Notch
C. elegans Notch homologs Notch is homologous to C. elegans lin-12 (Kidd, 1986) and glp-1 (Mello, 1994). There are three mammalian homologs as well (Larsson, 1994, Lardelli, 1994 and Higuchi, 1995).
Interactions mediated by Notch and Delta homologs contribute to the establishment of the
dorsal-ventral axis in the early C. elegans embryo.
The sister blastomeres ABp and ABa are equipotent at the beginning of the 4-cell stage in C.
elegans embryos, but soon become committed to different fates. The glp-1 gene, a
homolog of the Notch gene of Drosophila, functions in two distinct cell-cell interactions that specify
the ABp and ABa fates. These interactions both require maternal expression of glp-1. A second maternal gene, apx-1, functions with glp-1 only in the specification of the ABp fate.
apx-1 can encode a protein homologous to the Delta protein of Drosophila (Mello, 1994).
Cell-cell interactions mediated by LIN-12 and GLP-1, members of the LNG (LIN-12, Notch,
GLP-1) family of receptors, are required to specify numerous cell fates during development
of the nematode Caenorhabditis elegans. Maternally expressed glp-1 participates in two of
at least four sequential inductive interactions that specify the fates of early embryonic
descendants of the AB founder cell. GLP-1 and LIN-12, and apparently their
ligand, LAG-2, as well as a downstream component, LAG-1, are required in the latter two
inductions. lag-2 is expressed in the signaling cells and lin-12 is expressed
in cells receiving the inductions, consistent with their proposed respective roles as ligand and receptor. Maternal GLP-1 activity is required (1) to repress
early zygotic lag-2 expression and (2) to activate zygotic lin-12 expression in the early
embryo. The patterning of both receptor and ligand expression by maternal GLP-1 signaling
establishes competence for the zygotic LNG-mediated cellular interactions and localizes
these interactions to the appropriate cells. It is proposed that activation of maternal GLP-1
regulates zygotic lin-12 and lag-2 expression by a regulatory mechanism analogous to that
described for the post-embryonic gonad (Moskowitz, 1996).
C. elegans germ-line proliferation is controlled by an inductive interaction between the somatic distal tip cell and the germ line. GLP-1, a member of the Notch family of transmembrane receptors, is required continuously in the germ line to transduce the proliferative signal. In the
absence of GLP-1, all proliferative germ cells exit the mitotic cell cycle and enter meiotic prophase. oz112gf, an activating mutation in glp-1 has been characterized as having the opposite phenotype. Homozygous mutant hermaphrodites and mutant males have a completely tumorous germ line in which
germ cells never leave the mitotic cycle. In heterozygotes,
germ-line polarity is established correctly, but as adults age, the distal
proliferative population expands leading to a late-onset tumorous phenotype. The mutant receptor is constitutively active, promoting proliferation in the absence of ligand. The normal distal-proximal spatial restriction of GLP-1
expression is lost in tumorous and late-onset tumorous animals; ectopically proliferating germ cells contain membrane-associated GLP-1. The correlation between proliferation and expression suggests that GLP-1 signaling positively regulates GLP-1 expression: this is true for both wild-type, where GLP-1 signaling is limited by localized ligand, and in mutants, where signaling is ligand-independent. In addition to germ-line defects, mutation causes inappropriate vulval cell fate specification. A missense mutation in a
conserved extracellular residue, Ser642, adjacent to the transmembrane domain, is sufficient to confer the mutant phenotype. Two
mammalian Notch family members, TAN-1 and int-3, are proto-oncogenes. Thus, activating mutations in both invertebrate and vertebrate Notch family
members can lead to tumor formation (Berry, 1997).
A LIN-12::GFP fusion protein was used to examine LIN-12 accumulation during cell fate decisions
important for vulval development. During the naturally variable anchor cell (AC)/ventral uterine precursor
cell (VU) decision of the somatic gonad, a transcription-based feedback mechanism biases two equivalent
cells so that one becomes the AC while the other becomes a VU. LIN-12::GFP accumulation reflects lin-12
transcription: LIN-12::GFP is initially present in both cells, but disappears from the presumptive AC and
becomes restricted to the presumptive VU. During vulval precursor cell (VPC) fate determination, six
equipotential cells uniformly transcribe lin-12 and have invariant fates that are specified by multiple cell-cell
interactions. The pattern of LIN-12::GFP accumulation in VPCs and in the VPC lineages is dynamic and
does not always reflect lin-12 transcription. In particular, LIN-12::GFP is expressed initially in all six
VPCs, but appears to be reduced specifically in P6.p as a consequence of the activation of the Ras pathway
by an EGF-like inductive signal from the AC. It is proposed that downregulation of LIN-12 stability or
translation in response to inductive signalling helps impose a bias on lateral signalling and contributes to the
invariant pattern of VPC fates (Levitan, 1998a).
In C. elegans, the GLP-1 receptor acts with a downstream transcriptional regulator,
LAG-1, to mediate intercellular signaling. GLP-1 and LAG-1 are homologs of Drosophila Notch and
Su(H), respectively. The functions of two regions of the GLP-1 intracellular
domain were investigated: the ANK repeat domain, which includes six cdc10/ankyrin repeats plus flanking amino acids,
and the RAM domain, which spans approximately 60 amino acids just inside the transmembrane
domain. Both ANK and RAM domains interact with the LAG-1
transcription factor. The interaction between the ANK domain and LAG-1 is only observed in
nematodes by a co-localization assay and, therefore, may be either direct or indirect. By contrast, the
interaction between the RAM domain and LAG-1 is likely to be direct, since it is observed by
co-precipitation of the proteins in vitro as well as by yeast two-hybrid experiments. The RAM domain, when expressed in nematodes without a functional ANK repeat
domain, does not mimic the unregulated receptor in directing cell fates nor does it interfere with signaling by
endogenous components. In yeast the ANK repeats are strong transcriptional
activators. Furthermore, missense mutations that eliminate receptor activity also abolish transcriptional
activation by the GLP-1 ANK repeats in yeast. One possible function for the ANK
repeat domain is to act as a transcriptional co-activator with LAG-1 (Roehl, 1996).
Mutations that influence lin-12 activity in Caenorhabditis elegans may identify conserved factors that
regulate the activity of lin-12/Notch proteins. Genetic evidence is described indicating that sel-10 is a
negative regulator of lin-12/Notch-mediated signaling in C. elegans. Sequence analysis shows that SEL-10
is a member of the CDC4 family of proteins and has a potential human ortholog. Coimmunoprecipitation
data indicate that C. elegans SEL-10 complexes with LIN-12 and with murine Notch4. It is proposed that
SEL-10 promotes the ubiquitin-mediated turnover of LIN-12/Notch proteins (Hubbard, 1997).
The ectodomain of LIN-12/Notch proteins is cleaved and shed upon ligand binding. In Caenorhabditis elegans, genetic evidence has implicated SUP-17, the ortholog of Drosophila Kuzbanian and mammalian ADAM10, as the protease that mediates this event. In mammals, however, biochemical evidence has implicated TACE, a different ADAM protein. This study investigated potential functional redundancy of sup-17 and the C. elegans ortholog of TACE, adm-4, by exploring their roles in cell fate decisions mediated by lin-12/Notch genes. It was found that reduced adm-4 activity, like reduced sup-17 activity, suppresses an allele of glp-1 that encodes a constitutively active receptor. Furthermore, concomitant reduction of adm-4 and sup-17 activity causes the production of two anchor cells in the hermaphrodite gonad, instead of one—a phenotype associated with loss of lin-12 activity. Concomitant reduction of both sup-17 and adm-4 activity in hermaphrodites results in highly penetrant synthetic sterility, which appears to reflect a defect in the spermatheca. Expression of a truncated form of LIN-12 that mimics the product of ectodomain shedding rescues this fertility defect, suggesting that sup-17 and adm-4 may mediate ectodomain shedding of LIN-12 and/or GLP-1. The results are consistent with the possibility that sup-17 and adm-4 are functionally redundant for at least a subset of LIN-12/Notch-mediated decisions in C. elegans (Jarriault, 2005: full text of article).
The vulval precursor cells (VPCs) of C. elegans are polarized epithelial cells that adopt a precise pattern of fates through regulated activity of basolateral LET-23/EGF receptor and apical LIN-12/Notch. During VPC patterning, there is reciprocal modulation of endocytosis and trafficking of both LET-23 and LIN-12. sel-2 was identified as a negative regulator of lin-12/Notch activity in the VPCs; SEL-2 is the homolog of two closely related human proteins, neurobeachin (also known as BCL8B) and LPS-responsive, beige-like anchor protein (LRBA). In Drosophila, mutations in the single neurobeachin/LRBA homolog rugose (also referred to as DAKAP550) cause defects in eye development consistent with abnormalities in Notch and EGFR signaling (Schreiber, 2002; Shamloula, 2002; Wech, 2005), but the basis for these defects is unclear. SEL-2/neurobeachin/LRBA appears to form a subfamily within a larger family of BEACH-WD40 domain-containing proteins. There are three other C. elegans BEACH-containing proteins. The most closely related to SEL-2 is VT23B5.2, the ortholog of human ALFY and Drosophila Blue Cheese (Finley, 2003), consisting of little else but the BEACH domain and five WD40 motifs, plus a C-terminal FYVE domain. Loss of sel-2 activity leads to basolateral mislocalization and increased accumulation of LIN-12 in VPCs in which LET-23 is not active, and to impaired downregulation of basolateral LET-23 in VPCs in which LIN-12 is active. Downregulation of apical LIN-12 in the VPC in which LET-23 is active is not affected. In addition, in sel-2 mutants, the polarized cells of the intestinal epithelium display an aberrant accumulation of the lipophilic dye FM4-64 when the dye is presented to the basolateral surface. These observations indicate that SEL-2/neurobeachin/LRBA is involved in endosomal traffic and may be involved in efficient delivery of cell surface proteins to the lysosome. These results also suggest that sel-2 activity may contribute to the appropriate steady-state level of LIN-12 or to trafficking events that affect receptor activation (Wech, 2005).
The Notch signaling pathway controls growth, differentiation and patterning in divergent animal phyla; in humans, defective Notch signaling has been implicated in cancer, stroke and neurodegenerative disorders. Despite its developmental and medical significance, little is known about the factors that render cells to become competent for Notch signaling. This study shows that during vulval development in the nematode C. elegans the HOX protein LIN-39 and its EXD/PBX-like cofactor CEH-20 are required for LIN-12/Notch-mediated lateral signaling that specifies the 2° vulval cell fate. Inactivation of either lin-39 or ceh-20 resulted in the misspecification of 2° vulval cells and suppresses the multivulva phenotype of lin-12(n137) gain-of-function mutant animals. Furthermore, both LIN-39 and CEH-20 are required for the expression of basal levels of the genes encoding the LIN-12/Notch receptor and one of its ligands in the vulval precursor cells, LAG-2/Delta/Serrate, rendering them competent for the subsequent lin-12/Notch induction events. These results suggest that the transcription factors LIN-39 and CEH-20, which function at the bottom of the RTK/Ras and Wnt pathways in vulval induction, serve as major integration sites in coordinating and transmitting signals to the LIN-12/Notch cascade to regulate vulval cell fates (Takács-Vellai, 2007).
Convergent intercellular signals must be precisely coordinated in order to elicit specific biological responses. The C. elegans vulva provides an excellent experimental microcosm for studying how cell fate is specified according to the combined effects of different signaling pathways. This paper has studied the role of the Hox gene lin-39 and the Exd ortholog ceh-20 in vulval development. Genetic and molecular evidence is presented that the HOX protein LIN-39 and its putative cofactor CEH-20 are required for basal expression levels of lin-12 and lag-2 in the VPCs prior to vulval induction; this regulation may be important to render the VPCs competent for the subsequent lin-12/Notch induction events at the L3 larval stage. Identifying transcriptional regulators of lateral signaling in C. elegans vulval development will be essential for understanding how the Notch signaling pathway specifies cell fate in divergent animal species, and how compromised Notch signaling leads to human diseases (Takács-Vellai, 2007).
LIN-39 and CEH-20 are both required at the first larval stage to prevent fusion of the VPCs to the surrounding hypodermis. The data lead to the attractive possibility that LIN-39 and its putative cofactor CEH-20 regulate the competence of the VPCs to respond to any of the patterning signals during vulval formation. Along this line, it is challenging to speculate that, besides regulating lin-12 and lag-2 expression, they might also promote the expression of components of the inductive pathway (such as let-23) or other Notch pathway genes in the VPCs (Takács-Vellai, 2007).
It has been shown that CEH-20 binds in vitro, together with LIN-39, to the promoter of the twist transcription factor ortholog hlh-8 to regulate its expression in postembryonic mesodermal cells. ChIP experiments demonstrate that LIN-39 associates with the lag-2promoter, suggesting that the regulation of lag-2 expression by LIN-39 may be direct. It is proposed that LIN-39 forms a heterodimer with CEH-20 to promote the basal transcription of lag-2 and lin-12 in the VPCs. Based on their different expression pattern in the Pn.p lineages, ceh-20 is assumed to have some functions that are independent of lin-39. Indeed, mab-5 has been shown to be expressed in the descendants of the posterior VPCs, P7.p and P8.p, and to prevent them from adopting an induced vulval fate. Thus, it is possible that CEH-20 also interacts and functions with MAB-5 in controlling certain aspects of vulval fate specification. Furthermore, it is noted that ceh-20(ay9) mutant animals sometimes displayed a dual AC phenotype, whereas lin-39 mutants never did. RNAi-mediated depletion of mab-5 sometimes resulted in 2 ACs, suggesting that the correct AC specification requires the combined activity of mab-5 and ceh-20 (Takács-Vellai, 2007).
