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CHA P T E R E IG HT
Notch Targets and Their Regulation
Sarah Bray and Fred Bernard
Contents
1. Introduction 253
2. Number and Diversity of Notch Targets 254
3. How Does the Notch Switch Work? 258
4. Different Enhancer Logics 262
5. Context Dependence of Notch Responses 263
6. Concluding Comments 266
References 266
Abstract
The proteolytic cleavages elicited by activation of the Notch receptor release an
intracellular fragment, Notch intracellular domain, which enters the nucleus to
activate the transcription of targets. Changes in transcription are therefore a
major output of this pathway. However, the Notch outputs clearly differ from
cell type to cell type. In this review we discuss current understanding of Notch
targets, the mechanisms involved in their transcriptional regulation, and what
might underlie the activation of different sets of targets in different cell types.
1. Introduction
Notch signaling has widespread roles in development and adult home-
ostasis, as well as a pathogenic role, when misregulated in human disease.
The transcription factor CSL (CBF1-Suppressor of Hairless) plays a central
role in transducing Notch signals into transcriptional outputs. Following
activation, the formation of a ternary complex containing CSL, the Notch
intracellular domain (NICD) and Mastermind (Mam), is essential for upre-
gulating transcription from Notch target genes (Bray, 2006; Kopan and
llagan, 2009). This underscores the importance of transcriptional regulation
Department of Physiology Development and Neuroscience, University of Cambridge, Cambridge, UK
Current Topics in Developmental Biology, Volume 92 � 2010 Elsevier Inc.
ISSN 0070-2153, DOI 10.1016/S0070-2153(10)92008-5 All rights reserved.
253
254 Sarah Bray and Fred Bernard
in the Notch pathway. Here we consider our current understanding about
the transcriptional response to Notch, both the types of genes that are
regulated and the mechanisms underlying this regulation. The focus is on
the direct targets of NICD/CSL, using the criterion that they contain
validated CSL binding sites. Although our examples draw heavily from
studies of Notch function in Drosophila, because of familiarity, our aim is
to illustrate mechanisms that are generally relevant to Notch signaling in all
species. However, for simplicity we refer to all Notch receptors as "Notch"
and we do not discuss the added implications of the different Notch
paralogues that are present in many species including humans (Kopan and
llagan, 2009). We have also not discussed in detail the partners that interact
with CSL, which have been well summarized in a recent review (Borggrefe
and Oswald, 2009).
2. Number and Diversity of Notch Targets
The best-characterized Notch targets are the bHLH genes of the HES/
HEY families, exemplified by the E(spl) genes in Drosophila and HES1 in
mouse. These were the first genes whose transcription was shown to change
following Notch activation and provided a key paradigm for unraveling
Notch pathway activity (Fischer and Gessler, 2007). Induction of E(spl)
genes can be detected within 20-30 min of Notch activation (Krejci and
Bray, 2007). Their expression is usually transient and reflects the dynamic
nature of Notch signaling. In addition there is evidence for autoregulation
such that oscillations of HES expression have been observed and are thought
to contribute to clocks that regulate somitogenesis, limb segmentation, and
neural progenitor maintenance (Brend and Holley, 2009; Kageyama et al.,
2007; Lewis et al., 2009; Pascoal et al., 2007; Shimojo et al., 2008). Altogether
HES/HEY have now been shown to function downstream of Notch in
many critical processes and to contribute to oncogenesis. For example, in
tumor cells HES1 may participate in the regulatory circuitry sustaining cell
growth by repressing expression of PTEN (Palomero et al., 2008).
All HES/HEY proteins appear to function as transcriptional repressors.
For example, they share a C-terminal tetrapeptide motif WRPW/Y, which
is sufficient to recruit transcriptional corepressors of the Groucho family
(Fisher et al., 1996; Paroush et al., 1994), but note interacts less well with
Groucho and may recruit alternative factors (Fischer et al., 2002). Interac-
tions with Sir2 class of proteins (Rosenberg and Parkhurst, 2002; Takata and
Ishikawa, 2003) and with CtBP have also been demonstrated, the latter
requiring a PLSLV/PVNLA motif (Poortinga et al., 1998; Zhang and
Levine, 1999). Indeed, on a genome-wide scale it appears that that
binding of the HES protein Hairy overlaps to a larger extent with CtBP
255 Notch Targets and Their Regulation
and Sir2 than with Groucho (Bianchi-Frias et al., 2004). Similarly, the
closest related Notch targets in nematodes, the Ref-1 family, also appear
to function as repressors by recruiting CtBP (Neves and Priess, 2005).
