DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 2633
Development 137, 2633-2642 (2010) doi:10.1242/dev.053181
© 2010. Published by The Company of Biologists Ltd
Specificity of Notch pathway activation: Twist controls the
transcriptional output in adult muscle progenitors
Fred Bernard1, Alena Krejci1, Ben Housden1, Boris Adryan2 and Sarah J. Bray1,*
SUMMARY
Cell-cell signalling mediated by Notch regulates many different developmental and physiological processes and is involved in a
variety of human diseases. Activation of Notch impinges directly on gene expression through the Suppressor of Hairless [Su(H)]
DNA-binding protein. A major question that remains to be elucidated is how the same Notch signalling pathway can result in
different transcriptional responses depending on the cellular context and environment. Here, we have investigated the factors
required to confer this specific response in Drosophila adult myogenic progenitor-related cells. Our analysis identifies Twist (Twi)
as a crucial co-operating factor. Enhancers from several direct Notch targets require a combination of Twi and Notch activities for
expression in vivo; neither alone is sufficient. Twi is bound at target enhancers prior to Notch activation and enhances Su(H)
binding to these regulatory regions. To determine the breadth of the combinatorial regulation we mapped Twi occupancy
genome-wide in DmD8 myogenic progenitor-related cells by chromatin immunoprecipitation. Comparing the sites bound by
Su(H) and by Twi in these cells revealed a strong association, identifying a large spectrum of co-regulated genes. We conclude
that Twi is an essential Notch co-regulator in myogenic progenitor cells and has the potential to confer specificity on Notch
signalling at over 170 genes, showing that a single factor can have a profound effect on the output of the pathway.
INTRODUCTION
Signalling through the Notch receptor controls a broad spectrum of
cell fates and developmental processes in organisms ranging from
sponges to human and is associated with various pathologies
including different types of cancer (Radtke and Raj, 2003; Lasky
and Wu, 2005; van Es et al., 2005). This multitude of roles is
associated with different outputs, depending upon the cellular
context. For example, in certain contexts Notch activation promotes
proliferation and maintenance of progenitor populations [e.g.
myogenesis (Kopan et al., 1994; Delfini et al., 2000; Hirsinger et
al., 2001)], whereas in others it promotes cell type differentiation
[e.g. T-cell formation (Robey, 1999) or neural fate (Gaiano and
Fishell, 2002)]. Transduction of the Notch pathway is relatively
simple: activation of Notch elicits proteolytic cleavage, releasing
the Notch intracellular domain (NICD), which enters the nucleus
and collaborates directly with DNA-binding proteins of the CSL
family {CBF1 (RBPJ) in mammals, Suppressor of Hairless [Su(H)]
in Drosophila and LAG-1 in worms (Bray, 2006; Kopan and
Ilagan, 2009)}. Effects on gene expression are thus a primary
consequence of activating the pathway, implying that the different
outputs of Notch activation require a selective transcriptional
response. This is highlighted by the observations that the sites of
Su(H) occupancy after Notch activation differ according to cell
type (Krejci and Bray, 2007) and that only a fraction (slightly more
than 10%) of the potential Notch-regulated targets are shared
between two cell types (Krejci et al., 2009). The identification of
1
Department of Physiology, Development and Neuroscience, University of
Cambridge, Downing Street, Cambridge CB2 3DY, UK. 2Cambridge Systems Biology
Centre and Department of Genetics, University of Cambridge, Tennis Court Road,
Cambridge CB2 1QR, UK.
*Author for correspondence ([email protected])
Accepted 27 May 2010
co-regulators that confer such specificity on the Notch response is
of primary importance for understanding the context-specific
responses.
To date only a few cell type-specific transcription factors have
been shown to combine with the Notch pathway to regulate
induction of specific targets. These include GATA-like factors,
which act in combination with Notch to regulate ref-1 in the C.
elegans endoderm (Neves et al., 2007), and NFB family
members, which co-regulate HES and deltex 1, which are both
Notch targets during B-lymphocyte development (Moran et al.,
2007). The best-characterised co-regulators are the proneural
bHLH transcription factors that combine with Notch to activate
targets of the E(spl)/HES gene family during neurogenesis in a
wide range of species (Nakao and Campos-Ortega, 1996; Parras et
al., 1996; Culi and Modolell, 1998). Proneural co-regulation is
proposed to depend on a particular binding site architecture,
consisting of paired CSL binding sites and a proneural protein
binding site (Cave et al., 2005). As this code has been derived from
the evolutionarily related genes of the E(spl) family it is not clear
whether such specific binding site architecture extends more
broadly to other Notch targets, nor whether other co-regulators
would involve similar binding site organisation at their targets.