Finally, CEH-20 has been shown to be required as a cofactor for autoregulatory expression of the anterior Hox paralog (labial-like) ceh-13 in embryonic cells. Because ceh-13 is expressed all along the anteroposterior body axis in the ventral mid-line during the L1–L4 larval stages and a few percent of the ceh-13(sw1) mutant animals that are able to develop into fertile adults exhibit various defects in vulval formation, it is possible that CEH-13 acts with CEH-20 to control cell fate in the anterior VPC lineages. The future analysis of a potential role of ceh-13 in vulval development would help to establish the role of all of the major body Hox genes in this important process (Takács-Vellai, 2007).
The Notch pathway is the key signal for many cell fate decisions in the
nematode Caenorhabditis elegans including the uterine pi cell fate,
crucial for a proper uterine-vulval connection and egg laying. Expression of
the egl-13 SOX domain transcription factor is specifically
upregulated upon induction of the pi lineage and not in response to other
LIN-12/Notch-mediated decisions. Dual regulation by LIN-12
and FOS-1 is required for egl-13 expression at specification and for
complete rescue of egl-13 mutants. fos-1 mutants exhibit uterine defects and fail to express pi markers. FOS-1 is expressed at pi cell specification was demonstrated and that FOS-1 can bind in vitro to egl-13 upstream regulatory sequence (URS) as a heterodimer with C. elegans Jun (Oommen, 2007).
The C. elegans hermaphrodite egg-laying system comprises several tissues, including the uterus and vulva. lin-11 encodes a LIM domain transcription
factor needed for certain vulval precursor cells to divide asymmetrically. Lin-11 is homologous to a known, but unnamed and uncharacterized Drosophila Lim domain protein. Based on lin-11 expression studies and the lin-11 mutant phenotype, it has been found that lin-11 is
also required for C. elegans uterine morphogenesis. Specifically, lin-11 is expressed in the ventral uterine intermediate precursor (pi) cells and their progeny (the
utse and uv1 cells), which connect the uterus to the vulva. Like pi cell induction, the uterine lin-11 expression responds to the uterine anchor cell and the
lin-12-encoded receptor (Notch). In wild type animals, the utse, which forms the planar process at the uterine-vulval interface, fuses with the anchor cell. In
lin-11 mutants, utse differentiation is abnormal; the utse fail to fuse with the anchor cell and a functional uterine-vulval connection is not made. These findings
indicate that lin-11 is essential for uterine-vulval morphogenesis.
Activation of LIN-12 results in a variety of responses
depending on the cell, raising the issue of how cell-specific responses to a
general signal are programmed. lin-11 is
activated by lin-12 in both the vulval secondary cells and the uterine
po cells. However, lin-11 does not appear to be a general
executor of lin-12-mediated responses. According to
studies of the lin-11-lacZ fusion gene, lin-11 is not expressed
in the VU cell early, or in the SMs or G2 cell, all of which
require lin-12 function. Also, there is
no evidence for a role of lin-12 in VC neuron specification.
LIN-11 may be a component of a partially specific response to
LIN-12. The ability of a cell to activate LIN-11 in response to
LIN-12 might be one of the factors that contribute to the cell
specifity of such response (Newman, 1999).
In animal development, numerous cell-cell interactions are mediated by the GLP-1/LIN-12/NOTCH family of transmembrane receptors. These proteins function in a signaling pathway that appears to be conserved from nematodes to humans. The aph-2 gene is a new component of the GLP-1 signaling pathway in the early Caenorhabditis elegans embryo, and proteins with sequence similarity to the APH-2 protein are found in Drosophila (CG7012) and vertebrates. During the GLP-1-mediated cell interactions in the C. elegans embryo, APH-2 is associated with the cell surfaces of both the signaling, and the responding, blastomeres. Analysis of chimeric embryos that are composed of aph-2 plus and aph-2 minus blastomeres suggests that aph-2 plus function may be provided by either the signaling or responding blastomere (Goutte, 2000).
In C. elegans, the Notch receptor GLP-1 is localized within the germline and early embryo by translational control of glp-1 mRNA. RNA elements in the glp-1 3'untranslated region (3' UTR) are necessary for repression of glp-1 translation in germ cells, and for localization of translation to anterior cells of the early embryo. The direct regulators of glp-1 mRNA are not known. A 34 nucleotide region of the glp-1 3' UTR is shown to contain two regulatory elements, an element that represses translation in germ cells and posterior cells of the early embryo, and an element that inhibits repressor activity to
promote translation in the embryo. Furthermore, the STAR/KH
domain protein GLD-1 (Drosophila homolog: Held out wings) binds directly and specifically to the repressor element. Depletion of GLD-1 activity by RNA interference causes loss of endogenous glp-1 mRNA repression in early meiotic germ cells, and in posterior cells of the early embryo. Therefore, GLD-1 is a direct repressor of glp-1 translation at two developmental stages. These results suggest a new function for GLD-1 in regulating early embryonic asymmetry. Furthermore, these observations indicate that precise control of GLD-1 activity by other
regulatory factors is important to localize this Notch receptor. Such control contributes to the spatial organization of Notch signaling (Marin, 2003).
The vulval precursor cells (VPCs) are spatially patterned by a LET-23/EGF receptor-mediated inductive signal and a LIN-12/Notch-mediated lateral signal. The lateral signal has eluded identification, so the mechanism by which lateral signaling is activated has not been known. Ten genes have been
computationally identified that encode potential ligands for LIN-12; three of these genes, apx-1, dsl-1, and lag-2, are functionally redundant components of the lateral signal. Transcription of all three genes is initiated or upregulated in VPCs in response to inductive signaling, suggesting that direct transcriptional control of the lateral signal by the inductive signal is part of the mechanism by which these cell signaling events are coordinated. In addition, DSL-1, which lacks a predicted transmembrane domain, is a natural secreted ligand and can substitute for the transmembrane ligand LAG-2 in different functional assays (Chen, 2004).
Sequence analysis indicates that three DSL (Delta/Serrate/Lag-2) proteins have highly probable predicted transmembrane domains; these are encoded by the three genes that had been identified previously, lag-2, apx-1, and arg-1. Two of the proteins identified by computational analysis, DSL-2 and DSL-6, may also have transmembrane domains, although the potential transmembrane domains were assessed as lower probability in prediction programs. Surprisingly, the five remaining genes (dsl-1, dsl-3, dsl-4, dsl-5, and dsl-7) encode DSL proteins that are predicted to lack transmembrane domains and hence are likely to be secreted (Chen, 2004).
In terms of VPC specification, the cell biology of lateral signaling offers a rationale for why a secreted or peripheral membrane protein ligand might be a component of the lateral signal. The VPCs are polarized epithelial cells: they have an apical region and a basolateral domain, separated by adherens junctions. Since the apical regions of adjacent VPCs appear to be in contact only in the vicinity of the cell junctions, and LIN-12 is distributed over the whole apical surface, a transmembrane ligand on the surface of one VPC might have access to LIN-12 on the apical surface of its neighbor only in a relatively limited area. A ligand that can diffuse may be available to activate LIN-12 over a greater region of the apical domain, affording one solution to such a topographical problem (Chen, 2004).
The four-cell C. elegans embryo contains two sister cells called ABa and ABp that initially have equivalent abilities to produce ectodermal cell types. Multiple Notch-mediated interactions occur during the early cell divisions that diversify the ABa and ABp descendants. The first interaction determines the pattern of ectodermal cell types produced by ABp. The second interaction induces two ABa granddaughters to become mesodermal precursors. T-box transcription factors called TBX-37 and TBX-38 are essential for mesodermal induction, and these factors are expressed in ABa, but not ABp, descendants. Evidence is provided that the first Notch interaction functions largely, if not entirely, to prevent TBX-37, TBX-38 expression in ABp descendants. Neither the second Notch interaction nor TBX-37, TBX-38 alone are sufficient for mesodermal induction, indicating that both must function together. It is concluded that TBX-37, TBX-38 play a key role in distinguishing the outcomes of two sequential Notch-mediated interactions (Good, 2004).
At least four distinct interactions occur during the first few cell divisions of the C. elegans embryo, providing a relatively simple experimental system to analyze a network of Notch-mediated cell fate decisions. The anterior cell in the two-cell stage embryo is called AB, and all of the early descendants of AB express the receptors GLP-1/Notch or LIN-12/Notch. Various AB descendants contact one of several ligand-expressing cells that are born at different times and places during the early divisions, and change their fate accordingly. In genetic studies of Notch-mediated, binary cell fate decisions, one cell fate can often be considered as 'primary' and a second cell fate as 'secondary'; Notch function is required for the secondary, but not primary, fate (Artavanis-Tsakonas, 1999). In the absence of all Notch-mediated interactions in C. elegans embryos, AB descendants adopt highly patterned ectodermal fates that will thus be described here as primary fates (Good, 2004 and references therein).
The first Notch interaction occurs at the four-cell stage when the posterior daughter of AB, called ABp, contacts a cell called P2 that expresses a Notch ligand. The interaction between P2 and ABp causes the ABp descendants to adopt new fates that are described in this study as secondary fates; cells with secondary fates remain ectodermal precursors, but have a pattern of differentiation that is distinct from cells with primary fates. The anterior daughter of AB, called ABa, does not contact P2 and thus produces descendants that initially retain their potential for primary fates. At the 12-cell stage, however, two of the ABa granddaughters contact a new ligand-expressing cell called MS. This second Notch interaction induces those two ABa granddaughters to adopt novel, tertiary fates and become mesodermal precursors. During the next few cell divisions, there are third and fourth Notch interactions that further diversify the fates of some ABp descendants. Coincident with the Notch-mediated specification of cell fates, a separate anteroposterior polarity system generates additional differences between sister cells that are born from anteroposterior cell divisions. Thus, there are two types of primary fates (1a and 1p) depending on whether a cell is an anterior sister (1a) or posterior sister (1p). Similarly, there are two types of secondary fates and two tertiary fates. These anteroposterior differences appear to involve POP-1, a transcription factor that is localized asymmetrically after all anteroposterior divisions of the AB descendants (Good, 2004 and references therein).
The mesodermal precursors that are induced by the second Notch interaction form the anterior half of the pharynx, a large muscular structure used in feeding. The posterior half of the pharynx contains many of the identical mesodermal cell types found in the anterior half, but is derived from non-AB descendants through a Notch-independent pathway. Preventing the second Notch interaction results in embryos that lack the anterior pharynx, but that have a posterior half-pharynx produced by non-AB descendants (the Aph phenotype; anterior pharynx defective). Through genetic screens for Aph mutants, two functionally redundant T-box transcription factors called TBX-37 and TBX-38 have been identified that are essential for the development of the anterior pharynx. Notch signaling occurs at the 12-cell stage in tbx-37 tbx-38 mutant embryos, but does not result in mesodermal specification. Evidence suggests that the primary, if not sole, function of Notch signaling at the four-cell stage is to repress TBX-37, TBX-38 expression in ABp descendants. Thus, the first Notch interaction restricts the expression of T-box proteins that are essential to couple the second Notch interaction to mesoderm development (Good, 2004).
During the 12-cell stage of C. elegans embryogenesis,
Notch-mediated signaling from MS induces a subset of ABa descendants to
express forkhead-class transcription factor PHA-4 and become mesodermal precursors. TBX-37 and TBX-38
are essential for the ABa descendants to become mesodermal precursors. Several
examples of T-box proteins with roles in mesodermal development have been
described, including the prototype member Brachyury. The present study provides evidence that TBX-37, TBX-38 must act in conjunction with unidentified targets of the Notch signal transduction pathway for mesodermal induction in C. elegans. (1) It has been shown that TBX-37, TBX-38 expression and Notch signal transduction are regulated independently in ABa descendants. Killing the signaling cell, MS, or removing LAG-1, the transcriptional effector of the Notch pathway, does not prevent TBX-37, TBX-38 expression in ABa descendants. Conversely, removing TBX-37, TBX-38 does not prevent Notch-mediated repression of LAG-2 in the ABara granddaughters. (2) It has been shown that neither Notch signal transduction nor TBX-37, TBX-38 alone are sufficient for mesodermal induction. All of the
early ABa descendants express TBX-37, TBX-38 in wild-type embryos, but only those ABa descendants that are signaled by MS become mesodermal precursors. Conversely, activation of Notch represses LAG-2 expression in the ABara granddaughters of tbx-37 tbx-38 embryos, but does not induce PHA-4 expression in those same cells (Good, 2004).
