhancer deletion does not. Dependence on the nature of the heterozygous lesion in the regulatory region strongly suggests that Chip directly regulates cut. More strikingly, it indicates that in a Chip heterozygote, a gypsy insertion is more deleterious to enhancer function than deletion of the enhancer. The simplest explanation is that su(Hw) bound to gypsy in one cut allele acts in a transvection-like manner (interchromosomally) to block the wing enhancer in the wild-type cut allele on a second chromosome. This implicates Chip in
enhancer-promoter communication (Morcillo, 1997 and references).
Chip was cloned and found to encode a homolog of the recently
discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins, which bind nuclear LIM domain proteins.
Chip protein interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts
genetically with apterous, showing that these interactions are important for Apterous function in vivo.
Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many
stages of development. It is suggested that Chip cooperates with different LIM domain proteins and other
factors to structurally support remote enhancer-promoter interactions (Morcillo, 1997).
Integrins are evolutionarily conserved transmembrane alpha,beta heterodimeric receptors involved in
cell-to-matrix and cell-to-cell adhesions. In Drosophila, the position-specific (PS) integrins (see Myospheroid) mediate the
formation and maintenance of junctions between muscle and epidermis and between the two epidermal
wing surfaces. Besides integrins, other proteins are implicated in integrin-dependent adhesion. In
Drosophila, somatic clones of mutations in PS integrin genes disrupt adhesion between wing surfaces
to produce wing blisters. To identify other genes whose products function in adhesion between wing
surfaces, a screen was conducted for autosomal mutations that produce blisters in somatic wing clones.
76 independent mutations were isolated in 25 complementation groups, 15 of which contained more than
one allele. Chromosomal sites were determined by deficiency mapping, and genetic interactions with
mutations in the beta PS integrin gene myospheroid were investigated. Mutations in four known genes
(blistered [Drosophila's Serum response factor implicated in the specification of intervein cells], Delta, dumpy and mastermind) were isolated. Mutations were isolated in three new genes
(piopio, rhea and steamer duck) that affect myo-epidermal junctions or muscle function in embryos.
Mutations in three other genes (kakapo, kiwi and moa) may also affect cell adhesion or muscle
function at hatching. These new mutants provide valuable material for the study of integrin-dependent
cell-to-cell adhesion. It is thought that blisters arise in Delta and mastermind clones because of a failure to maintain the normal properties of ectodermal cells within the clonal boundaries (Prout, 1997).
The mechanisms that allow enhancers to activate promoters from thousands of base pairs away are
disrupted by the Drosophila Suppressor of Hairy-wing protein (Su[Hw]). Su[Hw] binds a DNA
sequence in the gypsy retrotransposon and prevents activation of promoter-enhancers that are distal to a gypsy insertion in a
gene without affecting proximal promoter-enhancers. Several observations indicate that
SUHW does not affect enhancer-binding activators. Instead, SUHW may interfere with factors that
structurally facilitate interactions between an enhancer and promoter. To identify putative enhancer
facilitators, a screen for mutations that reduce activity of the remote wing margin enhancer in the cut
gene was performed. Mutations in scalloped, mastermind, and a previously unknown gene, Chip, were
isolated. A TEA DNA-binding domain in the Scalloped protein binds the wing margin enhancer.
Interactions among scalloped, mastermind and Chip mutations indicate that Mastermind and Chip act
synergistically with Scalloped to regulate the wing margin enhancer. Chip is essential and also affects
expression of a gypsy insertion in Ultrabithorax. Relative to mutations in either scalloped or mastermind, a
Chip mutation hypersensitizes the wing margin enhancer in cut to gypsy insertions. Therefore, Chip
might encode a target of su(Hw) enhancer-blocking activity (Morcillo, 1996).
Sanpodo regulates Notch-mediated sibling cell fate decisions but
is not involved in Notch-mediated lateral inhibition. Notch functions in the neurogenic ectoderm to limit the number of cells adopting a neural fate. spdo mutation does not alter the number of neuroblasts that delaminate from the ectoderm, but instead is involved only in regulating sibling cell fate in the progeny of neuroblasts. Although the spdo sibling neuron phenotype is identical to the Notch sibling neuron phenotype, none of the 11 spdo alleles show the excess neuroblast formation
characteristic of Notch mutations. Mutations in two other genes, Delta (10 alleles)
and mastermind (1 allele) have been identified that yield similar equalization of sibling neuron fates. Because both Delta and mastermind are in the well-characterized Notch signaling pathway, null and hypomorphic alleles of several 'Notch pathway'
genes have been tested: Delta, Notch, mam, neuralized and E(spl). Mutations in all these genes result in an excess of neuroblasts due to failure of lateral inhibition within the neuroectoderm.