Furthermore, in none of the partnerships analysed, including that
involving proneural proteins, is it clear whether the regulatory
relationship is specific for the targets analysed or whether it extends
to a broad spectrum of genes.
In order to discover the scale and breadth of transcriptional
responses to Notch, a combination of genome-wide chromatin
immunoprecipitation (ChIP) and expression array analysis was used
to identify direct Notch targets in different Drosophila cell lines,
including DmD8 cells, related to adult muscle progenitors (AMPs)
(Krejci et al., 2009). In this context, Notch is required to maintain the
progenitors and prevent their differentiation, a function that appears
similar to the role of Notch in vertebrate myogenesis, in which
activation of Notch signalling prevents muscle differentiation both
DEVELOPMENT
KEY WORDS: Drosophila, Gene regulation, Myogenesis, Notch, Twist
2634 RESEARCH ARTICLE
MATERIALS AND METHODS
Drosophila experiments
All alleles and stocks are described in FlyBase unless indicated otherwise.
The following reporters were used: m6-GFP (Lai et al., 2000); 4.0HimGFP (Rebeiz et al., 2002); aosNRE-lacZ, aosNREmutSu(H)-lacZ, edlNRE-lacZ
(Krejci et al., 2009); and m8-lacZ (Kramatschek and Campos-Ortega,
1994). In overexpression and RNAi experiments, Gal4 driver stocks
[1151:Gal4 (Anant et al., 1998), Ptc:Gal4 Tub:Gal80ts, sca:Gal4] were
combined with UAS lines [UAS:twi (Baylies and Bate, 1996), UAS-Ni79.2]
and/or RNAi lines [UAS-Notch-RNAi (BL7078), UAS-twi-RNAi
(GD37092), UAS-da-RNAi (GD51297) (Dietzl et al., 2007), UAS-twiRNAi2x (Wong et al., 2008)] and larvae were shifted to 30°C 48 hours
before dissections. Immunofluorescence was performed as described
previously (Cooper and Bray, 1999) with the following antibodies: mouse
anti-NICD (DSHB, 1/20), mouse anti-Cut (DSHB, 1/20), mouse anti--gal
(DSHB, 1/20), rabbit anti-GFP (Invitrogen, 1/1000), guinea-pig anti-Myc
[1:500; gift of F. Martin and G. Morata (Herranz et al., 2008)], rabbit
anti-Roughest [1/50; gift of K. Fishbach (Schneider et al., 1995)].
-galactosidase expression was detected histochemically.
Cell experiments and lacZ reporters
E(spl)m3, NME (–ve control) and aosNRE luciferase reporters have been
described (Krejci et al., 2009). A fragment encompassing the region –2098
to +37 of E(spl)m6 (Nellesen et al., 1999) was amplified and cloned into a
luciferase vector containing a minimal promoter from Hsp70 (pGL3::Min).
Twi sites were mutated using oligonucleotides with three mismatches
(introducing T at positions 3, 4 and 8) as described (Krejci et al., 2009). After
mutagenesis, aosNRE fragments were cloned into either pGL3::Min or
pBlueRabbit (which contains lacZ downstream of a minimal Hsp70
promoter). Cell culture conditions and transfections were as described
previously (Nagel et al., 2005; Narasimha et al., 2008). To activate Notch,
cells were treated with EDTA as described (Krejci and Bray, 2007). For twi
RNAi treatment of DmD8 cells, double-stranded RNA duplexes were
produced from a 750 bp fragment of the twi exon with T7 promotercontaining primers and subsequently transcribed using a T7 Transcription Kit
(Ambion). Cells were incubated with twi RNAi for 72 hours before Notch
activation. Three biological replicates were performed in all experiments.
RNA isolation, quantitative PCR and ChIP experiments were performed
as described (Krejci and Bray, 2007). Antibodies for ChIP were goat antiSu(H) (Santa Cruz Biotechnology, sc15813) and rabbit anti-Twi [from M.
Leptin and S. Roth (Roth et al., 1989); or from E. Furlong (Sandmann et
al., 2007)].