Although the second Notch interaction (MS signaling) induces cells to
become mesodermal precursors that form the pharynx, the first Notch
interaction (P2 signaling) prevents cells from becoming mesodermal precursors. If the first Notch interaction does not occur, embryos have a hyperinduction of pharyngeal tissue. In normal development, MS signaling induces ABa (but not ABp) descendants to become mesodermal precursors. However, MS and its
sister cell, called E, both have the ability to signal, and one or both of
these cells contact some ABp descendants in addition to contacting ABa
descendants. When P2 signaling is blocked, either by
physically removing P2 or by mutations in the P2 ligand encoded by the Delta gene apx-1, MS and E induce these additional ABp descendants to become mesodermal precursors. Mutations in apx-1 cause the inappropriate expression of TBX-37,
TBX-38 in ABp descendants. In addition, it has been shown that removing TBX-37, TBX-38 activities from apx-1 mutant embryos prevents the
hyperinduction of pharyngeal cells. Thus,
the competence of both ABa and ABp descendants to become mesodermal precursors in response to the second Notch interaction is determined by the pattern of expression of TBX-37, TBX-38 (Good, 2004).
In summary, these results provide insight into two of the four Notch-mediated interactions that occur in rapid succession in early embryogenesis, and that modify ABa and ABp descendants in distinct ways. It is proposed that the transcription factors TBX-37, TBX-38 can promote 'primary' cell fates independent of Notch. The first Notch-mediated interaction blocks expression of TBX-37, TBX-38 in ABp descendants, thus allowing those cells to adopt novel, 'secondary' fates. Next, TBX-37, TBX-38 are expressed in ABa descendants independently of Notch, but shortly after the second Notch interaction. ABa descendants that do not undergo the second Notch interaction assume primary fates, in part through the action of TBX-37, TBX-38. In the ABa descendants that undergo the second Notch-interaction, TBX-37, TBX-38 collaborate with unidentified Notch targets to promote tertiary fates and mesoderm development (Good, 2004).
In the C. elegans germline, GLP-1/Notch signaling and two nearly identical RNA binding proteins, FBF-1 and FBF-2, promote proliferation. Here, the fbf-1 and fbf-2 genes are largely redundant for promoting mitosis but they have opposite roles in fine-tuning the size of the mitotic region. The mitotic region is smaller than normal in fbf-1 mutants but larger than normal in fbf-2 mutants. Consistent with gene-specific roles, fbf-2 expression is limited to the distal germline, while fbf-1 expression is broader. The fbf-2 gene, but apparently not fbf-1, is controlled by GLP-1/Notch signaling, and the abundance of FBF-1 and FBF-2 proteins is limited by reciprocal 3′UTR repression. It is proposed that the divergent fbf genes and their regulatory subnetwork enable a precise control over size of the mitotic region. Therefore, fbf-1 and fbf-2 provide a paradigm for how recently duplicated genes can diverge to fine-tune patterning during animal development (Lamont, 2004).
RNA binding proteins are key regulators of the germline decision between proliferation and differentiation. Of particular importance to this paper are FBF-1 and FBF-2 (for fem-3 Binding Factor) -- two nearly identical regulators of the PUF (Pumilio and FBF) protein family. The FBF-1 and FBF-2 proteins are collectively called FBF, and similarly, fbf-1 and fbf-2 are collectively called the fbf genes. The nucleotide sequences of fbf-1 and fbf-2 are 93% identical, and the amino acid sequences are 91% identical, suggesting that fbf-1 and fbf-2 are recently duplicated genes. During early larval stages, germline proliferation is normal in fbf-1 fbf-2 double mutants, but in the fourth larval stage, the germline precociously leaves the mitotic cell cycle to enter meiosis and differentiate as sperm. In addition, depletion of both fbf-1 and fbf-2 eliminates the hermaphrodite switch from spermatogenesis to oogenesis. Therefore, FBF is required for continued mitotic divisions in the germline as well as for the hermaphrodite sperm/oocyte switch (Lamont, 2004).
PUF proteins bind specifically to regulatory elements, usually in the 3' untranslated region (UTR) of a target mRNA, and repress that mRNA, either by promoting mRNA degradation or inhibiting translation. Pumilio, for example, inhibits translation of hunchback mRNA in the early Drosophila embryo, whereas PUF-5/Mpt5 destabilizes HO mRNA in yeast. In C. elegans, FBF-1 and FBF-2 promote mitosis by repressing mRNAs that encode regulators critical for entry into the meiotic cell cycle, and they promote the sperm/oocyte switch by repressing the fem-3 sperm-promoting mRNA. Both FBF-1 and FBF-2 bind specifically to the same RNA target sequence, which differs from the Pumilio binding site. The molecular mechanism by which FBF represses mRNAs in the C. elegans germline remains unknown, but by analogy with its homologs in yeast and Drosophila, FBF is likely to control the stability or translation of its target mRNAs (Lamont, 2004).
Previous studies have suggested that FBF-1 and FBF-2 are redundant: fbf-1 single mutants are grossly normal, albeit with smaller mitotic regions and more hermaphrodite sperm than wild-type. This study confirms the fbf-1/fbf-2 redundancy but also identify individual roles for each gene in regulating the size of the mitotic region. Like fbf-1, the fbf-2 single mutants are grossly normal, but in contrast to fbf-1, fbf-2 mutant germlines have a larger mitotic region than normal and can be feminized. Consistent with fbf-1 and fbf-2 having individual roles, their mRNAs and proteins are expressed in distinct patterns. Furthermore, the fbf-2 gene appears to be a direct target of GLP-1/Notch signaling, a finding that forges the first molecular link between GLP-1/Notch signaling and the RNA regulatory circuit. fbf-1 and fbf-2 repress each other's expression and this reciprocal repression is likely to be direct via FBF binding sites in the fbf-1 and fbf-2 3' UTRs. It is suggested that GLP-1/Notch signaling and FBF autoregulation work together to control the distribution and amount of FBF and thereby fine-tune the size of the mitotic region (Lamont, 2004).
The coordination of signals from different pathways is important for cell fate specification during animal development. A novel mode of crosstalk between the epidermal growth factor receptor/Ras/mitogen-activated protein kinase cascade and the LIN-12/Notch pathway during Caenorhabditis elegans vulval development has been defined. Six vulval precursor cells (VPCs) are initially equivalent but adopt different fates as a result of an inductive signal mediated by the Ras pathway and a lateral signal mediated by the LIN-12/Notch pathway1. One consequence of activating Ras is a reduction of LIN-12 protein in P6.p, the VPC believed to be the source of the lateral signal. A 'downregulation targeting signal' (DTS) has been identified in the LIN-12 intracellular domain, which encompasses a di-leucine-containing endocytic sorting motif. The DTS seems to be required for internalization of LIN-12, and on Ras activation it might mediate altered endocytic routing of LIN-12, leading to downregulation. If LIN-12 is stabilized in P6.p, lateral signalling is compromised, indicating that LIN-12 downregulation is important in the appropriate specification of cell fates in vivo (Shaye, 2002).
The DTS contains two adjacent leucine residues and several serines that are conserved in all known nematode LIN-12/Notch proteins. 'Di-leucine motifs' are well-characterized sorting signals that usually take the form (-)(2-4)XLL, where (-) is often an acidic residue or phosphoserine, although basic residues have also been seen at this position, and X is usually a polar or bulky residue. Functional motifs that contain M, V or I instead of L have also been found. Di-leucine motifs regulate different aspects of protein trafficking, including the constitutive or ligand-stimulated internalization of transmembrane receptors, and the routing of proteins within the endocytic and/or secretory pathways. Different residues within the same di-leucine motif might modulate different aspects of motif activity (namely internalization versus routing), and the activity of di-leucine motifs that contain serines might be regulated by phosphorylation. In several cases the di-leucine motif leads to routing of the protein to lysosomes for degradation.Mutating the two leucine residues of the DTS to two alanines disrupts downregulation of sTM::LIN-12(intra)::GFP. Because downregulation of GFP-tagged LIN-12 fragments requires both membrane association and a di-leucine motif, it is inferred that the mechanism of downregulation involves increased internalization and/or altered endocytic routing of LIN-12 when Ras is activated (Shaye, 2002).
The gene sur-2 encodes a component of the Mediator complex, which activates transcription in response to Ras/MAPK signalling in mammalian cells. SUR-2 also seems to be activated by the Ras pathway in C. elegans, and hermaphrodites lacking sur-2 activity display a failure in lateral signalling. In sur-2(-) hermaphrodites, LIN-12(+)::GFP is not downregulated. This result suggests that the failure of lateral signalling in sur-2(-) hermaphrodites might result at least in part from the failure to downregulate endogenous LIN-12(+). Furthermore, in sur-2(-) hermaphrodites, as in the wild type, LIN-12(+)::GFP accumulates in puncta, suggesting that SUR-2/Mediator-promoted transcription is not necessary for the initial internalization, but instead might affect the rate of internalization and/or routing of endocytosed LIN-12(+)::GFP (Shaye, 2002).
A model is presented for cross-talk between the Ras and LIN-12 pathways in P6.p. LIN-12, like other transmembrane proteins, seems to be constitutively internalized and might be routed to recycling endosomes or to lysosomes. It is proposed that Ras activation leads to transcription of at least one gene whose product regulates the rate of internalization and/or subcellular routing of LIN-12, leading to its degradation. Internalization (and perhaps, in addition, endocytic routing) of LIN-12 is mediated by the DTS: when the DTS is removed, LIN-12 accumulates in the apical membrane of P6.p. Persistence of LIN-12, achieved by deleting the DTS or by removing sur-2 activity, is correlated with inhibition of lateral signalling, demonstrating that downregulation is functionally important (Shaye, 2002).
The novel mode of crosstalk between the Ras and LIN-12/Notch pathways may be conserved. Vertebrate Notch proteins contain a highly conserved potential di-leucine-like motif (1822KKFRFEEPVVL1832 in human Notch1) that, like the LIN-12 DTS, lies between the transmembrane domain and the first ankyrin motif, and furthermore the large intracellular domain of notch proteins contains additional potential endocytic motifs. Whether these motifs function as such, and whether their function is regulated by Ras or other signals, can only be answered through experiments in other systems. Observations of Wingless endocytosis has led to the speculation that Ras might modulate the endocytic routing of Wingless bound to its receptor Frizzled2 via a di-leucine motif on Frizzled2, dependent on the transcription of an unknown factor in response to epidermal growth factor receptor/Ras/MAPK signalling. If this proves to be so, the future identification of the putative factors that recognize the targeting signals of LIN-12/Notch and Frizzled2 will reveal whether Ras works through a common or distinct mechanism to modulate the activity of these different receptors (Shaye, 2002).
During C. elegans development, LIN-12 (Notch) signaling specifies
the anchor cell (AC) and ventral uterine precursor cell (VU) fates from two
equivalent pre-AC/pre-VU cells in the hermaphrodite gonad. Once specified, the
AC induces patterned proliferation of vulva via expression of LIN-3 (EGF) and
then invades into the vulval epithelium. Although these cellular processes are
essential for the proper organogenesis of vulva and appear to be temporally
regulated, the mechanisms that coordinate the processes are not well
understood. egl-43 was computationally identified as a gene likely to
be expressed in the pre-AC/pre-VU cells and the AC, based on the presence of
an enhancer element similar to the one that transcribes lin-3 in the
same cells. Genetic epistasis analyses reveal that egl-43 acts
downstream of or parallel to lin-12 in AC/VU cell fate specification
at an early developmental stage, and functions downstream of fos-1 as
well as upstream of zmp-1 and him-4 to regulate AC invasion
at a later developmental stage. Characterization of the egl-43
regulatory region suggests that EGL-43 is a direct target of LIN-12 and HLH-2
(E12/47), which is required for the specification of the VU fate during AC/VU
specification. EGL-43 also regulates basement membrane breakdown during AC
invasion through a FOS-1-responsive regulatory element that drives EGL-43
expression in the AC and VU cells at the later stage. Thus, egl-43
integrates temporally distinct upstream regulatory events and helps program
cell fate specification and cell invasion (Hwang, 2007).
The Notch - Presenilin connection in C. elegans and mammals sel-12 (sel means suppressor/enhancer of lin-12) is a transmembrane protein that facilitates Notch signaling in C. elegans. It is related to Presenilin-1 (S182), a mammalian gene that when mutated causes aggressive, early-onset Alzheimer's via an unknown mechanism. In C. elegans, Notch signaling is involved in the anchor cell/ventral uterine precursor cell (AC/VU) decision and vulval precursor cell (VPC) specification during gonadogenesis. The AC/VU decision involves an interaction between two initially equivalent cells of the somatic gonad. When lin-12, which codes for a Notch family member, is eliminated, both precursor cells become ACs. When lin-12 is constitutively activated, both precursor cells become VUs. sel-12 was isolated as a suppressor of a lin-12 gain of function mutation. That is, sel-12 mutation acts to reduce lin-12 signaling. Reducing sel-12 activity reduces lin-12 activity in lateral signaling that specifies the secondary fate of VPCs. Cell ablation experiments show that sel-12 functions within a VPC to lower lin-12 activity. The predicted SEL-12 protein contains multiple potential transmembrane domains, consistent with its function as a receptor, ligand, channel or membrane structural protein. SEL-12 might be directly involved in lin-12-mediated reception, functioning for example as a co-receptor or as a downstream effector that activatesupon LIN-12 activation. Alternatively, sel-12 may be involved in a more general cellular process, such as receptor localization or recycling, and hence influence lin-12 activity indirectly (Levitan, 1995).