However, mutations in neuralized and E(spl) have no effect on the
identity of the sibling neurons that were assayed, despite strong
defects in the earlier process of neuroblast formation. In contrast, Delta, Notch and mam mutations all yield similar sibling neuron phenotypes, in addition to excessive neuroblast
formation. These results can be illustrated using embryos homozygous for a hypomorphic mam allele in which neuroblast formation is essentially normal but sibling neuron fates are equalized. Loss of mam does not affect eve expression in GMCs, but leads to the duplication of RP2, Usib, aCC and dMP2 fates at the expense of the RP2sib, U, pCC and vMP2 fates, respectively. Thus, mutations in three genes (Delta, Notch and mam) have precisely the same sibling neuron phenotype as spdo mutations, suggesting that spdo, Delta, Notch and mam act together to specify asymmetric sibling neuron fate (Skeath, 1998).
Asymmetric cell division is a widespread mechanism in developing tissues that leads to the generation of cell diversity. For the most part the basis of asymmetric cell division has been analyzed in neuroblasts in the process by which neuroblast division yields another neuroblast and a secondary precursor cell: the ganglion mother cell (GMC). In the embryonic central nervous system of Drosophila melanogaster, GMCs divide and produce postmitotic neurons that take on different cell fates. The current study analyses the process of binary fate decision of two pairs of sibling neurons that occurs during cell division in GMCs. This process is accomplished through the intrinsic fate determinant, Numb. GMCs have apical-basal polarity; Numb localization and the orientation of division are coordinated to segregate Numb to only one sibling cell. The correct positioning of Numb and the proper orientation of division require Inscuteable (Insc). Loss of insc results in the generation of equivalent sibling cells. These results provide evidence that sibling neuron fate decision is nonstochastic and normally depends on the presence of Numb in one of the two siblings. Moreover, the data suggest that the fate of some sibling neurons may be regulated by signals that do not require lateral interaction between the sibling cells (Buescher, 1998).
The focus for the analysis of the roles of insc, numb, and components of the N-signaling pathway in fate specification, was on the only two pairs of GMC-derived neurons for which sibling relationships have been established: the RP2/RP2sib and the aCC/pCC neurons. These neurons are derived from two GMCs that can be identified unambigously by their specific expression of the nuclear protein Even-skipped (Eve). GMC1-1a divides into the aCC/pCC neurons that have approximately equal size and continue to express Eve. However, at later stages of development, aCC is distinguished from pCC by the expression of Zfh-1 and 22C10 (a membrane associated antigen). aCC is a motoneuron and forms an ipsilateral projection that pioneers the intersegmental nerve. GMC4-2a divides to form the sibling neurons RP2/RP2sib that are morphologically distinguishable. In 88% of the hemisegments, the newborn siblings show a significant difference in the size of their nuclei and cell bodies. This asymmetry appears to be initiated during cell division. In GA1019 mutant embryos, in which GMC4-2a fails to complete cytokinesis, cells are formed that contain one large and one small nucleus. This strongly suggests that the difference in size is generated early, prior to the completion of cytokinesis. The larger cell always adopts the RP2 fate, which is characterized by the expression of Eve, Zfh-1, and 22C10. RP2 forms an antero-ipsilateral projection. The smaller sibling always adopts the RP2sib fate, which is characterized by a further decrease in cell and nuclear size and the loss of Eve immunreactivity. Zfh-1 and 22C10 expression have not been shown in RP2sib. These observations suggest that the cell and nuclear size difference may serve as an early physical marker that will allow one to differentiate between the two progeny of GMC4-2a, irrespective of the molecular markers they express later (Buescher, 1998).
Mutations in mastermind (mam),sanpodo, and Notch equalize aspects of sibling cell fate but retain the difference in cell and nuclear size of sibling neurons. In mam mutant embryos, both progeny of GMC4-2a can adopt the RP2 fate with respect to Eve, Zfh-1, and 22C10 expression. However, despite this apparent change from the RP2sib to the RP2 cell fate, the unequal sizes of the GMC4-2a daughter cells remain; that is, their sizes are unaffected. mam is required for the correct fate specification of RP2sib and pCC but not for that of RP2 and aCC. The requirement for mam suggests that N signaling may be involved in the resolution of distinct sibling neuron cell fate. Mutations in mam and N result in similar defects and support the notion that N signaling is required for the resolution of sibling neuron fate. In inscuteable mutant embryos, GMC1-1a and GMC4-2a are correctly formed and express normal levels of Eve (and in the case of GMC4-2a, also Pdm-1). However, GMC1-1a divides to form two sibling neurons that both adopt the aCC fate (94%) with respect to marker gene expression. Similarly, GMC4-2a division results in two sibling cells, both of which adopt the RP2 fate (96%) with respect to expression of Eve, Zfh-1, and 22C10, as well as axon morphology. This strongly suggests that in wild-type embryos, the divisions of GMC1-1a and GMC4-2a are asymmetric in an insc-dependent manner and produce sibling cells that are intrinsically different; loss of insc function leads to the generation of sibling neurons with equivalent cellular identities. Moreover, in contrast to mam, sanpodo, and Notch mutant embryos, the duplicated RP2s seen in insc mutants are equal with respect to their cell and nuclear size. These observations are consistent with the idea that the size difference seen in wild-type embryos is generated by an insc-dependent process during the GMC cell division and occurs prior to the events mediated by mam, spdo, and N that presumably act at the level of the postmitotic sibling cells. No size asymmetry between the sibling neurons should be generated in an insc background regardless of whether the other functions (e.g., spdo) are present or not (Buescher, 1998).