Bioinformatics and Twi genome-wide ChIP in DmD8 cells
Data sets
ChIP-enriched regions in DmD8 cells were determined de novo using
Nimblegen HD2 tiling arrays following the procedures and post-hybridisation
analysis described previously for Su(H) (Krejci et al., 2009). The array
data have been deposited at NCBI Gene Expression Omnibus (GEO,
http://www.ncbi.nlm.nih.gov/geo/) with accession number GSE22650. Peaks
were defined using Tamalpais (Bieda et al., 2006), with P<0.0001. ChIPenriched regions for Su(H) (292), Twi (2512), Mef2 (1464) or Biniou (292)
in embryos were taken from previously published experiments (Sandmann et
al., 2006; Jakobsen et al., 2007; Sandmann et al., 2007; Krejci et al., 2009;
Zinzen et al., 2009). In the case of Biniou, Twi and Mef2, for which analysis
of binding had been performed at multiple time-points (Sandmann et al.,
2006; Jakobsen et al., 2007; Sandmann et al., 2007; Zinzen et al., 2009), a
non-redundant data set was generated with all the regions that were occupied
in at least one stage. Where necessary, the UCSC Genome Browser (Kuhn et
al., 2009) Liftover tool was used to transpose all genomic positions to
coordinates of release 5 of the D. melanogaster genome.
Overlap
Custom-written Perl scripts were used to determine the overlap between
ChIP-enriched regions and their statistical significance. The significance
of overlap was determined by 1000 rounds of random simulation. In each
round, every Su(H) site was allowed to deviate around its original position
(Fig. 1A and see Table S2 in the supplementary material; uniform
distribution within a range around the original site). The overlap between
random Su(H) and Twi sites was determined in each round and recorded
to derive a Z-score [(actual overlap – average random overlap) / standard
deviation of random overlap] and empirical P-value (0.001 increment for
each random overlap ≥ actual overlap).
Position weight matrix (PWM) matches
Alignment matrices for Su(H) (Krejci et al., 2009), Twi (Down et al., 2007)
and Mef2 were built based on a compilation of previously published binding
sites (see Fig. S3 in the supplementary material). The motif scanner nmscan
from the NestedMICA package (Down and Hubbard, 2005) was used for
genome-wide motif matching, with empirically defined cut-off values for
each transcription factor (roughly yielding the same number of genomic
binding sites). For sites shown in Fig. 1, matches to PWMs were computed
using Patser (Hertz and Stormo, 1999) with a threshold of 5.5 (the
approximate cut-off used above is indicated on the resulting graph by a
dashed line).
DEVELOPMENT
in embryonic precursors and in satellite cells (Delfini et al., 2000;
Hirsinger et al., 2001; Conboy and Rando, 2002; Kopan and Ilagan,
2009). Among the 235 direct Notch targets identified in DmD8
muscle progenitor-like cells, several have been specifically linked to
roles in muscle development. However, many others are involved in
fundamental aspects of cell differentiation such as signalling and
morphogenesis (Krejci et al., 2009). In addressing how the genome
integrates Notch activation differentially according to cell type, one
question therefore is whether one or many transcription factors are
required to specify this diverse cohort of targets and to confer an
output that is distinct for myogenic precursors. A second question is
how these factors affect CSL binding.
Several key transcription factors are known to have important
roles in regulating myogenesis. These include the bHLH proteins
of the MyoD/myogenin family and the MADS box proteins of the
Mef2 family (Parker et al., 2003; Tajbakhsh, 2003). The latter are
important in promoting muscle differentiation in all species (Black
and Olson, 1998). In Drosophila, the bHLH protein Twist (Twi)
acts prior to Mef2 in both embryonic and adult myogenesis (Cripps
et al., 1998; Baylies and Michelson, 2001; Castanon and Baylies,
2002). Initially, Twi is expressed broadly throughout the embryonic
mesoderm and subsequently it regulates whether or not the cells
are routed into somatic muscle fates. Its expression is then lost in
larval muscles when they differentiate (Baylies and Bate, 1996),
but persists in adult muscle precursors (Bate et al., 1991), only
declining during pupation when the myoblasts fuse and
differentiate into the muscles of the adult (Anant et al., 1998). As
both the Notch pathway and Twi are important in maintaining
AMPs in an undifferentiated state (Anant et al., 1998), Twi is a
potential candidate to co-operate with Notch in muscle precursors.
Taking advantage of the Notch targets identified in DmD8 cells,
we have investigated how specificity is conferred on the Notch
response in the myogenic cell type. Focussing initially on three
targets, we find that their expression in vivo is dependent on Twi
acting in combination with Notch. Twi binding to target enhancers
precedes Su(H) recruitment, which is detected only after activation.
To assess whether Twi is likely to confer specificity on a broader
range of targets, we analysed whether Twi binding regions
identified genome-wide in the DmD8 myogenic precursor-related
cells are found in proximity to the Su(H) binding regions identified
in the same cells. This revealed a significant association and
identified more than 170 genes that are potentially co-regulated by
both factors. These results demonstrate that one co-factor, Twi, is
likely to regulate on a large scale the output from the Notch
pathway in muscle progenitor cells.