The data presented in this paper suggest that the effect of SEL-12/presenilin on LIN-12/Notch is analogous to its effect on APP. LIN-12/Notch proteins are transmembrane proteins with hallmark motifs: epidermal growth factor-like; LIN-12/Notch repeat, and cdc10/SWI6 (ankyrin) motifs. Like APP, LIN-12/Notch proteins must be correctly sorted and transported to the cell surface, and undergo proteolytic cleavage events. There appears to be at least one constitutive proteolytic cleavage event that occurs in the extracellular domain during the transport to the plasma membrane; the cleaved form produced by this constitutive cleavage event may be the major species present at the cell surface. In addition, binding of ligand appears to induce a cleavage event in or near the transmembrane domain; this apparent cleavage event enables the intracellular domain to translocate to the nucleus, where it participates directly in regulating downstream gene expression. It is conceivable that SEL-12/presenilin is involved in promoting one or more of these cleavage events, either by activating protease(s) or promoting trafficking of either LIN-12 or proteases to an appropriate compartment. The strong accumulation of SEL-12::GFP in the ER/Golgi is consistent with a role for SEL-12 in a constitutive cleavage event involved in maturation of LIN-12/Notch proteins. The fact that less LIN-12::GFP was observed at the cell surface in a sel-12 mutant background could be explained in the context of this model by proposing that abnormal processing of LIN-12 leads to its failure to be transported to the plasma membrane or to its degradation. The putative ligand-dependent cleavage of activated LIN-12 might occur at the plasma membrane or in internalized vesicles. The failure to observe SEL-12::GFP in the plasma membranes of the VPCs does not preclude a role for SEL-12 in ligand-dependent cleavage. It is possible that SEL-12::GFP is present at low abundance in the plasma membrane; the ligand-induced event appears to affect a very small proportion of receptor molecules, suggesting that the agent that promotes the cleavage may not be very abundant. Although the issue of the biochemical mechanism of presenilin function is not resolved in any system, the parallels between APP and LIN-12/Notch trafficking and processing suggest that a common mechanism is involved. An important challenge for the future will be to identify the primary effect of SEL-12/presenilin on APP and LIN-12/Notch, since proteolytic processing, intracellular trafficking and degradation are intimately linked, and altering one process can affect another (Levitan, 1998b).
Mutant presenilins have been found to cause Alzheimer disease. This paper describes the identification
and characterization of HOP-1, a Caenorhabditis elegans presenilin that displays much more lower
sequence identity with human presenilins than does the other C. elegans presenilin, SEL-12. Despite
considerable divergence, HOP-1 appears to be a bona fide presenilin, because HOP-1 can rescue the
egg-laying defect caused by mutations in sel-12 when hop-1 is expressed under the control of sel-12
regulatory sequences. HOP-1 also has the essential topological characteristics of the other presenilins.
Reducing hop-1 activity in a sel-12 mutant background causes synthetic lethality and terminal
phenotypes associated with reducing the function of the C. elegans lin-12 and glp-1 genes. These
observations suggest that hop-1 is functionally redundant with sel-12 and underscore the intimate
connection between presenilin activity and LIN-12/Notch activity inferred from genetic studies in C.
elegans and mammals (Li, 1997).
C. elegans gene sel-1 functions as a negative regulator of Notch homolog lin-12
activity. It has been predicted that SEL-1 is a secreted or membrane associated
protein. Cell ablation experiments that suggest
sel-1 mutations elevate lin-12 activity cell autonomously. The predicted signal sequence of
SEL-1 can direct secretion and is important for function, while a C-terminal
hydrophobic region is not required for SEL-1 function. SEL-1 is
localized intracellularly, with a punctate staining pattern suggestive of
membrane bound vesicles. A related yeast protein, L8167.5, a product of the HRD3 gene, acts as a component of the mechanism for degrading a yeast metabolic enzyme. It is suggested that SEL-1 might be involved in down-regulating the activated LIN-12 receptor (Grant, 1997).
The Notch signaling pathway regulates specification and proliferation in a variety of cell lineages in invertebrates and vertebrates. A murine homolog of SEL-1, a key negative regulator of the Notch pathway in Caenorhabditis elegans, has been cloned. C. elegans SEL-1 functions as a negative regulator of Notch homolog Lin-12 activity. It has been predicted that SEL-1 is a secreted or membrane associated protein. Murine SEL-1L (mSEL-1L) protein exhibits a high degree of similarity to SEL-1, including a signal peptide and the C-terminal region required for SEL-1 function in C. elegans. This mammalian homolog of sel-1 is widely expressed in adult mouse and human tissues, with particularly high levels in the pancreas. RNA in situ analysis of developing mouse embryos indicates that mSEL-1L is moderately expressed throughout the neural tube and dorsal root ganglia, with particularly high levels in the floor plate of the neural tube beginning at E10.5 and increasing at E11.5. Expression is high at E14.5 and E17.5 in the acini of the pancreas, and moderate in the epithelial cells of the gut villi. The SEL-1L protein has been localized to the cytosol, possibly in intracellular vesicles, in a beta-islet-derived tumor cell line (Donoviel, 1998).
Mutations in the human presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease. Studies in
Caenorhabditis elegans and in mice indicate that one function of presenilin genes is to facilitate
Notch-pathway signaling. Notably, mutations in the C. elegans presenilin gene sel-12 reduce signaling
through an activated version of the Notch receptor LIN-12. To investigate the function of a second C.
elegans presenilin gene hop-1 and to examine possible genetic interactions between hop-1 and sel-12, a reverse genetic strategy was used to isolate deletion alleles of both loci. Animals bearing both hop-1 and
sel-12 deletions display new phenotypes not observed in animals bearing either single deletion. These
new phenotypes (germ-line proliferation defects, maternal-effect embryonic lethality, and somatic gonad
defects) resemble those resulting from a reduction in signaling through the C. elegans Notch receptors
GLP-1 and LIN-12. Thus SEL-12 and HOP-1 appear to function redundantly in promoting
Notch-pathway signaling. Phenotypic analyses of hop-1 and sel-12 single and double mutant animals
suggest that sel-12 provides more presenilin function than does hop-1. There is as yet no evidence that Notch-pathway signaling is
involved in the pathophysiology of Alzheimer's disease (AD). Thus, the relationship between the roles of presenilins in
proteolytic processing of APP and in facilitating Notch receptor function remains unclear. Intriguingly,
recent evidence suggests that multiple proteolytic processing events are required for intracellular trafficking
and signal transduction of the Notch receptor: two cleavage events are proposed to occur in the
extracellular domain and a third proposed cleavage occurs within or just carboxyl-terminal to the
transmembrane region. The apparent similarities between the processing of APP and Notch,
particularly the prospect that both are cleaved within the transmembrane domain, raise the possibility that
presenilins affect proteolytic processing of APP and Notch in analogous ways. Presenilins might regulate
proteolytic processing directly or might do so indirectly, for example, by promoting normal intracellular
trafficking of APP or Notch. In support of a role for presenilins in the processing or trafficking of Notch, LIN-12::GFP levels at the plasma membrane are seen to be reduced in a sel-12 mutant background. An understanding of how presenilins affect Notch-receptor
activity may be relevant to an understanding of the way in which presenilins affect APP cleavage and to the
identification of targets for preventing the pathophysiological effects of presenilin dysfunction in AD (Westlund, 1999).
Presenilin-1 (PS1), a polytopic membrane protein primarily localized to the endoplasmic reticulum, is required for efficient proteolysis of both Notch and
beta-amyloid precursor protein (APP) within their trans-membrane domains. The activity that cleaves APP (called gamma-secretase) has properties of an
aspartyl protease; mutation of either of the two aspartate residues located in adjacent transmembrane domains of PS1 inhibits gamma-secretase
processing of APP. These aspartates are required for Notch processing, since mutation of these residues prevents PS1 from inducing the
gamma-secretase-like proteolysis of a Notch1 derivative. Thus PS1 might function in Notch cleavage as an aspartyl protease or di-aspartyl protease
cofactor. However, the ER localization of PS1 is inconsistent with that hypothesis, since Notch cleavage occurs near the cell surface. Using pulse-chase and
biotinylation assays, evidence is provided that PS1 binds Notch in the ER/Golgi and is then co-transported to the plasma membrane as a complex. PS1
aspartate mutants are indistinguishable from wild-type PS1 in their ability to bind Notch or traffic with it to the cell surface, and do not alter the secretion of
Notch. Thus, PS1 appears to function specifically in Notch proteolysis near the plasma membrane as an aspartyl protease or cofactor (Ray, 1999b).
The C. elegans intestine is a simple tube consisting of a
monolayer of epithelial cells. During embryogenesis, cells
in the anterior of the intestinal primordium undergo
reproducible movements that lead to an invariant,
asymmetrical 'twist' in the intestine. The
development of this twist has been examined to determine how left-right and
anterior-posterior asymmetries are generated within the
intestinal primordium. The twist requires the LIN-12/
Notch-like signaling pathway of C. elegans. All cells
within the intestinal primordium initially express LIN-12,
a receptor related to Notch; however, only cells in the left
half of the primordium contact external, nonintestinal cells
that express LAG-2, a ligand related to Delta. LIN-12 and
LAG-2 mediated interactions result in the left primordial
cells expressing lower levels of LIN-12 than the right
primordial cells. It is proposed that this asymmetrical
pattern of LIN-12 expression is the basis for asymmetry in
later cell-cell interactions within the primordium that lead
directly to intestinal twist. Like the interactions that
initially establish LIN-12 asymmetry, the later interactions
are mediated by LIN-12. The later interactions, however,
involve a different ligand related to Delta, called APX-1. The anterior-posterior asymmetry in intestinal
twist involves the kinase LIT-1, which is part of a signaling
pathway in early embryogenesis that generates anterior-posterior
differences between sister cells (Hermann, 2000).
aph-2 (Drosophila homolog: CG7012) encodes a novel extracellular protein required for GLP-1-mediated signaling (Goutte, 2000). Aph-2, termed Nicastrin (see Drosophila nicastrin) in this study, is a transmembrane glycoprotein that forms high molecular weight complexes
with presenilin 1 and presenilin 2. Suppression of nicastrin expression in
C. elegans embryos induces a subset of notch/glp-1
phenotypes similar to those induced by simultaneous null mutations in both
presenilin homologs of C. elegans (sel-12 and hop-1).
Nicastrin also binds carboxy-terminal derivatives of beta-amyloid precursor
protein (betaAPP), and modulates the production of the amyloid beta-peptide
(Abeta) from these derivatives. Missense mutations in a conserved hydrophilic
domain of nicastrin increase Abeta42 and Abeta40
peptide secretion. Deletions in this domain inhibit Abeta production. Nicastrin
and presenilins are therefore likely to be functional components of a multimeric
complex necessary for the intramembranous proteolysis of proteins such as
Notch/GLP-1 and betaAPP (Yu, 2000).
In the absence of homology to other proteins, sequence databases
were screened for orthologous genes in other species. A full-length C. elegans
nicastrin ortholog was found in public databases (accession no. Q23316; identity = 22%; similarity = 41%). Full-length murine and Drosophila nicastrin orthologs
from appropriate cDNA libraries were cloned
and sequenced using partial cDNA sequences from these databases
as start points (mouse nicastrin accession no. AF24069,
identity = 89%, similarity = 93%; D. melanogaster nicastrin accession
no. AF240470, identity = 30%, similarity
= 48%). The four animal nicastrins have similar
predicted topologies and have three domains with significant sequence conservation
near residues 306-360, 419-458, and 625-662 of human nicastrin. Within
the first conserved domain, all four proteins contained the motif DYIGS (residues
336-340), which is also partially conserved in an Arabidopsis
protein. All four animal nicastrins also contain four cysteines spaced at
16 to 17-residue intervals in the N terminus (Cys 195, Cys 213,
Cys 230 and Cys 248) (Yu, 2000).
To explore whether nicastrin, like the presenilins, might have a role in Notch signaling in vivo, RNA interference (RNAi) was used in
C. elegans. Wild-type worms injected with C. elegans nicastrin
double-stranded (ds) RNA produce dead embryos, many of which lack an anterior
pharynx. This phenotype is highly
reproducible and specific. Except for embryonic lethality, none of the other phenotypes
associated with a lack of C. elegans presenilin (sel-12) activity
were observed. However, this phenotype is identical to that induced when the
activity of genes in the notch/glp-1 pathway (glp-1, aph-1
or aph-2) are reduced, or when the activities of both C. elegans
presenilin homologs (sel-12 and hop-1) are reduced simultaneously. Thus nicastrin
contributes to some aspects of notch/glp-1 signaling in C. elegans
embryos (Yu, 2000).
The Notch signaling pathway plays essential roles during
the specification of the rostral and caudal somite halves and
subsequent segmentation of the paraxial mesoderm. The role of presenilin 1 (Ps1; encoded by Psen1) during segmentation has been investigated using newly generated
alleles of the Psen1 mutation. In Psen1-deficient mice,
proteolytic activation of Notch1 is significantly affected
and the expressions of several genes involved in the Notch
signaling pathway are altered, including Delta-like3, Hes5, lunatic fringe (Lfng) and Mesp2, which encodes a bHLH
transcriptional regulator expressed in the rostral region of
the presomitic mesoderm. Thus, Ps1-dependent
activation of the Notch pathway is essential for caudal
half somite development. Defects were observed in Notch
signaling in both the caudal and rostral region of the
presomitic mesoderm. In the caudal presomitic mesoderm,
Ps1 is involved in maintaining the amplitude of cyclic
activation of the Notch pathway, as represented by
significant reduction of Lfng expression in Psen1-deficient
mice. In the rostral presomitic mesoderm, rapid
downregulation of the Mesp2 expression in the presumptive
caudal half somite depends on Ps1 and is a prerequisite for
caudal somite half specification. Chimaera analysis
between Psen1-deficient and wild-type cells reveals that
condensation of the wild-type cells in the caudal half somite
is concordant with the formation of segment boundaries,
while mutant and wild-type cells intermingle in the
presomitic mesoderm. This implies that periodic activation
of the Notch pathway in the presomitic mesoderm is still
latent to segregate the presumptive rostral and caudal
somite. A transient episode of Mesp2 expression might be
needed for Notch activation by Ps1 to confer rostral or
caudal properties. In summary, it is proposed that Ps1
is involved in the functional manifestation of the
segmentation clock in the presomitic mesoderm (Koizumi, 2001).