Although highly diverged from the HES family, Ref-1 and relatives contain
two bHLH domains, which have moderate similarity to the basic regions
of HES proteins, and a terminal FRPWE motif shown to be a weak CtBP
binding domain (Alper and Kenyon, 2001). Thus, the regulation by Notch
of E(spl)/HES/HEY/Ref1 bHLH repressors (which we will refer to colle-
ctively as "HESR") appears to be an ancient phenomenon and these
proteins are essential in many Notch-dependent processes where they
repress key cell fate determinants and cell cycle regulators (Fischer and
Gessler, 2007).
Although HESR genes fulfill multiple pivotal roles in Notch-dependent
processes, it is evident that they are not sufficient to explain all Notch
functions. For example, elimination of E(spl) genes in the Drosophila wing
fails to mimic the classic wing "notching" caused by reductions in Notch
function. Here and elsewhere other targets are essential. Initially a relatively
small number of other direct targets were identified. These included vestigial
(Kim et al., 1996), required for wing development in Drosophila, single-
minded (Morel and Schweisguth, 2000), a midline determinant in Drosophila,
GATA 3, required for physiological Th2 responses to parasite in mammals
(Amsen et al., 2007; Fang et al., 2007), and egl-43, an EVI1 homologue with
crucial roles in the Caenorhabditis.elegans reproductive system (Hwang et al.,
2007). More recently genome-wide studies in human T-ALL cells and in
Drosophila myogenic precursor-related cells have revealed that, even within
these specific cell types, Notch regulates a diverse array of direct targets
(Krejci et al., 2009; Palomero et al., 2006).
Apart from the HESR genes, so far there are relatively few genes that
have been found to be Notch regulated in both vertebrates and inverte-
brates. This may be because studies have not focused on the same processes
but it may also reflect species divergence in the outputs. Nevertheless,
several consistent messages have emerged (Fig. 8.1). First, Notch has been
found to directly regulate genes involved in proliferation and apoptosis.
For example, the myc gene is a direct target of Notch in several types of
cancer cells and in Drosophila cells (Klinakis et al., 2006; Krejci et al., 2009;
Palomero et al., 2006; Weng et al., 2006). Knock down of myc in these
contexts compromised the extent of proliferation, arguing that myc is an
important intermediate in the proliferative response to Notch activation.
Other direct targets involved in promoting proliferation include CyclinD
(Jeffries et al., 2002; Joshi et al., 2009; Ronchini and Capobianco, 2001),
string/CDC25 (Krejci et al., 2009; Palomero et al., 2006), and CDK5
(Palomero et al., 2006). Although Notch activates these proproliferative
genes in several contexts, in others it activates cell cycle inhibitors like p21
(Rangarajan et al., 2001) reflecting the differing consequences on
256 Sarah Bray and Fred Bernard
Proliferation
Mam
Target genes
active
Nicd
CSL
e.g., myc, cyclinD
Apoptosis
e.g., reaper, hid
Cell fates
e.g., HESR, GATA3, Pax2
Signaling pathways
e.g., Lip-1, ErbB2, EGFR,
Notch
“Realizators”
e.g., metabolism genes,
cytoskeletal regulators
Figure 8.1 Diversity in Notch outputs. Simplified diagram of the Notch pathway.
Interaction between the ligand (green) and the Notch (purple) leads to cleavage by
ADAM metaiioproteases (yellow) and gamma secretase complex (brown) to release the
NICD. In the nucleus, NICD binds to CSL (orange) and recruits Mam (green) to activate
target genes. Arrows indicate different types of output that have been observed, with
examples of some of the direct targets identified. CSL consensus binding site is depicted
below, relative sizes are indicative of frequency for a given base occupying that position in
the 56 validated Su(H) sites used to compile the logo. (See Color Insert.)
proliferation (Koch and Radtke, 2007). Notch has also been shown to
directly control apoptosis effector genes. Hence reaper and Wrinkled/hid in
Drosophila have been found as direct targets (Krejci et al., 2009). Similarly bcl-
2 in mammals has been reported to respond rapidly to Notch activation
consistent with being a direct target (Deftos et al., 1998), but direct CSL
binding to its promoter remains to be proved. Finding out what underlies
the selection of apoptotic and proliferative targets is of major importance for
understanding the diverse roles of Notch in development and cancers.