The Notch receptor is the central element in a cell signaling mechanism controlling a broad spectrum of
cell fate choices. Genetic modifier screens in Drosophila and subsequent molecular studies have
identified several Notch pathway components, but the biochemical nature of signaling is still elusive.
The results are described of a genetic modifier screen of the bristle phenotype of a gain-of-function
Notch allele, Abruptex16. Abruptex mutations interfere with lateral inhibition/specification events that
control the segregation of epidermal and sensory organ precursor lineages, thus inhibiting bristle
formation. Mutations that reduce Notch signaling suppress this phenotype. This screen of
approximately 50,000 flies led to the identification of a small number of dominant suppressors in seven
complementation groups. These include Notch, mastermind, Delta,
and Hairless
, known components in the pathway, as well as two novel mutations: A122 and M285. A122, appears to interact with Notch only during bristle development. M285, displays extensive genetic interactions with the Notch pathway elements and appears, in general, capable of suppressing Notch gain-of-function phenotypes while enhancing Notch loss-of-function phenotypes, suggesting that it plays an important role in Notch signaling. The profile of the genetic interactions
documented with M285 is quite similar to that of mutations in other known components of the Notch
pathway. Three kismet alleles were isolated as weak suppressors of the Ax16 bristle phenotype. Interestingly,
mutations in kismet have been isolated independently as enhancers of the eye phenotype associated
with the expression of constitutively activated forms of the Notch receptor. kismet, which may encode a structural component of chromatin, does not display broad genetic interactions with Notch. It has therefore been suspected that the identification of these alleles through the eye screen may reflect its effect on the expression of the transgene by perturbing normal chromatin function rather than significant
interactions with Notch signaling. The fact that
such alleles were isolated in the bristle screen may be indicative of a link between Notch signaling and kismet function; however, further analysis is necessary before such a relationship can be established (Go, 1998).
In the mesoderm of Drosophila embryos, a defined number of cells segregate as progenitors of individual
body wall muscles. Progenitors and their progeny founder cells display lineage-specific expression of
transcription factors but the mechanisms that regulate their unique identities are poorly understood. The homeobox genes ladybird early and ladybird late are shown to be expressed in only one muscle progenitor and its progeny: the segmental border muscle (SBM) founder cell and two precursors of adult muscles. lb activity is associated with all stages
of SBM formation, namely the promuscular cluster, progenitor cell, founder cell, fusing myoblasts and syncytial fiber. The segregation of the ladybird-positive progenitor requires coordinate action of neurogenic genes and an
interplay of inductive Hedgehog and Wingless signals from the overlying ectoderm. The SBM progenitor corresponds to the most
superficial cell from the promuscular cluster,
thus suggesting a role for the overlying
ectoderm during its segregation. . Since epidermal Wg and Hedgehog (Hh) signaling has been shown to
influence muscle formation, the SBM-associated lb expression was examined in embryos
carrying hh and wg thermosensitive mutations. Wg and Hh signalings, mutually dependent at this time, are shown to be required for the promuscular lb activity and/or the segregation of SBM
progenitors. The initial influence of these signals is no longer observed later in
development. In addition to signals from the epidermis, the
activity of the mesodermal gene tinman, initially expressed in the whole trunk mesoderm, is involved in the early events of myogenesis. In tin - embryos, the formation of SBM promuscular clusters and segregation of lb-positive progenitor cells are strongly affected, leading to the absence of
the majority of SBM fibers. During promuscular cluster
formation, since tin expression becomes restricted to the dorsal mesoderm, its influence on ventrolaterally located SBMs is likely to be indirect and mediated via an
unknown factor. The lack of neurogenic gene function, known to be
involved in cell-cell interactions during lateral inhibition, generates the opposite phenotype. Mastermind - and Enhancer of split - embryos fail to restrict promuscular lb expression to only one cell; in consequence, they display a hyperplastic lb pattern in later stages (Jagla, 1998).