Mouse Notch1, which plays an important role in cell fate
determination in development, is proteolytically processed within its
transmembrane domain by unidentified gamma-secretase-like activity that
depends on presenilin. To study this proteolytic event,
a cell-free Notch cleavage assay system was established using the membrane fraction of fibroblast transfectants of various Notch constructs with deletion of
the extracellular portion (NotchDeltaE). The cytoplasmic portion of
Notch1DeltaE is released from the membrane upon incubation at 37°C; this is inhibited by the specific gamma-secretase inhibitor, MW167, or
by overexpression of dominant negative presenilin1. Likewise, other
members of mouse Notch family are proteolytically cleaved in a
presenilin-dependent, MW167-sensitive manner in vivo as
well as in the cell-free Notch DeltaE cleavage assay system. All four
members of the mouse Notch family migrate to the nucleus and activate
the transcription from the promoter carrying the RBP-J consensus
sequences after they are released from the membrane. These results
demonstrate the conserved biochemical mechanism of signal transduction
among mammalian Notch family members (Mizutani, 2001).
Nicastrin is a multi-pass transmembrane protein that has recently been identified as a member of high-molecular weight complexes
containing presenilin. The C. elegans homolog of nicastrin, aph-2, is required for GLP-1/Notch signaling in the early embryo. In
addition to the maternal-effect embryonic lethal phenotype, aph-2 mutant animals also display an egg-laying defect. This latter defect is related to the SEL-12/presenilin egg-laying defect. aph-2 and sel-12 genetically interact and cooperate to regulate LIN-12/Notch signaling in the development of the somatic gonad. In addition, aph-2 and lin-12/Notch genetically interact. A new role for aph-2 in facilitating lin-12 signaling in the somatic gonad is illustrated, thus providing evidence that APH-2 is involved in both GLP-1/Notch- and LIN-12/Notch-mediated signaling events. Nicastrin can partially substitute for aph-2, suggesting a
conservation of function between these proteins (Levitan, 2001).
Enzymatic cleavage of Notch The Notch receptor on the plasma membrane is cleaved. The
cleavage of human Notch2 is an evolutionarily conserved, general property of Notch and occurs in the trans-Golgi network as the receptor traffics toward the plasma membrane. Although full-length Notch2 is detectable in the cell, it does not reach the surface. Cleavage results in a C-terminal fragment, N(TM), that appears to be cleaved N-terminal to the transmembrane domain, and an N-terminal fragment, N(EC), that contains most of the extracellular region. These fragments are tethered together on the plasma membrane by a link that is sensitive to reducing conditions, forming a heterodimeric receptor. It is likely that the cleavage occurs between the EGF and Lin-12/Notch (LN) repeats, producing two fragments with a calculated molecular mass of 112 kDa and 180 kDa (Blaumueller, 1997).
A gamma-secretase-like proteolysis at site 3 (S3), within the transmembrane domain, releases the Notch intracellular domain (NICD) and activates CSL-mediated
Notch signaling. S3 processing occurs only in response to ligand binding; however, the molecular basis of this regulation is unknown. Ligand
binding facilitates cleavage at a second, novel site (S2), within the extracellular juxtamembrane region, which serves to release ectodomain repression of NICD production.
Cleavage at S2 generates a transient intermediate peptide termed NEXT (Notch extracellular truncation). NEXT accumulates when NICD production is blocked by
point mutations or gamma-secretase inhibitors or by loss of presenilin 1, and inhibition of NEXT eliminates NICD production. These data demonstrate that S2
cleavage is a ligand-regulated step in the proteolytic cascade leading to Notch activation (Mumm, 2000).
Peptide sequencing shows that S2 cleavage occurs between Ala-1710 and Val-1711 residues, approximately 12 amino acids outside the
transmembrane domain. Thus, NEXT is a naturally occurring equivalent of constitutively active, membrane-tethered, NDeltaE proteins. Pulse-chase analysis
has demonstrated that NDeltaE proteins undergo S3 processing and are converted to NICD. Consistent with this observation, evidence is presented that production of NEXT and NICD is linked: NEXT is enriched by blocking NICD production via point mutation, gamma-secretase inhibitors,
and loss of presenilin 1 (PS1), while inhibition of NEXT production eliminates NICD accumulation. An initial inhibitor screen implicates metalloprotease activity in
S2 proteolysis. In support of this, TACE (TNFalpha-converting enzyme) has been identified as a protease capable of S2 proteolysis in vitro. These data suggest that a ligand-induced proteolytic cascade activates Notch1; ligand binding serving to promote S2 cleavage, which
is required for S3 cleavage (Mumm, 2000).
For NEXT to be a true signaling intermediary, it should be regulated in a ligand-dependant manner. In order to demonstrate that S2 is relevant to activation of the
full-length receptor, NFL6MT constructs were cotransfected with CSLRBP-Jkappa and the DSL family member Jagged. NEXT and NICD both accumulate in the presence of Jagged. This result demonstrates that S2
proteolysis occurs in response to ligand activation of the full-length Notch1 receptor. However, one caveat of this and previous cotransfection experiments stems
from the question of whether Notch is activated by a ligand presented by the same cell (in cis) or from a neighboring cell (in trans).
Therefore, in order to confirm that trans interactions at the cell surface between Notch and its ligands lead to NEXT and NICD production, HeLa cells expressing
Notch and CSLRBP-Jkappa were cocultured with HEK 293T cells or HEK 293T expressing exogenous Jagged. Both S2 and S3 proteolysis are strongly induced when receptor-ligand interactions occur exclusively in trans. This result provides
further support for the hypothesis that the S2 cleavage occurs extracellularly at the plasma membrane. Taken together, these results support the hypothesis that ligand binding serves to relieve extracellular inhibition of S3 cleavage by promoting S2
cleavage. This ectodomain shedding-like process creates NEXT, a NDeltaE-like molecule, which undergoes S3 cleavage, producing NICD and leading to Notch activation (Mumm, 2000).
TACE is among a number of enzymes (alternatively termed secretases or sheddases) known to mediate a proteolytic process termed ectodomain shedding, whereby
transmembrane proteins are cleaved extracellularly and released into the extracellular milieu. Ectodomain shedding is known to play
key roles in cancer invasion, metastasis, activation of soluble ligands, and protein turnover. Sheddases generally exhibit low substrate specificity, and some are
believed to be more dependent on structural motifs than primary residues, often cleaving at a fixed distance from the transmembrane domain (e.g., TACE). Disruption
of the TACE locus in mice unexpectedly results in embryonic lethality, implicating TACE and ectodomain shedding in essential developmental events and suggesting
TACE normally cleaves substrates other than pro-TNF. While no direct evidence is presented here that S2 cleavage results in
shedding of Notch per se, recent reports have demonstrated that dissociation of the extracellular domain is sufficient for activation. Further, it has
been observed that the extracellular domain of Notch alone is trans-endocytosed (thus 'shed') into ligand-expressing cells when Notch is activated during pupal
wing vein and retinal pigment cell development in Drosophila. If correct, this model would predict that truly 'soluble' ligands will fail to
activate Notch, and that Notch will be activated by membrane-tethered or extracellular matrix associated ligands (Mumm, 2000).
A final consideration must be taken of the involvement of metalloprotease(s) in Notch activation. Notch pathway activation has been shown to
serve as a secondary event contributing to the oncogenic transformation of either Myc- or E1A- expressing
cells. Elevated metalloprotease activity, which is often linked to transformation and metastasis, may lead to ectopic, ligand-independent activation of the endogenous
Notch receptor. This may explain why nuclear Notch staining is often found in various tumors and suggests that ectopic,
metalloprotease-mediated Notch activation may be a common event during oncogenesis (Mumm, 2000).
The Notch1 receptor is presented at the cell membrane as a heterodimer after constitutive processing by a furin-like convertase. Ligand binding induces the
proteolytic release of Notch intracellular domain by a gamma-secretase-like activity. This domain translocates to the nucleus and interacts with the DNA-binding
protein CSL, resulting in transcriptional activation of target genes. An additional processing event occurs in the extracellular part of the receptor,
preceding cleavage by the gamma-secretase-like activity. Purification of the activity accounting for this cleavage in vitro shows that it is due to TACE (TNFalpha-converting enzyme), a member of the ADAM (a disintegrin and metalloprotease domain) family of metalloproteases. Furthermore, experiments carried
out on TACE-/- bone marrow-derived monocytic precursor cells suggest that this metalloprotease plays a prominent role in the activation of the Notch pathway (Brou, 2000).
The TACE cleavage site in the Notch1 sequence is located between Ala-1710 and Val-1711, 13 amino acids upstream of the transmembrane domain. This site is perfectly conserved in murine or human Notch1 and in Xenopus Notch. In murine Notch2 and Notch3 and Drosophila Notch, similar sites can be found (SV in murine Notch2 and Drosophila Notch at the same position upstream of the transmembrane domain; AV 24 amino acids upstream of the transmembrane domain in murine Notch3). It is difficult to identify a similar site in Lin12 or Glp1, because of the weak general conservation. However, a database search indicates that a TACE ortholog exists in C. elegans. Thus, it is possible that TACE could recognize a degenerated site in C. elegans Notch homologs; alternatively, other mechanisms may exist to lead to a conformational change after ligand binding. Interestingly, some constitutively active mutants of C. elegans Lin12 carry mutations in the region surrounding the putative S2 site. The AV cleavage site in Notch matches perfectly known TACE preferred recognition sites such as those found in pro-TNF or pro-TGF (Brou, 2000).
Notch is a conserved cell surface receptor that is activated through direct contact with neighboring ligand-expressing cells.
The primary 300-kDa translation product of the Notch1 gene (p300) is cleaved by a furin-like convertase to generate a
heterodimeric, cell-surface receptor composed of 180- (p180) and 120- (p120) kDa polypeptides. Heterodimeric Notch is
thought to be the only form of the receptor that is both present on the cell surface and able to generate an intracellular
signal in response to ligand. Disruption of furin processing of Notch1, either
by coexpression of a furin inhibitor or by mutation of furin target sequences within Notch1 itself, perturbs ligand-dependent
signaling through the well-characterized mediator of Notch signal transduction, CSL [CBF1, Su(H), and LAG-1].
Yet contrary to these reports, the full-length p300 Notch1 product can be detected on the cell surface. Moreover, this
uncleaved form of Notch1 can suppress the differentiation of C2C12 myoblasts in response to ligand. Taken together,
these data support characterizing a CSL-independent Notch signaling pathway and identify this uncleaved isoform of Notch as a potential mediator of this pathway. These results suggest a novel paradigm in signal
transduction, one in which two isoforms of the same cell-surface receptor can mediate two distinct signaling pathways in response to ligand (Bush, 2001).
The biological activity of the soluble form of the Notch ligand (sNL) and requirement of the intracellular domain (ICD) of the Notch ligand have been debated. Soluble Delta1 (sD1) has been shown to activate
Notch2 (N2), but much more weakly than full-length Delta1 (fD1). Furthermore, tracing the N2 molecule after sD1 stimulation reveals that sD1 has a defect in the cleavage releasing ICD of N2 (intracellular cleavage), although it triggers cleavage in the extracellular domain of N2. This represents the molecular basis of the lower activity of sD1 and suggests the presence of an unknown mechanism regulating activation of the intracellular cleavage. The fact that Delta1 lacking its ICD (D1ICD) exhibits the phenotype similar to that exhibited by sD1
indicates that the ICD of D1 (D1DeltaICD) is involved in such an as yet unknown mechanism. Furthermore, the findings that D1DeltaICD acts in a dominant-negative fashion against fD1 and that the signal-transducing activity of sD1 is enhanced by antibody-mediated cross-linking suggest that the multimerization of Delta1 mediated by D1ICD may be required for activation of the N2 intracellular cleavage (Shimizu, 2002).
Following ectodomain shedding, Notch-1 undergoes presenilin (PS)-dependent constitutive intramembranous endoproteolysis at site-3. This cleavage is similar to the PS-dependent γ-secretase cleavage of the ß-amyloid precursor protein (ßAPP). However, topological differences in cleavage resulting in amyloid ß-peptide (Aß) or the Notch-1 intracellular domain (NICD) indicate independent mechanisms of proteolytic cleavage. The secretion of an N-terminal Notch-1 Aß-like fragment (Nß) is reported in this study. Analysis of Nß by MALDI-TOF MS revealed that Nß is cleaved at a novel site (site-4, S4) near the middle of the transmembrane domain. Like the corresponding cleavage of ßAPP at position 40 and 42 of the Aß domain, S4 cleavage is PS dependent. The precision of this cleavage is affected by familial Alzheimer’s disease-associated PS1 mutations similar to the pathological endoproteolysis of ßAPP. Considering these similarities between intramembranous processing of Notch and ßAPP, it is concluded that these proteins are cleaved by a common mechanism utilizing the same protease, i.e. PS/γ-secretase (Okochi, 2002).