Second, many components of the Notch pathway are themselves direct
targets. DELTEX1, encoding a ubiquitin ligase that regulates Notch traf-
ficking, was first shown to be positively regulated by Notch in C2C12 cells
(Kishi et al., 2001) and has subsequently emerged as a target in multiple
vertebrate tissues but not yet in invertebrates [e.g., Campese et al. (2006),
Chang et al. (2000), Deftos et al. (1998), and Deftos et al. (2000)]. NRARP,a
Notch inhibitor, appears to be a target in a range of vertebrate cell types
[e.g., Krebs et al. (2001), Lamar et al. (2001), Phng et al. (2009), Pirot et al.
(2004) and Weerkamp et al. (2006)]. Other pathway members have so far
only emerged as direct targets in invertebrates [e.g., Serrate, (Martinez et al.,
2009; Yan et al., 2004); Su(H), (Barolo et al., 2000; Christensen et al., 1996);
neuralized, numb, Kuzbanian/Adam10, (Krejci et al., 2009), although indirect
evidence suggest that some are also targets in mammalian processes [e.g.,
Cheng et al. (2003, 2007)]. In addition Notch autoregulates its own expres-
sion in some mammalian (Weng et al., 2006; Yashiro-Ohtani et al., 2009)
257 Notch Targets and Their Regulation
and Drosophila cells (Krejci et al., 2009) as well as in C. elegans (Christensen
et al., 1996), providing a feedback mechanism that reinforces signaling
(Christensen et al., 1996).
Third, common targets include components of other signaling pathways.
Multiple Ras pathway regulators were identified through bioinformatics and
genetic screens in C. elegans, where the MAP kinase phosphatase (MKP)
lip-1 is a direct target along with five other negative regulators of the RAS-
MAPK pathway (Berset et al., 2001;Yoo et al., 2004). A similar elaborate
cross talk with EGF receptor signaling network and with other signaling
pathways is evident in Drosophila and direct Notch targets include positive as
well as negative regulators (Hurlbut et al., 2009; Krejci et al., 2009). Hints at
similar cross talk in mammalian cells are seen with the identification of ErbB-
2 as a direct target (Chen et al., 1997), with upregulation of MAPK regulators
in hematopoietic progenitors (Weerkamp et al., 2006) and with the oscilla-
tory network related to Notch signaling in somitogenesis (although in this
case there is as yet no proof that the cross talk involves direct regulation). The
precise nature of the Notch targets and the consequences for the cross-
regulation of signaling pathways are likely to differ depending on the context
of the cell. In the C. elegans vulva and Drosophila wing veins the consequences
on Ras signaling are inhibitory (Berset et al., 2001;Yoo et al., 2004), but
elsewhere Notch can cooperate with Ras (e.g., R7 development in Drosophila
eye) suggesting a requirement for different cohorts of targets (Hurlbut et al.,
2009; Mittal et al., 2009; Sundaram, 2005). As more studies of direct targets
are carried out, it may prove possible to extract underlying rules.
Fourth, it is evident that Notch also directly regulates expression of
genes encoding proteins that actually implement cell functions ("realizator"
genes). For example, in T-ALL cells many of the direct targets are involved
in metabolism (Margolin et al., 2009; Palomero et al., 2006). And in several
developmental contexts direct targets include cytoskeletal regulators such as
cytoskeletal crosslinkers Short stop and Gas2 and the genes encoding Ig cell
adhesion receptors Roughest and Hibris (Apitz et al., 2005; Artero et al.,
2003; Fuss et al., 2004; Krejci et al., 2009; Pines et al., 2010). Likewise,
Tenascin-C is a target of Notch2 in glioblastoma cells, where it may con-
tribute to invasiveness of the tumor cells (Sivasankaran et al., 2009).
Finally, several regulatory motifs are beginning to emerge from syste-
matic studies of Notch targets. These include positive feed-forward loops,
exemplified by the role of Myc in T-ALL cells (Palomero et al., 2006), and
incoherent (IFL), characteristic of the response in Drosophila myogenic
precursors (Krejci et al., 2009). In this type of IFL, the stimulus (Notch)
regulates both a gene and a repressor of the gene. Classic examples involve
members of the HESR family. For example, PTEN, atonal and twist are all
directly responsive to CSL/Notch, and in each case these genes can also be
repressed by HESR proteins (Ligoxygakis et al., 1998; Palomero et al., 2008;
Tapanes-Castillo and Baylies, 2004; Whelan et al., 2007).Genome-wide
258 Sarah Bray and Fred Bernard
studies revealed further targets that form IFL independent of HESR mem-
bers including String/CDC25-hindsight and myc-brat (Krejci et al., 2009).