Development 137 (16)
Twist confers specificity on Notch
RESEARCH ARTICLE 2635
RESULTS
Functional relevance of Twist for Notch target
gene expression
We first focussed our investigations on a selection of DmD8 Notch
targets with distinct cellular functions: E(spl)m6, an immediate
early Notch target involved in regulating Notch pathway activity
(Bardin and Schweisguth, 2006); argos (aos), an inhibitor of EGF
receptor signalling (Howes et al., 1998); and Him, a repressor that
inhibits Mef2 (Liotta et al., 2007). The first question we addressed
was whether the cell-context specificity was retained in enhancer
fragments isolated from two of these target genes, by testing
whether the enhancers responded differently to NICD according to
cell type. Fragments encompassing Su(H) ChIP peak regions from
E(spl)m6 and aos (Krejci et al., 2009) (see Fig. 1A,B) were tested
in comparison to E(spl)m3, which is inducible by Notch in all cells
that we have tested (Krejci and Bray, 2007). The resulting reporter
genes were co-transfected into DmD8 muscle progenitor type cells
and into S2 blood-related cells. E(spl)m6 and aos enhancer
fragments both recapitulated the cell specificity, responding to
NICD much more robustly in DmD8 cells (Fig. 1D). The
magnitude of the response was larger for aosS than for the longer
aosL, suggesting that the latter can recruit factors that antagonise
NICD or that affect accessibility. Nevertheless, the results suggest
that the enhancer fragments are sufficient to confer a contextspecific Notch response that is dependent on factors present in
DmD8 cells.
Transcription factors that might contribute to the specificity of
Notch in DmD8 cells and myogenic precursors include Twi and
Mef2 (Castanon and Baylies, 2002; Cripps et al., 2004; Lovato et
al., 2005). RNA sequencing profiles have shown that DmD8 cells
DEVELOPMENT
Fig 1. Requirement for Twist at Notch target enhancers. (A-C)Genomic regions surrounding Drosophila E(spl)m6 (B), Him (C) and aos (A).
Black lines and boxes (exons) represent transcribed regions. Graphs show matches to Twi PWM (green bars; Patser score 5-9.3), Su(H) PWM (red
bars; Patser score 5-9.79) or the region enriched in ChIP experiments by Su(H) (blue bars; enrichment relative to input; log2 0.3-2.3). Blue rectangles
represent regions cloned to create reporters used in subsequent experiments. Asterisks (A) represent binding sites that have been mutated in
subsequent experiments. Light-blue rectangle represents fragment aosS, the dark-blue rectangle fragment aosL. Note that aosS contains all Su(H)
sites and two of the three Twi sites, aosL=aosNRE and contains additional Su(H) sites. (D) Response of the indicated enhancers to NICD in transient
transfection assays in S2 cells (light-grey bars) or DmD8 cells (dark-grey bars). (E-G)twi RNAi treatment of DmD8 cells. (E)Efficiency of twi RNAi
treatment on twi levels in DmD8 cells. Values in control untreated cells are normalised to 1. (F,G)Fold change in the expression levels of the
indicated genes after Notch activation, as determined by quantitative RT-PCR in the absence (light grey) or presence (dark grey) of twi RNAi. The
average of three biological replicates is shown; the data are plotted on separate graphs because of the difference in magnitude of the fold
stimulation by Notch. (F)E(spl)m6 and Him, which show 175-fold and 113-fold stimulation by Notch, respectively, are reduced by 76% and 79%,
respectively, by twi RNAi, whereas the 140-fold stimulation of E(spl)m3 is not reduced by twi RNAi. (G)The 2-fold stimulation of aos by Notch
showed a 56% reduction in the presence of twi RNAi. Control genes (nimC1 and Ef2) were neither induced by Notch nor reduced by twi RNAi.
Expression levels in F and G were normalised to that of rp49 (RpL32). Error bars are s.e.m.
express high levels of twi mRNA (Celniker et al., 2009) and there
is a striking similarity between Notch and twi function in myogenic
precursors (Anant et al., 1998), making Twi a good candidate for
co-regulatory activity. As a first test of this hypothesis, we treated
DmD8 cells with twi RNAi and assayed its effects on a selection
of targets (using quantitative RT-PCR to detect transcript levels),
under conditions in which the efficacy of twi knockdown was
~50% to limit any concomitant effects on overall cell fate (Fig. 1E).
Even under these limited conditions, E(spl)m6, Him and aos all
showed a reduced response to Notch in twi RNAi-treated cells (Fig.
1F,G; 76% reduction for E(spl)m6, 79% for Him and 56% for aos).
Neither E(spl)m3, nimC1 (which encodes a phagocytic receptor)
nor Elongation factor 2 (Ef2) showed any reduction, arguing that
the reduced expression is specific and likely to reveal Twidependent targets.