Notch receptors undergo a cascade of endoproteolytic cleavages required for Notch signaling. Upon binding of membrane-anchored ligands from the DSL (Delta/Serrate/Lag-2) family, Notch receptors undergo consecutive cleavages at site-2 (S2) and site-3 (S3). Cleavage of mouse Notch-1 at S2 occurs in its ectodomain by TACE [tumor necrosis factor-α (TNF-α-converting enzyme], a member of the ADAM (a disintegrin and metalloprotease domain) family ~12 amino acids distant from the TM. This 'ectodomain shedding' event results in the generation of NEXT (Notch extracellular truncation), that is cleaved subsequently at S3 within the TM close to the cytoplasmic border. Cleavage of Notch at S3 liberates NICD (Notch intracellular domain) that translocates to the nucleus, where it is involved in target gene transcription. S3 cleavage strictly depends on the biological activity of the presenilin (PS) proteins, which may contribute the catalytic site of γ-secretase, an unusual intramembrane-cleaving aspartyl protease complex (Okochi, 2002 and references therein).
Beside the Notch-1-4 receptors, several other type I TM proteins have been identified as substrates for PS-dependent endoproteolysis, including the Alzheimer's disease (AD)-associated ß-amyloid protein precursor (ßAPP), ErbB-4, E-cadherin and LRP. These proteins undergo ‘ectodomain shedding’ in their large extracellular domains, prior to the consecutive PS-dependent cleavage within the TM. In the case of ßAPP, these cleavages are mediated by α-secretase and ß-secretase. Cleavage of ßAPP by α- and ß-secretase (BACE) results in the generation of the respective ßAPP C-terminal fragments (CTFs), CTFα and CTFß, which are the direct substrates for γ-secretase cleavage. Cleavage of CTFß and CTFα by γ-secretase occurs in the middle of the TM and leads to the liberation of Aß and p3 peptides), respectively. Aß is deposited in the brain of AD patients in 'senile plaques', an invariant pathological hallmark of AD. Recently, the elusive C-terminal cleavage product of γ-secretase, AICD (ßAPP intracellular domain), has been identified and characterized. Surprisingly, AICD results from PS-dependent γ-secretase cleavage of ßAPP-CTFs predominantly after Leu49 (Aß numbering). This cleavage is almost identical to the S3 cleavage of Notch-1 and does not occur after Val40 and Ala42 (Aß numbering) as predicted. Thus, γ-secretase cleaves the ßAPP TM at several sites: one in the middle after position 40 (γ40) and 42 (γ42) (with major γ40 and minor γ42 cleavage) and one close to the cytoplasmic border after position 49 (γ49) of the Aß domain. Interestingly, AICD may translocate to the nucleus where it could have a role in transcriptional regulation similar to NICD (Okochi, 2002 and references therein).
Because of these striking similarities between Notch and ßAPP endoproteolysis, it was hypothesized that an Aß/p3-like species (called Notch ß-peptide, Nß) derived from NEXT intramembranous proteolysis may be secreted into the extracellular space. This study reports the identification and characterization of secreted Nß peptides derived from endoproteolysis of NEXT derivatives. Sequence analysis revealed that Nß is derived from endoproteolytic cleavage near the middle of the Notch-1 TM at site-4 (S4), which is 12 amino acid residues upstream of S3. Like S3 cleavage, S4 cleavage occurs in a PS- and γ-secretase-dependent manner. Strikingly, familial AD (FAD)-associated PS mutants known to cause the increased production of C-terminally elongated pathogenic Aß42 also affect the generation of C-terminally elongated Nß variants, supporting a direct role for PS in the proteolytic cleavage of Notch-1 and ßAPP (Okochi, 2002).
The role of Notch signaling in general and presenilin in particular
during mouse somitogenesis was analyzed. Cyclical production of
activated Notch (NICD) was visualized and it was established that somitogenesis requires less NICD than
any other tissue in early mouse embryos. Indeed, formation of cervical somites
proceeds in Notch1; Notch2-deficient embryos. This is in contrast to mice
lacking all presenilin alleles, that have no somites. Since
Nicastrin-, Pen-2-, and APH-1a-deficient embryos have anterior somites
without γ-secretase, presenilin may have a
γ-secretase-independent role in somitogenesis. Embryos triple homozygous
for both presenilin null alleles and a Notch allele that is a poor
substrate for presenilin (N1V→G) experience fortuitous
cleavage of N1V→G by another protease. This restores
NICD, anterior segmentation, and bilateral symmetry but does not rescue
rostral/caudal identities. These data clarify multiple roles for Notch signaling
during segmentation and suggest that the earliest stages of somitogenesis are
regulated by both Notch-dependent and Notch-independent functions of presenilin (Hupper, 2005).
Several type I integral membrane proteins, such as the Notch receptor or the amyloid precursor protein, are cleaved in their intramembrane domain by a gamma-secretase enzyme, which is carried within a multiprotein complex. These cleavages generate molecules that are involved in intracellular or extracellular signaling. At least four transmembrane proteins belong to the gamma-secretase complex: presenilin, nicastrin, Aph-1, and Pen-2. It is still unclear whether these proteins are the only components of the complex and whether a unique complex is involved in the different gamma-secretase cleavage events. A genetic screen was set up based on the permanent acquisition or loss of an antibiotic resistance depending on the presence of an active gamma-secretase able to cleave a Notch-derived substrate. Clones were selected deficient in gamma-secretase activity using this screen on mammalian cells after random mutagenesis. Two of these clones were examined and previously undescribed mutations were identified in the nicastrin gene. The first mutation abolishes nicastrin production, and the second mutation, a point mutation in the ectodomain, abolishes nicastrin maturation. In both cases, gamma-secretase activity on Notch and APP is impaired (Olry, 2005).
The four highly conserved Notch receptors receive short-range signals that
control many biological processes during development and in adult vertebrate
tissues. The involvement of Notch1 signaling in tissue self-renewal is less
clear, however. This study developed a novel genetic approach N1IP-CRE
(Notch1 Intramembrane Proteolysis) to follow, at high resolution, the
descendents of cells experiencing Notch1 activation in the mouse.
To generate a genetic sensor of Notch1 proteolysis the mouse Notch1 intracellular domain (NICD1), immediately downstream of the transmembrane domain, was replaced with the site-specific recombinase Cre, such that the Cre activity is now governed by ligand-induced proteolysis of the Notch1 transmembrane domain tether. In Cre-reporter strains, Notch activation is visualized by β-galactosidase expression. Because Notch1 proteolysis releases Cre that leads to a cell-heritable expression of lacZ, Notch1 signaling in actively cycling stem/progenitor cells will mark all their descendents producing a 'clone', whereas Notch1 activation in transit amplifying or differentiating cells will result in small clones (2-4 cells) or in salt-and-pepper patterns of individually labeled cells. By combining
N1IP-CRE with loss-of-function analysis, Notch activation patterns
were correlated with function during development, self-renewal and malignancy
in selected tissues. Identification of many known functions of Notch1
throughout development validated the utility of this approach. Importantly,
novel roles for Notch1 signaling were identified in heart, vasculature, retina
and in the stem cell compartments of self-renewing epithelia. The
probability of Notch1 activation in different tissues does not always indicate
a requirement for this receptor, and gradients of Notch1 activation are
evident within one organ. These findings highlight an underappreciated layer
of complexity of Notch signaling in vivo. Moreover, NIP-CRE represents a
general strategy applicable for monitoring proteolysis-dependent signaling in
vivo (Vooijs, 2007).
The cell fate determinant Numb influences developmental decisions by antagonizing the Notch signaling pathway. However, the underlying molecular mechanism of this inhibition is poorly understood. The mammalian Numb protein promotes the ubiquitination of membrane-bound Notch1 receptor. Furthermore, Numb expression results in the degradation of the Notch intracellular domain following activation -- this correlates with a loss of Notch-dependent transcriptional activation of the Hes1 promoter as measured by a Hes1 luciferase reporter assay. The phosphotyrosine-binding (PTB) domain of Numb is required for both Notch1 ubiquitination and down-regulation of Notch1 nuclear activity. Numb-mediated ubiquitination of Notch1 is not dependent on the PEST region, which was previously shown to mediate Sel10-dependent ubiquitination of Notch in the nucleus, suggesting a distinct E3 ubiquitin ligase is involved. In agreement, Numb is shown to interact with the cytosolic HECT domain-containing E3 ligase Itch; Numb and Itch act cooperatively to promote ubiquitination of membrane-tethered Notch1. These results suggest that Numb recruits components of the ubiquitination machinery to the Notch receptor thereby facilitating Notch1 ubiquitination at the membrane, which in turn promotes degradation of the intracellular domain circumventing its nuclear translocation and downstream activation of Notch1 target genes (McGill, 2003).
The beta-amyloid precursor protein (APP) and the Notch receptor undergo intramembranous proteolysis by the Presenilin-dependent gamma-secretase. The cleavage of APP by gamma-secretase releases amyloid-beta peptides, which have been implicated in the pathogenesis of Alzheimer's disease, and the APP intracellular domain (AID), for which the function is not yet well understood. A similar gamma-secretase-mediated cleavage of the Notch receptor liberates the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates the transcription of genes that regulate the generation, differentiation, and survival of neuronal cells. Hence, some of the effects of APP signaling and Alzheimer's disease pathology may be mediated by the interaction of APP and Notch. Membrane-tethered APP binds to the cytosolic Notch inhibitors Numb and Numb-like in mouse brain lysates. AID also binds Numb and Numb-like, and represses Notch activity when released by APP. Thus, gamma-secretase may have opposing effects on Notch signaling; positive by cleaving Notch and generating NICD, and negative by processing APP and generating AID, which inhibits the function of NICD (Roncarati, 2002).
Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells (Itoh, 2003).
There are two models that could explain why an E3 that is responsible for ubiquitylation and internalization of Delta would be required for effective Notch signaling. One possibility is based on the proposition that Mib is required in the cell that delivers signals; the other assumes that it is required in the cell that receives them. In the first model, Mib promotes the transendocytosis of the Notch extracellular domain by promoting endocytosis of Delta and, in doing so, facilitates proteolytic events that generate the transcriptionally active NotchICD fragment. This proposal comes from studies of the neurogenic phenotype of shibire and neur mutants in Drosophila, suggesting that transendocytosis of the Notch extracellular domain by the adjacent Delta-expressing cell is essential for efficient Notch activation. In the zebrafish system, transplantation experiments show that cells with reduced mib function are less likely to become neurons when surrounded by wild-type cells. This supports the idea that loss of mib function primarily reduces a cell's ability to produce an effective inhibitory signal in the competition to become a neuron (Itoh, 2003).
The other model that explains why ubiquitylation and internalization of Delta might be essential for Notch signaling postulates a cell-autonomous role for mib in signal reception, as has also been suggested previously for neur. According to this model, mib-mediated Delta turnover would limit Delta's ability to inhibit Notch function cell autonomously. However, cell transplantation results argue against a significant deficit in reception of the inhibitory signal. Furthermore, the luciferase experiments, in which cells were cotransfected with notch and various delta constructs, show that, while Delta does indeed have a cell-autonomous effect in blocking signal reception, mib does not significantly influence this action of Delta. Moreover, this action of Delta does not seem to be ubiquitin dependent: the recombinant addition of ubiquitin does not significantly reduce DeltaDeltaICD's ability to inhibit Notch function. It is possible that in these assays, Mib is ineffective at reducing cell-autonomous inhibition of Notch by Delta because very high levels of artificially expressed Delta in the transfected cells in vitro may overwhelm the capacity of the Mib-dependent machinery. However, in studies in COS7 cells, at least, Mib is effective in removing artificially expressed Delta from the cell surface, suggesting that the inhibitory effect of Delta may be independent of delivery of Delta to the cell surface: it may result from Delta-Notch interactions within the secretory pathway. In short, observations do not support a significant role for Mib in limiting Delta's ability to cell autonomously inhibit Notch function as has been described for Neur, but such a role cannot be completely ruled out (Itoh, 2003).
The role of Mib in signal delivery is strongly supported and tightly correlated with Delta ubiquitylation. Ectopic expression of XDelta1DeltaICD, which cannot be ubiquitylated, permits too many cells to become neurons, while XDelta1 and XDelta1DeltaICD-Ub, ectopically expressed in the embryo in a similar way, both inhibit cells from becoming neurons. These effects correlate with the ubiquitin-dependent reduction of cell surface Delta. The internalization of XDelta1DeltaICD-Ub is consonant with previous studies that have shown that in-frame addition of ubiquitin to stable plasma membrane proteins can serve to target their entry into the endocytic pathway. The obvious suggestion, therefore, is that Mib-induced ubiquitylation drives internalization of Delta by endocytosis, and that this process is critical for effective signaling by Delta (Itoh, 2003).