The overall output of IFL is difficult to predict since it is dependent on
several criteria such as the rate of synthesis and the thresholds required for
activation and repression, but in some conditions it has been shown to
create pulse of target activities (Alon, 2007) and it is proposed to render
the response proportional to the fold change in the input signal
(Goentoro et al., 2009)
3. How Does the Notch Switch Work?
Binding of NICD to the DNA-binding CSL mediates the "transcrip-
tional switch" to activate gene expression from the target promoters. CSL
binds to DNA as a monomer and initial studies identified high-affinity
binding sites for both Drosophila and mammalian CSL proteins with the
core consensus YGTGRGAA (Bailey and Posakony, 1995; Lecourtois
and Schweisguth, 1995; Tun et al., 1994). The verification of more target
binding sites implied a less stringent consensus [e.g., Nellesen et al. (1999)]as
illustrated by the logo in Fig 8.1. Matches to the CSL consensus are detected
throughout the genome: one estimate places a high affinity site in the
vicinity of �40% of Drosophila genes (Rebeiz et al., 2002). It is unclear
how many such sites are functional and what determines functionality.
Certainly in one cell type only �260 genes (<2%) were directly responsive
to Notch demonstrating that at any one time only a subset of binding sites
are utilized (Krejci et al., 2009).
One factor contributing to target selection could be the arrangement
of sites. In the best-characterized targets, E(spl) genes and Hes 1, there is a
specific site architecture comprising two CSL binding sites arranged
in a head-to-head manner with an approximately 16 base pair A/T-rich
spacer sequence (SPS motif). It is proposed that SPS could confer the
ability to respond at lower levels of NICD, explaining their presence in
the strongly responding HES1 and E(spl) genes. Cooperative interactions
between NICD-containing complexes have been detected when SPS
have the appropriate spacing (15–22 nucleotides), suggesting a mecha-
nism that would ensure a sensitive and tight response at promoters at such
targets (Nam et al., 2006; 2007). However, relatively few targets contain
SPS motifs implying that additional mechanisms contribute to binding
site selection and activity, as discussed further.
The interaction of NICD with CSL creates an interface that is recog-
nized by Mam, a critical adaptor in the activation of targets. Recent
structural analysis revealed that there is a stepwise assembly of the complex,
with binding by the N-terminal part of NICD (RAM domain) providing a
259 Notch Targets and Their Regulation
tether for the interaction and causing a conformational change that also
favors Mam recruitment [reviewed in Gordon et al. (2008)]. Mam in turn
can recruit histone acetyltransferase (HAT) complexes such as p300-PCAF
and GCN5 (Kurooka and Honjo, 2000; Oswald et al., 2001).Mam is
required for p300-dependent acetylation of nucleosomes at a minimal
Notch enhancer in vitro (Fryer et al., 2002) and enhances p300 acetylation
(Hansson et al., 2009). Additional HAT complexes, such as the Tip60
complex containing TRRAP/Nipped-A, may also facilitate target gene
transcription in some contexts (Gause et al., 2006), although Tip60 has also
been reported to suppress Notch activity (Kim et al., 2007). The recruitment
of HAT complexes explains the increased H4 acetylation seen at actively
transcribed Notch targets in Drosophila cells (Krejci and Bray, 2007).
Other histone modifications, such as ubiquitination of H2B and asso-
ciated methylation of H3K4, are important for expression of Notch targets
in Drosophila (Bray et al., 2005; Buszczak et al., 2009; Moshkin et al., 2009;
Tenney et al., 2006). Mutations in the H2B ubiquitinating enzyme Bre1
result in loss of target gene expression (Bray et al., 2005) while mutations
in scrawny, encoding an ubiquitin-specific protease that deubiquitinates
H2B, lead to premature expression of key differentiation genes, including
Notch targets, in stem cells (Buszczak et al., 2009). While these histone
modifications are likely important generally for transcription, Notch-regu-
lated genes appear particularly susceptible. This may be because the mod-
ifications at the CSL binding site are critical for activity (Liefke et al., 2010)
and/or because NICD, and hence the activation complex, is present only
transiently. In transfected HeLa cells Mam was found to promote phos-
phorylation of NICD by CDK8, rendering it a substrate for proteasomal
degradation by E3 ubiquitin ligases that include Sel10/Fbw7 (Fryer et al.,
2004; Gupta-Rossi et al., 2001; Tsunematsu et al., 2004). This suggests that
target gene activation is coupled to a mechanism that down regulates the
signal (Fryer et al., 2004). Given the dynamic requirements for Notch
signaling during development, and the oncogenic effects of mutations that
interfere with NICD turnover (O'Neil and Look, 2007; Welcker and
Clurman, 2008), this aspect of Notch regulation is of major importance.