We extended these assays to the muscle progenitors in the wing
disc. Enhancers from E(spl)m6, aos and Him all direct expression
in AMPs (Lai et al., 2000; Krejci et al., 2009). These cells are
apposed beneath the disc epithelium in the notal region (and hence
are also known as adepithelial cells) and can be distinguished from
the overlying epithelium by the expression of markers such as Cut.
All three enhancers directed expression in the Cut-expressing
AMPs (Fig. 2A,D,F), although E(spl)m6-GFP expression was
restricted to a localised region in the anterior (Fig. 2A). Using
several independent RNAi constructs to ablate twi in these cells
(expressed using the 1151-Gal4 driver), we consistently detected a
reduction of E(spl)m6-GFP, aosNRE-lacZ and 4.0Him-GFP
expression (Fig. 2B,C,E,G). Importantly, the muscle progenitors
were still present under these conditions and could be detected by
expression of markers such as Cut (e.g. Fig. 2B,C). Ablation of
Daughterless (Da), a dimerisation partner of Twi in certain contexts
(Castanon et al., 2001), had no effect on these targets [although
expression of da RNAi in proneural clusters with sca-Gal4 did
alter the expression of E(spl)m8 as expected] (see Fig. S1 in the
supplementary material). Likewise, no change in E(spl)m6-GFP
expression was seen with RNAi targeting nine other bHLH genes
(see Table S1 in the supplementary material). Thus, the conditional
knockdown of twi, but not da or other bHLH transcription factors,
is sufficient to prevent expression from E(spl)m6, aos and Him
enhancers.
Twist and Notch co-operate to activate target
genes
The reduced expression of several target genes following twi
ablation indicates that Twi contributes to their regulation. However,
these assays do not distinguish whether Twi alone is sufficient or
whether it requires collaboration with Notch. To address this
question, we assessed the ability of Twi to confer appropriate Notch
responsiveness when expressed in a different cell type in the
presence and absence of Notch. Genes such as E(spl)m6 or Him are
normally only expressed in AMPs, not elsewhere in the wing disc,
despite Notch activity at other locations. Similarly, expression from
enhancers identified by Su(H) binding in DmD8 cells is also
restricted to muscle progenitors (e.g. aos). We therefore investigated
whether Notch and/or Twi were able to activate these enhancers
when expressed ectopically in the wing pouch using ptc-Gal4 (Fig.
3T) in combination with thermosensitive Gal80, so that expression
was limited to a defined period at the end of larval development.
First we assessed whether simply increasing Notch activity in the
wing pouch domain would activate the muscle progenitor
enhancers. Although this was sufficient to produce ectopic
expression of genes that are direct Notch targets at the dorsal-ventral
Development 137 (16)
Fig 2. Twist regulation of Notch target enhancers in muscle
progenitors. (A-C)Effect of two independent twi RNAi constructs
(GD37092, B; twi-RNAi2x, C) on E(spl)m6-GFP expression (green in
A,B,C; white in A⬘). A patch of E(spl)m6-GFP is detectable in wild type
in Cut-expressing adult muscle progenitors (AMPs) (A, arrow; inset) but
is severely reduced (B, arrow) or abolished (C, arrow) in the presence of
twi RNAi. Cut expression (which marks all muscle progenitors; purple,
A-C) is unchanged. (D-G)Expression of the indicated Notch target
genes in wild type (D,F) and in muscle progenitors expressing twi RNAi
(E,G). Expression was monitored using the indicated reporters, detected
by antibodies (A-E) or histochemistry (F,G), and colocalisation with
AMPs was verified by co-staining with Cut (A-C) or by interference
microscopy (F,G).
boundary (e.g. Fig. 3B,G), neither E(spl)m6-GFP, 4.0Him-GFP nor
aosNRE-lacZ exhibited any ectopic activation (Fig. 3B,G,L). By
contrast, expression of Twi using the same driver elicited ectopic
expression from all three enhancers (Fig. 3C,H,M), giving strong
induction of 4.0Him-GFP and weak induction of E(spl)m6-GFP and
aosNRE-lacZ. Their ectopic induction indicates that the enhancers are
capable of responding to Twi. This response is highly selective, as
increased or ectopic expression of 20 other bHLH proteins,
including proneural proteins, failed to elicit any change in E(spl)m6
expression (see Table S1 in the supplementary material).