An additional possibility that cannot as yet be excluded is that Mib-dependent ubiquitylation of Delta also decreases the amount of Delta that reaches the cell surface by sorting Delta directly from the Golgi complex to late endosomes. Such a dual role has been shown in yeast for the E3 ligase Rsp5p, which ubiquitylates its substrate, Gap1p, to regulate the total amount of Gap1p at the cell surface. Ubiquitylation of Gap1p by Rsp5p promotes endocytosis of Gap1p and favors sorting of Gap1p from the Golgi to the vacuole, where it is degraded. Mib may also have dual roles in endocytosis of Delta and in direct sorting of Delta to late endosomes/lysosomes; however, it is not clear at this time how the later function might contribute to Notch signaling (Itoh, 2003).
Although mib mutants express unusually high levels of cell surface Delta, it is unlikely that this is per se the cause of the neurogenic phenotype, since artificial expression of even higher levels of Delta in mib mutants following injection of delta mRNA suppress the neurogenic phenotype (Itoh, 2003).
From these observations, it seems that while all the forms of Delta that were examined can cell autonomously inhibit Notch function, only the forms of Delta that are ubiquitylated and endocytosed can effectively activate Notch in neighboring cells. It is likely that when Delta is driven to high levels in a group of cells, the effect of Delta in trans, as an activator of the Notch pathway, dominates over its effect in cis, as an inhibitor, accounting for the ability of Xdelta1 and Xdelta1DeltaICD-Ub to inhibit neurogenesis in the embryo (Itoh, 2003).
The opposing cell-autonomous and nonautonomous effects on Notch signaling define two synergistic mechanisms by which a cell expressing more Delta than its neighbors gains an enhanced ability to become a neuron. By activating Notch in neighboring cells, Delta reduces the neighbors' ability to express the Notch ligand Delta at high levels. At the same time, Delta interferes with Notch function in the cell where Notch and Delta are coexpressed, making it harder for this cell to be inhibited from becoming a neuron by Delta in neighboring cells (Itoh, 2003).
The role for mib in promoting endocytosis in the signal-delivering cell, as demonstrated in this study, is similar to one role proposed for neur in Drosophila. In vertebrates, however, neur seems to have a much more limited role than has been demonstrated for it in Drosophila. Mice that are homozygous for a neur loss-of-function mutation have restricted defects: one study demonstrated defects in spermatogenesis and in mammary gland development, while another study has shown ethanol hypersensitivity and an olfactory discrimination defect. In Xenopus, interfering with neur function by overexpressing either wild-type Neur or a mutant form that lacks the RING finger domain increases the density of ciliated cells in the epidermis . But none of these studies revealed the dramatic neural phenotypes or defects in somitogenesis that are seen when there is broad loss of Notch signaling. In contrast, mib mutants do show widespread abnormalities, suggesting a deficit in many more Notch-dependent developmental events. Currently being investigated are whether mib has assumed some roles that were originally played by neur in Drosophila or whether a cooperative role for neur and mib in Notch signaling limits the deficit caused by loss of neur alone in vertebrates (Itoh, 2003).
In summary, the analysis of the zebrafish mib mutant has led to the identification of a gene that is essential for effective Notch signaling in many different tissues during development. The function of Mib as a ubiquitin ligase in the internalization of Delta provides new avenues for clarifying the mystery of how endocytosis may increase the ability of cell surface Delta to deliver lateral inhibition signals (Itoh, 2003).
Notch receptors modulate transcriptional targets following the proteolytic release of the Notch intracellular domain (NotchIC). Phosphorylated forms of NotchIC have been identified within the nucleus and have been associated with CSL members, as well as correlated with regions of the receptor that are required for activity. Genetic studies have suggested that Shaggy, the Drosophila homolog of glycogen synthase kinase-3ß (GSK3ß) may act as a positive modulator of the Notch signaling. GSK3ß is a serine/threonine kinase and is a component of the Wnt/wingless signaling cascade. GSK3ß is able to bind and phosphorylate Notch1IC in vitro, and attenuation of GSK3ß activity reduces phosphorylation of NotchIC in vivo. Functionally, ligand-activated signaling through the endogenous Notch1 receptor is reduced in GSK3ß null fibroblasts, implying a positive role for GSK3ß in mammalian Notch signaling. As a possible mechanistic explanation of the effect of GSK3ß on Notch signaling, it was observed that inhibition of GSK3ß shortens the half-life of Notch1IC. Conversely, activated GSK3ß reduces the quantity of Notch1IC that was degraded by the proteasome. These studies reveal that GSK3ß modulates Notch1 signaling, possibly through direct phosphorylation of the intracellular domain of Notch, and that the activity of GSK3ß protects the intracellular domain from proteasome degradation (Foltz, 2002).
Axon-derived molecules are temporally and spatially required as positive or negative signals to coordinate oligodendrocyte differentiation. Increasing evidence suggests that, in addition to the inhibitory Jagged1/Notch1 signaling cascade, other pathways act via Notch to mediate oligodendrocyte differentiation. The GPI-linked neural cell recognition molecule F3/contactin (See Drosophila Contactin)
is clustered during development at the paranodal region, a vital site for axoglial interaction. F3/contactin acts as a functional ligand of Notch. This trans-extracellular interaction triggers gamma-secretase-dependent nuclear translocation of the Notch intracellular domain. F3/Notch signaling promotes oligodendrocyte precursor cell differentiation and upregulates the myelin-related protein MAG in OLN-93 cells. This can be blocked by dominant negative Notch1, Notch2, and two Deltex1 mutants lacking the RING-H2 finger motif, but not by dominant-negative RBP-J or Hes1 antisense oligonucleotides. Expression of constitutively active Notch1 or Notch2 does not upregulate MAG. Thus, F3/contactin specifically initiates a Notch/Deltex1 signaling pathway that promotes oligodendrocyte maturation and myelination (Hu 2003).
Neurons and glia in the vertebrate central nervous system arise in temporally distinct, albeit overlapping, phases. Neurons are generated first followed by astrocytes and oligodendrocytes from common progenitor cells. Increasing evidence indicates that axon-derived signals spatiotemporally modulate oligodendrocyte maturation and myelin formation. F3/contactin is a functional ligand of Notch during oligodendrocyte maturation, revealing the existence of another group of Notch ligands. NB-3, a member of the F3/contactin family, acts as a novel Notch ligand to participate in oligodendrocyte generation. NB-3 triggers nuclear translocation of the Notch intracellular domain and promotes oligodendrogliogenesis from progenitor cells and differentiation of oligodendrocyte precursor cells via Deltex1. In primary oligodendrocytes, NB-3 increases myelin-associated glycoprotein transcripts. Thus, the NB-3/Notch signaling pathway may prove to be a molecular handle to treat demyelinating diseases (Cui, 2004).
Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. A modification, monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu. The lysine residue targeted by the monoubiquitination event has been identified, and its importance for activation of Notch receptor by its ligand, Delta-like 1, has been confirmed. A new model is proposed where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and its relevance for other gamma-secretase substrates is discussed (Gupta-Rossi, 2004).
The ubiquitination pathway involves a multiprotein cascade in which the substrate specificity is determined by the E3 component. Multi-ubiquitin chains at least four subunits long are required for efficient recognition and degradation of ubiquitinated proteins by the proteasome, but ubiquitin has more recently been shown to endorse new functions that do not always involve the proteasome (Gupta-Rossi, 2004).
The results show that a monoubiquitination event takes place on the DeltaE molecule, a constitutively active form of the Notch receptor that mimics the intermediate TACE-processing product generated after ligand binding. This modification is a prerequisite for gamma-secretase cleavage and targets one of the subunits of a dimeric membrane-anchored form of Notch DeltaE. The major site of monoubiquitination has been localized to a juxtamembrane, conserved lysine residue K1749 in mNotch1. Access to the monoubiquitinated form DeltaEu was gained by coimmunoprecipitation with endogenous PS1 when gamma-secretase activity was inhibited by a specific drug. Thus, DeltaEu is a labile intermediate appearing before gamma-secretase cleavage. This form could also be detected after coexpression of PS1 or DeltaC4, a PS2-derived construct. These molecules are probably not included in PS-containing high molecular weight complexes; neither are gamma-secretase components when transiently overexpressed. The existence of DeltaEu was varified by extracting and stabilizing it out of the active complexes. It is proposed that this ubiquitination step is required in the context of the full-length receptor activated by ligand binding. Although the modified intermediate species derived from full-length Notch could not be directly accessed, mutating the crucial lysine residue impaired Dll1-mediated Notch signaling, in accordance with the DeltaE results. It remains to be determined which E3 ubiquitin ligase is involved in this modification. Various proteins carrying such an activity have been associated with the Notch cascade and are candidates to be tested, e.g., Deltex, Suppressor of Deltex, and Cbl. Experiments are in progress to answer this question (Gupta-Rossi, 2004).
The results show that endocytosis of Notch DeltaE and of ligand-activated full-length Notch are necessary for gamma-secretase cleavage. The involvement of a clathrin-dependent endocytosis event for Notch activation complies with the mosaic analysis performed in Drosophila, which revealed that shibire function is required in Notch-expressing cells receiving a lateral inhibition signal. It is proposed that monoubiquitination on a juxtamembrane lysine (K1749) and endocytosis occur after ligand-induced cleavage of the Notch extracellular domain by TACE. The data are in apparent contradiction with a previous model, according to which gamma-secretase cleavage occurs at the plasma membrane. However, the previous data can be reinterpreted in light of the new model. The previous study argued that the TACE-processing product of Notch (similar to the DeltaE construct used in this study) remains associated with the apical membrane in Nicastrin or PS mutant cells, and in WT cells only a small amount of this molecule can be found in endocytic vesicles. This result can be explained by the fact that ubiquitination is one of the limiting steps in Notch signaling, or that active PS is needed to direct the final steps of endocytosis of the ubiquitinated forms. Probably for the same reason, DeltaE is very poorly cleaved by gamma-secretase when overexpressed, and the ubiquitination event can hardly be detected. The current results are also in apparent contradiction with those of another study, which postulates that in Drosophila, PS-mediated proteolysis does not appear to require a particular sequence nor the presence of active dynamin. However, the assay used appears unusually sensitive, as it even detects the cleavage of a Notch molecule carrying a G1743V mutation of the gamma-secretase cleavage site, a mutation that prevents activation in most other assays and, when introduced into mice, gives rise to an almost perfect Notch1 null phenotype. Therefore, a leakage due to overexpression might in some cases be responsible for the activity detected. Experiments are in progress to test the effect of mutating the juxtamembrane lysine of Drosophila Notch (Gupta-Rossi, 2004).
Various papers have described monoubiquitination as a signal for internalization of receptors such as EGFR or glycine receptor. The results do not allow one to discriminate between ubiquitination triggering endocytosis or being concomitant with the first steps of endocytosis. However, the observations show a more internal localization of LLFF (the site of gamma-secretase cleavage) compared with the K1749R mutant, and endocytosis of the K1749R DeltaE mutant seems to be blocked at an earlier stage when compared with the WT or LLFF mutant. These data suggest that ubiquitination is necessary for late events driving Notch to compartments where gamma-secretase cleavage can occur (Gupta-Rossi, 2004).
A novel mode of crosstalk between the EGFR-Ras-MAPK and LIN-12/Notch pathways occurs during the patterning of a row of vulval precursor cells (VPCs) in Caenorhabditis elegans: activation of the EGFR-Ras-MAPK pathway in the central VPC promotes endocytosis and degradation of LIN-12 protein. LIN-12 downregulation in the central VPC is a prerequisite for the activity of the lateral signal, which activates LIN-12 in neighboring VPCs. This study characterizes cis-acting targeting sequences in the LIN-12 intracellular domain; in addition to a di-leucine motif, serine/threonine residues are important for internalization and lysine residues are important for post-internalization trafficking and degradation. Two trans-acting factors are identified that are required for post-internalization trafficking and degradation: ALX-1, a homolog of yeast Bro1p and mammalian Alix and the WWP-1/Su(dx)/Itch ubiquitin ligase. By examining the effects of mutated forms of LIN-12 and reduced wwp-1 or alx-1 activity on subcellular localization and activity of LIN-12, evidence is provided that the lateral signal-inhibiting activity of LIN-12 resides in the extracellular domain and occurs at the apical surface of the VPCs (Shaye, 2005).
LIN-12 appears to be downregulated via multivesicular endosomes (MVEs). Although mutation of the conserved lysines near the extracellular 'downregulation targeting sequence' DTS does not affect internalization of LIN-12, degradation in P6.p is blocked, and in all VPCs this mutant form accumulates in large pleiomorphic internal vesicles. Furthermore, the ubiquitin ligase WWP-1 and the MVE-associated factor ALX-1 are required for LIN-12 degradation after internalization. Since sur-2 mutants (referring to mutations in the the MED23 subunit of the 'Mediator' transcription activation complex) display a similar phenotype, transcriptional targets of the EGFR-Ras-MAPK pathway may be involved in directing LIN-12 to MVEs (Shaye, 2005).
Mutating the conserved lysines near the DTS caused the 'Multivulva' phenotype associated with constitutive LIN-12 activation. This phenotype is consistent with an MVE sorting defect. If a transmembrane protein does not go through the MVE sorting step, then upon delivery to the lysosome its extracellular domain will be degraded whereas its intracellular domain will remain exposed to the cytosol. For LIN-12/Notch, the mechanism of signal transduction involves cleavage and release of the intracellular domain. Thus, if MVE sorting is disrupted, degradation of the extracellular domain in the lysosome could mimic ectodomain shedding, creating a substrate for Presenilin-dependent release of the intracellular domain of LIN-12, or perhaps such disruption would result in release the intracellular domain by an alternative mechanism (Shaye, 2005).