The role of CSL in mediating transcriptional activation of Notch targets
appeared initially at odds with its preceding characterization as a repressor in
mammals (Dou et al., 1994). Several different corepressors were identified
in mammalian cells, including CIR, SMRT, and SHARP (Hsieh et al.,
1999; Oswald et al., 2005; Zhou and Hayward, 2001). These interacted
directly with CSL and, when added in increasing amounts in cell transfec-
tion assays, antagonized the activation by NICD. From these data, a model
emerged where NICD displaced corepressors to convert DNA-bound CSL
to an activator (Borggrefe and Oswald, 2009; Bray, 2006; Kopan and llagan,
2009). This elegant "switch" model helped to explain many complex
observations, such as the fact that some target genes are still expressed in
260 Sarah Bray and Fred Bernard
Su(H) mutants, albeit at lower levels and with a broader domain (Koelzer
and Klein, 2003; Li and Baker, 2001; Morel and Schweisguth, 2000). There
is now increasing in vivo evidence that supports the repressive role of CSL
at Notch targets in flies (Bardin et al., 2010; Castro et al., 2005; Furriols
and Bray, 2001; Koelzer and Klein, 2006; Nagel et al., 2005), where it is
sometimes referred to as "default repression" (Barolo et al., 2002), but there
has been less definitive in vivo evidence from mammalian studies so far
(Kopan and llagan, 2009).
A key component of the repressor complex in Drosophila is the protein
Hairless, which serves as a platform or adaptor to recruit the corepressors
Groucho and CtBP (Barolo et al., 2002; Morel et al., 2001; Nagel et al.,
2005). These proteins contribute to Hairless-mediated repression to diffe-
ring extents, and it remains to be determined whether both act in combina-
tion or whether they are part of discrete complexes. Similarly, SHARP/
MINT may function in an analogous manner in mammals (Kuroda et al.,
2003; Oswald et al., 2002; Yabe et al., 2007) and has been shown to bind
several different corepressors including CtBP and SMRT/NCoR (Oswald
et al., 2005; Borggrefe and Oswald 2009). However, the diversity of CSL
corepressors identified in mammals (Borggrefe and Oswald, 2009)
and emerging examples also in Drosophila (Tsuda et al., 2006) raise questions
whether CSL is associated with distinct classes of repressor complex and how
this would impact on its relationship with Notch. It is also unclear whether
CSL has functions in gene repression where it is insensitive to Notch. Such
a possibility has emerged from studies of hlh-6 gene in C. elegans (Ghai and
Gaudet, 2008). Detailed analysis of its regulation demonstrated a requi-
rement for CSL (Lag-1) but its expression was unaffected by changes in
C. elegans Notch gene function (lin-12 or glp-1). And in mammalian cells,
CSL (RBPjk) is a potent repressor of the HIV-LTR promoter (Tyagi and
Karn, 2007). It remains to be determined whether Notch-insensitive CSL
repression is more widespread and, if so, what renders the targets Notch
insensitive.
In both flies and mammals one function of the CSL corepressor complex
is thought to be the recruitment of histone deacetylases (HDACs). SMRT,
CtBP, and Gro have all been shown to interact directly with class 1 HDACs
(Chen et al., 1999; Nagy et al., 1998; Subramanian and Chinnadurai, 2003).
Furthermore, elevated levels of HESR (ESR-1) gene expression were
detected following treatment of Xenopus caps with the HDAC inhibitor
(Kao et al., 1998). Likewise, the Notch-responsive HESR genes her6 and
her4 were ectopically expressed at distinct sites within the developing nervous
system in zebrafish hdacl mutant embryos (Cunliffe, 2004; Yamaguchi
et al., 2005). However, this does not appear to be the whole story as other
chromatin modifications appear to be important (Borggrefe and Oswald,
2009). For example, the Hairless/CSL repressor complex was found to
associate with large protein complexes, containing histone chaperones and
261 Notch Targets and Their Regulation
the histone H3K4 demethylase Lid/KDM5A, that contribute to target gene
repression (Goodfellow et al., 2007; Moshkin et al., 2009). Similarly specific
interactions were also detected between CSL (RBPjK) and KDM5A in
mammalian cells, where methylation of histone H3K4 was erased at CSL
sites upon Notch inhibition (Liefke et al., 2010). Thus a combination of
histone-modifying and remodeling activities are likely to contribute to the
silencing of targets in the absence of Notch activation. Transcription elonga-
tion may also be regulated; some Notch pathway genes have paused poly-
merase at their promoters in Drosophila embryos and may be affected by
mutations in elongation factors (Chopra et al., 2009; Zeitlinger et al., 2007).