Combining expression of Twi with NICD yielded much stronger
expression from all three enhancers in the wing pouch than
expression of Twi alone (Fig. 3D,I,N). These results suggest that
Notch and Twi co-operate to induce expression of these genes. To
further confirm whether this co-operation was essential, we also
tested the consequences of ablating Notch activity when Twi was
ectopically expressed. All three enhancers showed substantially
DEVELOPMENT
2636 RESEARCH ARTICLE
Twist confers specificity on Notch
RESEARCH ARTICLE 2637
Fig. 4. Twist binding sites are required for Notch activation of
argosNRE. (A)Response of the indicated enhancers to NICD in transient
transfection assays in DmD8 cells, expressed as fold change (dark-grey
bars) relative to expression levels in the absence of Notch (light-grey
bars). Mutating Twi binding sites as indicated abolishes aosNRE
responsiveness. (B,C)In vivo, mutations of the same sites lead to a
decrease in expression (C) compared with the control enhancer (B).
reduced expression when Twi was expressed in combination with
Notch RNAi (Fig. 3E,J,O), as compared with Twi alone, and two
of the three had no residual ectopic expression. This indicates that
the expression induced by Twi alone is largely dependent on an
underlying activity of Notch [sensitive Notch reporters reveal a low
level of Notch activity in most cells of the wing pouch (Furriols
and Bray, 2001)]. The dependence on Notch was further confirmed
by analysing expression from an aos enhancer in which the Su(H)
binding sites had been mutated. This mutated aosNREmutSu(H)
enhancer is no longer expressed in muscle progenitors (Krejci et
al., 2009) and was not able to respond to ectopic Twi expression or
to the combination of Twi and NICD (Fig. 3P-S). Together, these
results show that Twi confers the ability on these target enhancers
to respond to Notch activity, and vice versa.
Twist binding sites are required for the activity of
Notch target gene enhancers
Our results point to a pivotal role for Twi in the context-specific
Notch response, but do not distinguish whether this effect is direct.
In order to test whether direct binding by Twi is required, we
mutated putative Twi binding sites in one of the responsive
enhancers. aosNRE contains three matches to a position weight
matrix (PWM) for Twi binding sites (see Fig. 1A), with sites 2 and
3 having the best matches. We generated fragments with mutations
in site 2 (aosNRE⌬Twi2) and with mutations in both site 2 and the
adjacent site 3 (aosNRE⌬Twi2,3). When tested in DmD8 cells, both
enhancers had lost their ability to respond to Notch activation,
suggesting that these sites are essential (Fig. 4A). Similarly, when
tested in vivo, the mutated fragments had lost the ability to direct
expression in AMPs (Fig. 4B,C). Thus, Twi binding sites are
necessary for aosNRE activity in DmD8 cells and in vivo in
myogenic precursors.
Genome-wide association of Twist and Suppressor
of Hairless binding
As the tested enhancers all require Twi for their responsiveness to
Notch, this regulatory combination might be required for the
majority of Notch targets in DmD8 cells and muscle progenitors.
If this were the case, there should be a strong association of Su(H)
and Twi sites among the DmD8 target loci. To test this possibility,
we first used data from previously published genome-wide ChIP
experiments performed in embryos (Sandmann et al., 2006;
Jakobsen et al., 2007; Sandmann et al., 2007; Zinzen et al., 2009).
Using two approaches (Fig. 5A and see Fig. S2 in the
supplementary material), we found a significant association
between Su(H) and Twi binding regions (based on a null model
that constrained the position of the peaks to take account of their
non-random distribution across the genome; see Materials and
methods and Table S2 in the supplementary material). Furthermore,
when the partner combinations between Su(H) and several other
transcription factors were calculated on the basis of the embryonic
data, there was a strong pair-wise correlation of Su(H) with Twi
(see Fig. S2B in the supplementary material, red box) and ChIP
DEVELOPMENT
Fig. 3. Twist acts in combination with Notch to regulate
myogenic target enhancers. (A-S)Expression from reporters
E(spl)m6-GFP (green, A-E), 4.0Him-GFP (green, F-J), aosNRE-lacZ (grey,
K-O) or aosNRESu(H)mut-lacZ (grey, P-S) in wild type (A,F,K,P), and in discs
where the levels of Twi and Notch activity were manipulated in a stripe
(arrow, ptc-Gal4;Tub-Gal80ts; see T) by NICD expression (>NICD;
B,G,L,Q), by Twi overexpression (>Twi; C,H,M,R), by combining NICD
and Twi overexpression (>Twi;NICD; D,I,N,S), or by ablating Notch (with
Notch RNAi) in cells overexpressing Twi (>Twi;N-RNAi; E,J,O). (T)The
expression domain of the Ptc:Gal4 driver, detected with UAS:lacZ. In
A-J, Cut expression (magenta) is used as an indicator of Notch activity
at the dorsal-ventral boundary; in K-T, -galactosidase was detected
histochemically.