Recent reports have described 'ligand-independent' activation of Drosophila Notch in late endosomes. In these studies, overexpression of the protein Deltex was shown to promote internalization and accumulation of Notch in late endosomes, correlated with activation of Notch signaling. It was suggested that such endosomal activation of Notch might represent a novel and relevant mode of activating this pathway. However, the finding that an apparent block in MVE sorting can lead to LIN-12 activation suggests an alternative explanation for the effect of Deltex overexpression: the enhanced internalization and endosomal accumulation of Notch may saturate the MVE sorting machinery, so that some Notch is not correctly internalized into MVE lumenal vesicles, leading to degradation of the extracellular domain without concomitant degradation of the intracellular domain (Shaye, 2005).
Evidence is provided that internalization of LIN-12 is mediated by the di-leucine motif and basal phosphorylation of flanking serine/threonine residues. By contrast, for Drosophila Notch, recent evidence suggests that ubiquitination by the dNedd4 ubiquitin ligase is required for Notch internalization. Drosophila Notch does not have a di-leucine-based motif similar to the one described for LIN-12. Conversely, LIN-12 does not have a C-terminal PPXY signal, which in Drosophila Notch promotes interaction with dNedd4. It is suggested that C. elegans and Drosophila may utilize different mechanisms for targeting LIN-12/Notch for internalization (Shaye, 2005).
Both of these mechanisms may be utilized in vertebrate Notch proteins. Sequence analysis of vertebrate Notch proteins shows an intriguing inverse correlation between the presence of a di-leucine based motif and a PPXY signal. The corresponding juxtamembrane regions of vertebrate Notch1 and Notch2 proteins have a segment that is strikingly similar to the LIN-12 DTS, including conserved flanking lysines, but these proteins do not have a conserved PPXY signal at their C-termini. By contrast, most vertebrate Notch3 proteins appear more divergent in this region, but possess a PPXY signal at their C-termini; it is curious that zebrafish Notch3 lacks the PPXY motif, but has instead a canonical di-leucine motif. These observations raise the possibility that the two modes of internalizing Notch proteins (di-leucine based versus ubiquitination via a PPXY motif) have been conserved in different vertebrate Notch proteins through evolution. Perhaps other modes exist as well, as vertebrate Notch4 does not seem to have either of these conserved motifs. Mutational analysis of these potential internalization sequences in vertebrate Notch proteins will be necessary to test their roles (Shaye, 2005).
Notch receptors control differentiation and contribute to pathologic states such as cancer by interacting directly with a transcription factor called CSL (for CBF-1/Suppressor of Hairless/Lag-1) to induce expression of target genes. A number of Notch-regulated targets, including genes of the hairy/enhancer-of-split family in organisms ranging from Drosophila to humans, are characterized by paired CSL-binding sites in a characteristic head-to-head arrangement. Using a combination of structural and molecular approaches, it has been establish that cooperative formation of dimeric Notch transcription complexes on promoters with paired sites is required to activate transcription. These findings identify a mechanistic step that can account for the exquisite sensitivity of Notch target genes to variation in signal strength and developmental context, enable new strategies for sensitive and reliable identification of Notch target genes, and lay the groundwork for the development of Notch pathway inhibitors that are active on target genes containing paired sites (Nam, 2007).
Cocrystals of a human Notch transcriptional activation complex (NTC) core, which consists of an N-terminal MAML-1 peptide, the ANK domain of human Notch1, and CSL on a DNA duplex derived from the HES-1 promoter, contain contacts between the convex surfaces of ANK domains from adjacent unit cells that also are seen in crystals of the ANK domain solved in isolation in several different crystallization conditions (Nam, 2006). These contacts lie near a twofold symmetry axis in the crystals, such that the interacting complexes are positioned head-to-head at a distance roughly equal to that needed to occupy both recognition elements of an SPS. Primary sequence alignment of Notch ANK domains from different homologs shows that the key contacts are evolutionarily conserved. These conserved residues are not engaged in contacts within an individual MAML1/ANK/CSL/DNA complex, suggesting that the observed conservation reflects functional importance in mediating dimerization at SPS sites. The conservation among the four mammalian Notch receptors also predicts that each receptor should be capable of making interactions like those between the adjacent Notch1 complexes (Nam, 2007).
The ANK-ANK contacts primarily are electrostatic and lie in the second and third ankyrin repeats. Key interactions consist of contacts between the guanidino group of Arg-1985 and at least three backbone carbonyl oxygen atoms, as well as interactions between Glu-1950 and Lys-1946. Arg-1983 also forms hydrogen bonds with Ser-1952 and a backbone carbonyl. In addition to homotypic interactions between the ANK domains, unmodeled electron density in the MAML-1/ANK/CSL/DNA complex also suggests the existence of interactions between the ANK domain of one complex and the N-terminal end of MAML-1 in the second complex. Based on the architecture of the complex, and the evolutionary conservation of SPSs and the crystal contact residues, it is postulated that the ANK domains of Notch receptors mediate dimerization of ternary complexes on SPSs found in Notch target gene promoters (Nam, 2007).
To test whether residues engaged in ANK-ANK contacts in the crystal contribute to transcriptional activation of SPS-bearing promoters, the ability of different forms of ICN to induce transcription of a luciferase gene under control of the HES-1 promoter, which has a functionally important SPS element, was tested. In contrast to normal ICN1, mutations that disrupt the predicted dimerization interface either abrogate (R1985A) or diminish (K1946E and E1950K) the ability of ICN1 to induce expression of the HES-1 reporter gene. Combining the K1946E and E1950K mutations in cis, however, rescues the defect in transcriptional activation, indicating that the putative dimerization interface is functionally important in regulating transcriptional activity at a promoter that contains an SPS. In addition, when coexpressed with ICN1, the R1985A mutation dominantly interferes with activation of the HES-1 promoter element by normal ICN1. Importantly, when these mutants are scored on an artificial reporter that contains four CSL-binding sites oriented in the same direction and in tandem, there is no change in the ability of the mutants to activate transcription. Moreover, in cotransfected cells, all ICN1 polypeptides with mutations that disrupt the predicted dimerization interface are expressed at similar levels to normal ICN1, and they coimmunoprecipitate in similar amounts with CSL and MAML-1. Together, these findings indicate that the ability to form monomeric ternary complexes with MAML-1 and CSL is not affected by these mutations (Nam, 2007).
To establish directly whether NTCs (consisting of one molecule each of MAML-1, ICN, and CSL) can cooperatively dimerize on DNA, electrophoretic mobility shift assays (EMSAs) were carried on an oligonucleotide probe containing the HES-1 promoter SPS. Without Notch or MAML-1, CSL binds to each of the two sites independently. When present in excess, most probes bind a single CSL molecule, a finding consistent with previous studies showing that CSL binds its recognition element as a monomer without detectable cooperativity at paired sites. Adding RAMANK from Notch1 does not change the stoichiometric distribution of complexes bound per probe molecule. However, when MAML-1 is added, the stoichiometric distribution of the complexes changes dramatically: all of the probe is either free or bound by NTC dimers, indicating that NTC loading at one site leads to cooperative loading of the second site. As predicted, cooperative loading is abrogated by the R1985A mutation, which instead produces a smear corresponding to an ensemble of species that likely results from a weak residual tendency to self-associate. In contrast, the R1985A mutation does not detectably affect ternary complex formation on a probe containing only a single CSL-binding site, indicating that the R1985A mutation is a cooperativity mutant that specifically interferes with dimerization. The partial loss of activity of the K1946E and E1950K mutants in the HES-1 reporter assays is echoed in EMSA titrations, where the proteins undergo a cooperative transition to form dimers at a concentration ~4-fold greater than normal ICN1 or the K1946E/E1950K double mutant (Nam, 2007).
To test whether higher-order complexes exhibit specificity for the SPS architecture, additional EMSA assays were carried out on variant DNA sequences that eliminate the integrity of one of the SPS sites, flip the site orientation, or alter the spacing between the sites by a half-turn of helix. When either site A or site B is mutated so that it no longer corresponds to a CSL consensus site (YGTGDGAA), cooperative assembly of the dimer is no longer observed. Moreover, cooperative dimerization is no longer detected when the second site is inverted, and it is dramatically diminished when the second site is moved by a 5-base insertion. Because the intrinsic affinity of a single ternary complex for DNA is not altered under the conditions of inversion or insertion, these studies show that the proper spatial arrangement of the two individual binding sites is needed for cooperative dimerization to occur (Nam, 2007).
It was next asked what range of spacer lengths between sites is compatible with cooperative loading of dimeric complexes. The optimal spacing between consensus sites for cooperative dimerization is 16 bp, but cooperative dimerization still can occur on templates with spacers varying from 15 to 19 bp, implying that two NTCs can adjust their positions relative to each other to accommodate a modest range of spacer lengths between sites. This inferred flexibility is consistent with the different conformations of CSL seen in the crystal structures of the Notch ternary complexes formed with the human and worm proteins and with the enrichment of adenosine and thymidine in the spacer between the paired sites (Nam, 2007).
To determine whether the assembly of NTCs and their cooperative dimers is general among the human Notch homologues, the ability of the RAMANK domains of Notch1-4 to form complexes on single and sequence-paired sites was tested. Despite qualitative differences in mobility on the EMSA, all four purified RAMANK polypeptides bind to CSL independent of MAML-1 and then recruit MAML-1 to ternary complexes on a single site probe. When the longer, paired site probe is provided, all RAMANK polypeptides mediate cooperative dimerization, as predicted from the conservation in primary sequence at the dimerization interface. Thus, a similar series of events takes place to assemble single and dimeric NTCs in all four mammalian Notch homologues (Nam, 2007).
Notch receptors transduce essential developmental signals between neighboring cells by forming a complex that leads to transcription of target genes upon activation. This study reports the crystal structure of a Notch transcriptional activation complex containing the ankyrin domain of human Notch1 (ANK), the transcription factor CSL on cognate DNA, and a polypeptide from the coactivator Mastermind-like-1 (MAML-1). Together, CSL and ANK create a groove to bind the MAML-1 polypeptide as a kinked, 70 Å helix. The composite binding surface likely restricts the recruitment of MAML proteins to promoters on which Notch:CSL complexes have been preassembled, ensuring tight transcriptional control of Notch target genes (Nam, 2006).
Notch signaling mediates communication between cells and is essential for proper embryonic patterning and development. CSL is a DNA binding transcription factor that regulates transcription of Notch target genes by interacting with coregulators. Transcriptional activation requires the displacement of corepressors from CSL by the intracellular portion of the receptor Notch (NotchIC) and the recruitment of the coactivator protein Mastermind to the complex. This study reports the 3.1 Å structure of the ternary complex formed by CSL, NotchIC, and Mastermind bound to DNA. As expected, the RAM domain of Notch interacts with the beta trefoil domain of CSL; however, the C-terminal domain of CSL has an unanticipated central role in the interface formed with the Notch ankyrin repeats and Mastermind. Ternary complex formation induces a substantial conformational change within CSL, suggesting a molecular mechanism for the conversion of CSL from a repressor to an activator (Wilson, 2006)
Spatiotemporal modulation of the evolutionarily conserved, intercellular Notch signaling pathway is important in the development of many animals. Examples include the regulation of neural-epidermal fate decisions in neurogenic ectoderm of Drosophila and somitogenesis in vertebrate presomitic mesoderm. In both these and most other cases, it appears that Notch-class transmembrane receptors are ubiquitously expressed. Modulation of the pathway is achieved primarily by the localized expression of the activating ligand or by alteration of receptor specificity through a glycosyl transferase. In contrast, this report presents an instance where the abundance of the Notch-class mRNA itself is dynamically regulated. Taking advantage of the long cell cycle of the two-cell-stage embryo of the leech Helobdella robusta, it was shown that this regulation is achieved at the levels of both transcript stability and transcription. Moreover, MAPK signaling plays a significant role in regulating accumulation of the transcript by virtue of its effect on Hro-notch mRNA stability. Intracellular injection of heterologous reporter mRNAs shows that the Hro-notch 3' UTR, containing seven AU-rich elements (AREs), is key to regulating transcript stability. Thus, this study shows that regulation of the Notch pathway can occur at a previously underappreciated level, namely that of transcript stability. Given that AU-rich elements occur in the 3' UTR of Notch-class genes in Drosophila, human, and Caenorhabditis elegans, regulation of Notch signaling by modulation of mRNA levels may be operating in other animals as well (Gonsalves, 2007).
In conclusion, this study shows that transcript levels of a Notch-class gene (Hro-notch) oscillate antiphasically in the AB and CD blastomeres of the two-cell embryo in the leech H. robusta, i.e., high transcript levels in AB are associated with low levels in CD and vice versa. Moreover, the Hro-notch levels are controlled by dynamic activation of one or more of the MAPK (p38MAPK and ERK) signaling pathways. Initially, the Hro-notch level in each cell reflects primarily inherited maternal transcripts. Later, the production and turnover of zygotic transcripts becomes important. The 3' UTR of Hro-notch mRNA confers a relatively short half-life to the transcripts, apparently because of the presence of multiple AREs. This instability is counteracted by the p38MAPK pathway. Thus, the Hro-notch transcript levels are controlled by MAPK signaling at the level of transcript stability and possibly also at the level of transcription. This link between the p38MAPK and Notch pathways persists into later development, because coincident p38MAPK a