The most commonly depicted model of the switch suggests that CSL is
statically bound to DNA while regulating transcription from Notch target
genes (Fig. 8.2). More recent studies, including work from our lab, sug-
gested that CSL binding to DNA is dynamic rather than static and it is
Nicd
CoR
Mam
PRC1
Asf1
Nap1
Lid/LSD1
Scrawny/
Usp36
HDAC
complexes
Lola,
pipsqueak
Brm
Bre1
HAT
complexes:
p300/PCAF
GCN5
Tip60
Mam
Nicd
CoR
“Dynamic” model “Classic” model
Figure 8.2 Alternative models for the transcriptional switch and factors that enhance or
suppress Notch-mediated activation. Both models are based on the fundamental principal
that (1) CSL (orange) bound to corepressors (CoR, grey) contribute to target gene
repression (2) NICD (purple) interacts with CSL (orange) and recruits Mam (green)
and coactivators to activate transcription (red arrow). The boxes list other factors
known to suppress (left) or promote (right) activation of targets. The two models differ
in how stably the CSL is bound to the DNA. In the dynamic model we propose that there
is equilibrium between bound and unbound CSL repression complexes, that NICD
containing complexes can form off the DNA, and that the exchange is between
different CSL-containing complexes (repression and activation). The activation complex
becomes stabilized by interactions with the basal transcription machinery. In the classic
model, CSL remains bound to the DNA and exchange occurs between NICD and
co-repressors on a DNA-bound CSL. This implies that CSL has high affinity for DNA
and no other interactions are needed to stabilize this interaction. (See Color Insert.)
262 Sarah Bray and Fred Bernard
notable that, in both Drosophila and human cells, CSL occupancy on promo-
ters was enhanced when NICD was present (Fryer et al., 2004; Joshi et al.,
2009; Krejci and Bray, 2007; Zhou and Hayward, 2001). This raises the
possibility that complexes may be forming/exchanging in the nucleoplasm
and indeed makes it possible that there are distinct pools of CSL complexes
(Fig. 8.2). Recent measurements of the affinity of CSL for DNA support this
more dynamic model (Friedmann and Kovall, 2010). If correct, this model
also suggests that cooperative mechanisms will be required to recruit and/
or stabilize CSL to sites on the DNA. One mechanism is likely to involve
interactions between NICD-containing complexes at appropriately spaced
CSL sites (e.g., SPS motif), which would increase the stability of the activa-
tion complex (Cave et al., 2005; Gordon et al., 2008; Ong et al., 2006).
However, few enhancers contain the optimal pairing of sites, suggesting that
other mechanisms are also likely to be important.
4. Different Enhancer Logics
One prediction of the switch model is that target genes will be de-
repressed in the absence of CSL, as seen for a number of targets in Drosophila
which are ectopically expressed in Su(H) mutants [e.g., Bardin et al. (2010),
Koelzer and Klein(2006), and Morel and Schweisguth(2000)]. This ectopic
expression is limited and often weaker than normal but contributes to
phenotypic differences in Su(H) and Notch signaling mutants. For this reason,
defects in Notch signaling may in some cases be alleviated by mutations in
the co-repressors. For example, conditional inactivation of the corepressor
SHARP/MINT in developing nephrons resulted in a moderate rescue of
the Notch2 mutant phenotype (Surendran et al., 2010). CSL-mediated
repression is clearly essential in some processes (Bardin et al., 2010; Koelzer
and Klein, 2006), but in others there has been no clear evidence for target
gene de-repression contributing to the phenotypes of CSL-knockouts (Han
et al., 2002; Oka et al., 1995; Shen et al., 1997). It thus remains to be resolved
to what extent CSL-mediated repression is important at all target enhancers.
A second prediction is that targets will have reduced expression in the
absence of CSL in the places where they are normally responsive. This would
seem to be a fundamental expectation for Notch-regulated targets and is
certainly the case for many of the best characterized: in most cases HESR
gene expression is compromised by the loss of CSL binding [e.g., Bailey and
Posakony (1995), Lamar and Kintner (2005), Lecourtois and Schweisguth
(1995), and Nellesen et al. (1999)]. However, there are examples of direct
targets in Drosophila that show no loss of activation in Su(H) mutants, includ-
ing atonal in the eye imaginal disc and sox15 in the sensory organ lineage
(Li and Baker, 2001; Miller et al., 2009). Despite their continued expression
263 Notch Targets and Their Regulation
in Su(H) mutants, atonal and Sox15 are both Notch regulated and require
CSL for this regulation. Such observations have led to the proposal that the
function of NICD at some targets, referred to as Notch-permissive targets, is
primarily to alleviate the repression function of CSL complexes (Bray and
Furriols, 2001). Permissive targets would not require the activation function
of NICD and would achieve high levels of expression through other factors
bound to the enhancer. In contrast, so-called Notch-inductive targets, such as
HESR genes, require the activation function of NICD for high levels of
expression, and their expression is compromised by mutations in the CSL-
binding sites (Bailey and Posakony, 1995; Flores et al., 2000; Lecourtois and
Schweisguth, 1995; Nellesen et al., 1999; Neves and Priess, 2005). It is unclear
what mechanisms might underlie these differences in the target responses; it
may depend on whether the promoters are in a poised conformation, what
histone modifications are present and what type or amounts of cooperating
factors are already bound.