2638 RESEARCH ARTICLE
Development 137 (16)
peaks of other muscle transcription factors exhibited less
association with Su(H)-bound regions (Fig. 5A and see Fig. S2C in
the supplementary material), suggesting that the association with
Twi is specific.
It is likely that Twi binding differs between cell types and
developmental stages. Therefore, although the embryonic data are
indicative of regions that have the potential to bind Twi, these
might not be identical to the occupied regions in Notch-activated
DmD8 cells. We therefore performed ChIP in the DmD8 cells and
hybridised the bound DNA to a whole-genome tiling array to
determine the sites of Twi occupancy. This identified ~2500 Twienriched regions (Twi peaks called at P<0.0001) in the DmD8
cells. Here, we focus on the relationship of the Twi peaks with
Su(H)-bound regions to investigate Notch and Twi co-regulation
and identify putative co-regulated genes. First, we found that
184/260 (71%) Su(H) peaks directly overlapped with Twi-bound
regions (Z-score>15.35, based on the constrained random model;
Fig. 5A and see Table S2 in the supplementary material). To avoid
bias due to differences in the average size of the peaks in the two
data sets, we also analysed the proximity of Twi and Su(H) peaks
using a midpoint-centred method (Fig. 5B,C). This approach
identified 181 (70%) Su(H) peaks with midpoints within 1 kb of
Twi peak midpoints (Fig. 5B) and 200 (76%) that were within 2 kb
of Twi peak midpoints (see Fig. S2 in the supplementary material).
Again, these are highly significant when compared with a similar
analysis using an Su(H) peak data set from another (Kc) cell type
(Fig. 5B; P<<10–16). Thus, our comparisons reveal a striking
association between Su(H) and Twi in DmD8 AMP-like cells and
we note also that the majority of potential Twi sites (identified by
motif-finding with PWM; see Fig. S3 in the supplementary
material) in proximity to the Su(H) peaks are occupied by Twi in
these cells (Fig. 5B).
Extrapolating the Twi peaks to the previously assigned direct
Notch-regulated target genes in DmD8 cells (Krejci et al., 2009)
revealed that 84% (197/235) of the assigned Notch targets had Twi
peaks in their vicinity (see Table S3 in the supplementary material).
These included E(spl)m6, aos and Him, as well as other targets
previously shown to be expressed in muscle progenitors in vivo
(Fig. 5C) (Krejci et al., 2009). To further confirm that Twi was
required for normal expression of the associated Notch targets, we
DEVELOPMENT
Fig 5. Genome-wide association of Twist and Su(H) binding. (A)Observed and expected random frequency of overlap between Su(H) peaks
(TF1) from DmD8 or Kc cells and the indicated binding regions of other transcription factors (TF2). TF2 binding regions were Twi in DmD8 (this
study), Twi, Mef2 and Biniou in embryos (Sandmann et al., 2006; Jakobsen et al., 2007; Sandmann et al., 2007) and a second Twi embryonic data
set generated with finer scale peak resolution (Twi_meso) (Zinzen et al., 2009). The Z-score expresses the divergence of the observed result from the
random model, with scores above three (blue) indicating significant divergence. In the random model, Su(H) sites were allowed to deviate by five
times the average peak width around the actual position, and differences in the number of TF2 peaks are internally controlled for in this
randomisation strategy. We note, however, that Su(H)_Kc peaks were narrower than those of Su(H)_DmD8, so the replacement parameters were
not directly comparable. (B)Pie charts representing the percentage of Su(H)_DmD8 peaks (left) or Su(H)_Kc peaks (right) that have midpoints within
1 kb of Twi_DmD8 peaks with (dark blue) and without (light blue) matches to the PWM, of Twi PWM matches (light green) or neither association
(grey). The midpoint analysis is independent of the peak width; peak numbers in Su(H)_DmD8 (260) and Su(H)_Kc (241) are similar. (C)Examples of
genomic regions from representative DmD8 Notch targets showing Su(H) (blue) and Twi (green) ChIP-enriched regions in DmD8 cells. Note that
oligonucleotides were spaced at 300 nucleotides in the tiling arrays for Su(H) and at 55 nucleotides for Twi. Gene models are depicted in black.
Coloured bars above represent the windows used to calculate peak overlaps using the midpoint method and a 1 kb window.