The Notch pathway plays a key role in the formation of many tissues and cell types in Metazoans. Notch acts in two pathways to
determine muscle precursor fates. The first is the 'standard' Notch pathway, in which Delta activates the Notch receptor, which then translocates into the nucleus in
conjunction with Su(H) to reprogram transcription patterns and bring about changes in cell fates. The second pathway is poorly defined, but known to be
independent of the ligands and downstream effectors of the standard pathway. The standard pathway is required in many different developmental contexts; it was of interest to determine if there is a general requirement for the novel pathway. The novel Notch pathway is required for the development of each of five
examined cell types. Holonull Notch mutants (mutants null for maternal and zygotic Notch) have a more extreme phenotype
than null mutants for Su(H), Delta, neuralized or mastermind. In Notch holonull embryos, clusters of 10 or 15 eve expressing RP2-like cells are found in place of a normal single RP2. The phenotype for the other neurogenic genes is far less severe. Notch and other neurogenic genes are involved in the determination of the mesectoderm and the visceral mesoderm. The Notch holonull phenotype is more severe in both cases than that of other holonull embryos. These results indicate that the novel pathway is a widespread and fundamental component of Notch function (Rusconi, 1999).
In the Drosophila CNS glial cells
are known to be generated from glioblasts, which produce
exclusively glia or neuroglioblasts that bifurcate to produce
both neuronal and glial sublineages. The
genesis of a subset of glial cells, the subperineurial glia
(SPGs), involves a new mechanism and requires Notch. SPGs share direct sibling
relationships with neurons and are the products of
asymmetric divisions. This mechanism of specifying glial
cell fates within the CNS is novel and provides further
insight into regulatory interactions leading to glial cell
fate determination. Furthermore, Notch
signaling positively regulates glial cells missing
expression in the context of SPG development (Udolph, 2001).
In order to better understand how a complete lineage of a
specific NGB with all its progeny, including its glial cells,
might be created, NB1-1 was chosen for a detailed analysis. NB1-
1 has been extensively used for cell fate specification studies
and a sound basis of information about this NB lineage is
available. NB1-1 is a NB that develops differential lineages
in the thoracic versus the abdominal segments. Focus was placed on the abdominal NB1-1A because only
these abdominal NB1-1 lineages contain glia. In addition to the
aCC/pCC sibling neurons, which are the progeny of the first
GMC produced from this lineage, NB1-1A generates 2 to 3
glial cells and 4 to 5 clustered interneurons (cN), yielding a
total of 9 to 10 cells. The three glial cells belong to the group
of subperineurial glia (SPG) that lie at the periphery of the
nerve cord and enwrap the entire ventral nervous system. Two of the glia, the A- and B-SPGs, can be found in dorsal positions, with a third
glia, the LV-SPG, located at ventral positions of the nerve cord.
All SPGs, including the A- and B-SPG and LV-SPG of NB1-1A, are specifically labelled by two enhancer trap lines, M84
and P101 (Udolph, 2001 and references therein).
As a first step toward elucidating the origin of the glial cells
of the NB1-1A lineage, the effects of loss of
function mutants in several genes, Notch, mastermind (mam)
and numb, which are known to affect the resolution of distinct
sibling cell fates, were tested for their effect on the development of A-, B- and
LV-SPGs. Embryos hemizygous/homozygous for a conditional Notch allele, Nts1, and also carrying one copy each of M84 and P101 (Nts1/M84/P101) were subjected to the non-permissive temperature of 29°>C after 6 hours of development. This regime allows Notch to function during the singling out of NBs and removes Notch during the crucial period when it is required for sibling cell fate resolution. Double staining with
anti-Eve and anti-ß-gal was performed. As expected,
in most hemisegments, Nts1/M84/P101 embryos duplicate the RP2 neuron at the expense of its sibling cell. Moreover, in
96% of the hemisegments, M84/P101+ cells could not
be found in typical dorsal or ventral positions. It is concluded that Notch function is required for the specification of the M84/P101 positive A-, B- and LV-SPGs. In wild-type embryos, M84/P101 is expressed in about eight SPGs per hemisegment, including the A- and B-SPGs and the LV-SPG (Udolph, 2001).
mastermind, which has been linked to the Notch signaling pathway by its
genetic interactions with Notch and its strikingly similar
phenotype in early and late neurogenesis, was tested. mam acts downstream of Notch during sibling cell fate
specification in the embryonic nervous system. The hypomorphic mam345 allele used in this
study shows only a mild hypertrophy of the nervous system but
clearly has an effect on sibling cell fate specification. A severe reduction (94%) of P101+ cells was observed in mam345;P101 embryos
similar to that seen with Nts1/M84/P101 embryos.
These data suggest that both genes are strictly required for the
specification of SPGs, most likely in a linear pathway.
However, it is unclear how Notch acts in the specification of
the SPGs. The possibility is considered that SPG glial cells could
arise from a series of asymmetric cell divisions, with Notch
being required to specify the glial daughters of these divisions (Udolph, 2001).
mastermind:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| References
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