5. Context Dependence of Notch Responses
The context corresponds to the mechanisms that make a gene respon-
sive when the Notch pathway is activated. Thus, while most Notch-
dependent processes are associated with expression of HESR genes, the
specific HESR gene(s) activated varies according to the context, illustrating
that even these common targets acquire additional specificity-conferring
inputs. For example, in Drosophila there are seven closely related E(spl)
bHLH genes that have arisen through recent gene duplications and are
clustered on the chromosome (Schlatter and Maier, 2005). Despite their
relatively recent origins and close proximity, the different genes have dis-
tinct patterns of expression (especially during post embryonic stages) and can
only be activated by Notch in limited territories [e.g., Cooper et al. (2000),
de Celis et al. (1996),and Nellesen et al., (1999)]. Similar spatial restricted
patterns occur for the Hes and Hey genes in mouse, the Her genes in
zebrafish (Kageyama et al., 2007) and ESR genes in Xenopus (Lamar and
Kintner, 2005). Thus despite the fact that many target genes contain multi-
ple CSL binding sites, they are only able to respond in a subset of the places
where Notch is activated. Clearly there is other information that restricts
where the specific gene targets are responsive. Further complexity arises
with targets that show Notch-dependent and Notch-independent expres-
sion [e.g., Yeo et al. (2007)] and with targets that cross-regulate each other
(Fior and Henrique, 2005; Hatakeyama et al., 2004).
Combinatorial regulation with patterning transcription factors is one
way that genes acquire specificity in their response to Notch. An obligate
integration at enhancers of signaling inputs, such as Notch, with patterning
264 Sarah Bray and Fred Bernard
protein inputs was proposed from studies in the Drosophila wing and eye
(Flores et al., 2000; Guss et al., 2001). Synergy between Notch and the
proneural bHLH proteins emerged as critical in the regulation of HESR
genes during neurogenesis (Bailey and Posakony, 1995; Castro et al., 2005;
Cooper et al., 2000; Kramatschek and Campos-Ortega, 1994; Lamar and
Kintner, 2005; Singson et al., 1994). Other examples of combinatorial factors
include GATA factors, which synergize in regulating ref-1 in C. elegans
endoderm (Neves and Priess, 2005), NFKB family members, which co-
regulate HES, Deltex- 1, and cyclinD3 (Joshi et al., 2009; Moran et al., 2007),
the AML1 homologue Lozenge, which combines with Notch on the Pax2
enhancer (Flores et al., 2000) and Twist, which coregulates many targets in
muscle progenitors (Bernard et al., 2010).
At present the mechanisms underlying the combinatorial and synergistic
interactions between Notch and other factors are not fully understood. One
possibility is that direct interactions between synergizing factors and CSL
complexes could lead to their mutual stabilization on the target enhancer, so
favoring activation (Fig. 8.3A). Both ELT-2/GATA and Daughterless/E2A
have been shown to interact directly with CSL in support of this model
(Cave et al., 2005; Neves and Priess, 2005). NFKB has also been found to
augment binding of NICD to cyclinD3 (Joshi et al., 2009). Such interactions
may be facilitated by specific arrangements of binding sites. For example,
Daughterless/E2A combines with proneural proteins to bind a target site
that is found associated with paired CSL sites (SPS) in E(spl) gene enhancers
(Cave et al., 2005). Synergy is no longer observed if the geometry of these
sites is altered, arguing that their configuration is important in enabling
interactions. Although, many enhancers do not appear to contain a stereo-
typic site architecture [e.g., Guss et al., (2001), and Lamar and Kintner
(2005)] a recent analysis of the Drosophila Pax2 "sparkling" enhancer reveals
that the organization of CSL and other regulatory sites is important to
determine the correct expression pattern (Swanson et al., 2010). The con-
straints on spacing and organization suggest that expression is dependent on
short-range regulatory interactions that could be compatible with direct
protein–protein interactions.
A second possibility to explain synergy between Notch and cooperating
factors is that they make independent interactions with targets within the
transcriptional machinery such that the readout is an integration of their
inputs (Fig. 8.3B). Despite the importance of site organization in the spark-
ling enhancer, the composition and distribution of sites are not maintained in
a functionally conserved enhancer from distantly related species (Swanson
et al., 2010). This implies greater flexibility than is likely to be feasible for
direct interactions between specific transcription factors. Such binding site
flexibility could result from NICD/CSL and coregulating transcription
factors being able to contact the basal machinery or chromatin-modifying
cofactors, from many different configurations (Arnosti and Kulkarni, 2005).
265 Notch Targets and Their Regulation
Direct protein
interactions
(A) NICD
CSL
(B)
(C)
NICD
CSL
CSL
NICD
Indirect interactions
via transcriptional
machinery
Recruitment of
chromatin
remodeling/histone−
modifying enzymes
Figure 8.3 Possible mechanisms underlying combinatorial regulation at Notch targets.
Context-conferring factors (blue) could act by (A) directly contacting CSL (or NICD) to
stabilize interactions at the enhancer; (B) contacting intermediate targets, such as the basal
machinery (grey), allowing greater diversity in the configurations of sites; (C) recruiting
chromatin-modifying cofactors (grey) that allow accessibility to NICD/CSL by altering the
chromatin conformation (CSL, orange; NICD, purple; Mam, green). (See Color Insert.)
A third possibility is that context-conferring factors alter the chromatin
at Notch enhancers, making it accessible to CSL/NICD, a mechanism
that would not necessitate simultaneous binding. Instead these factors
could result in altered nucleosome placement or histone modifications
(Fig. 8.3C). There is mounting evidence that chromatin modifications
are important for Notch outputs. For example, two BTB/POZ proteins,
Lola and Pipsqueak, synergize with Notch activity in Drosophila through
a mechanism that appears to involve changes in histone and DNA methy-
lation at critical targets (Ferres-Marco et al., 2006). Similarly, the Brm/
Brahma ATPase component of the SWI/SNF chromatin-remodeling
complex interacts with the NICD in C2C12 cells and shows functional
interactions with the Notch pathway in Drosophila (Armstrong et al., 2005;
Kadam and Emerson, 2003). Conversely, recent studies suggest that Notch
pathway is repressed by PRC1, one of the Polycomb chromatin-silencing
266 Sarah Bray and Fred Bernard
complexes (or at least by proteins that are part of PRC1) (Martinez et al.,
2009; Tolhuis et al., 2006). It has been suggested that repression by the PcG
could raise the threshold Notch has to overcome to activate certain genes
(Merdes et al., 2004). However, it remains possible that PcG may regulate
transcription of the receptor and ligands, rather than altering the accessibility
of targets, especially since other studies reported different consequences on
target gene activation in the PRC1 mutant cells (Classen et al., 2009). Further
studies will be needed to unravel the contribution of these and other
epigenetic regulators at Notch targets.
6. Concluding Comments
Starting with an initial trickle and increasing to the current deluge from
genome-wide studies, the number of direct Notch-regulated targets has risen
exponentially since the initial discovery that NICD is a transcriptional acti-
vator. Here we have summarized some of the general conclusions that have
emerged from studies of Notch targets and their regulation so far. One of the
challenges in future will be to extract fundamental messages from the repe-
rtoire of targets identified in different tissues and diseases, in order to deter-
mine whether there are specific signatures and conserved patterns in the
responses. A second challenge will be to unravel the mechanisms that confer
different Notch responses: the cell context is fundamental to target gene
activation and the identification of factors conferring specificity remains of
primary importance. Possible mechanisms contributing to this specificity
include chromatin accessibility and, although it is evident that epigenetic
factors contribute to target gene activation, the critical changes that make
enhancers accessible to CSL/Notch complexes remain to be established. Also
unclear is how different the mechanisms of regulation are at individual targets,
for example, whether there are different modes of CSL repression complexes.
Finally, another factor that has largely been overlooked is whether the level or
duration of the signal could impact on the sets of genes activated. In
Drosophila, use of a thermosensitive Notch allele suggests that some genes
are more sensitive to a slight decrease of signal than others (Becam and Milan,
2008) and studies with hematopoietic progenitors indicate that quantitative
aspects of Notch signaling are relevant for cell fate outcomes (Delaney et al.,
2005). Clearly this will be another important question for the future.
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