Fig 6. Depletion of Twi compromises target expression and Twi is
present at target enhancers prior to Notch activation. (A)Fold
change in the expression levels of the indicated genes after Notch
activation as determined by quantitative RT-PCR in the absence (light
grey) or presence (dark grey) of twi RNAi. The average of three
biological replicates is shown. (B)Expression of the indicated Notch
target genes in wild type and in muscle progenitors expressing twi
RNAi. (C,D)Enrichment of fragments from the indicated genes in antiTwi ChIP from DmD8 cells before Notch activation. T, Twi binding/
enhancer region; ORF, open reading frame. Results are the average of
three independent experiments; error bars indicate s.e.m.
RESEARCH ARTICLE 2639
Fig 7. Twist enhances Su(H) binding. (A)Enrichment of fragments
from the indicated genes in an anti-Su(H) ChIP from control (con, blue)
or Twi-expressing (+Twi, green) cells, before (light bars) or after (dark
bars) Notch activation (N act). S, Su(H) binding/enhancer region; O, ORF.
(B)mRNA levels of the indicated genes. Colours indicate conditions as
in A.
monitored the expression of four genes in twi RNAi-treated cells
(under conditions that reduced Twi by ~50%, as previously). All
showed a decrease in activation (e.g. Fig. 6A). We therefore
selected three further target genes and monitored the effects of twi
RNAi on their expression in vivo using available reporter genes or
antibodies (Fig. 6B). All three genes [edl (mae), roughest and
diminutive (myc)] were expressed in the muscle progenitors in
wild-type discs and had reduced expression in the presence of twi
RNAi, supporting the model that they are co-regulated by Twi.
The genome-wide results suggest, therefore, that Twi has a
widespread role in conferring the myogenic-specific response to
Notch. We further queried whether the organisation of the Su(H)
and Twi sites in the target enhancers had any conserved features
such as spacing or topology, as seen for the proneural and Su(H)
binding region in E(spl) genes (the A-SPS motif). However, we
found no evidence for a consistent motif organisation within the
identified regions. We note, however, that in the majority (71%) of
the assigned co-regulated targets, the distance between the Su(H)
and Twi peak midpoints is less than 500 bp (see Table S3 in the
supplementary material).
occupancy precedes recruitment of the Notch-Su(H) complex.
Similar results were obtained with an independent anti-Twi
antibody (see Fig. S4 in the supplementary material). We therefore
asked whether Twi alters the recruitment of Su(H) to target
enhancers. For this purpose, we created S2-Notch cells in which
twi expression is under the control of an inducible metallothionein
(MT) promoter. We then compared Su(H) binding enrichment in
the presence and absence of Twi and/or Notch activation (Fig. 7A).
As previously, we detected increased binding of Su(H) close to
E(spl)m3 after Notch activation in S2 cells. By contrast, no binding
was detected at the sites associated with E(spl)m6 or aos. Induction
of Twi alone had little effect on the Su(H) binding at these
locations in the absence of Notch. However, when we activated
Notch in cells in which Twi expression had been induced, we
detected a significant enrichment for Su(H) binding (Fig. 7A) and
an induction of cognate mRNA expression (Fig. 7B). aos
expression was increased above basal levels by Twi alone,
supporting the observation that Twi can bind to target enhancers in
the absence of Notch activation. As aos is associated with several
Twi peaks (Fig. 5C), its increased expression in the absence of
Notch might be explained by Twi recruitment at other regulatory
modules. As a further control, we examined E(spl)m8, which is a
Notch target in the proneural clusters, where it is regulated by
proneural bHLH proteins (Achaete and Scute). E(spl)m8 was not
activated in the Twi-expressing S2 cells and Su(H) binding was not
enriched (Fig. 7A,B). These results suggest, therefore, that Twi
enhances Su(H) binding to myogenic Notch target enhancers under
conditions in which Notch is also activated.
Twist facilitates Suppressor of Hairless binding to
Notch target gene enhancers
Twi has the characteristics to confer cell context on the Notch
response. We therefore first investigated whether it is present on
target enhancers prior to Notch activation. Fragments that
encompass Twi sites in E(spl)m6, Him and aos were enriched, as
compared with the cognate open reading frame, in a Twi ChIP from
unactivated DmD8 cells (Fig. 6C,D), suggesting that Twi
DISCUSSION
A major outcome of Notch activation resides in the transcriptional
programme elicited in a given cell type. However, little is known
about the factors that are required to confer such specificity. This is
primarily because most studies have focussed on a small number of
HES/E(spl) gene targets. Here, we have taken advantage of novel
Notch targets identified in a genome-wide study of muscle
progenitor-related cells to uncover a key component in specifying the
DEVELOPMENT
Twist confers specificity on Notch
Development 137 (16)
Notch response in this context: the bHLH transcription factor Twi.
Starting initially with a diverse set of Notch targets that are expressed
in AMPs, we found that Twi was crucial for their expression in these
cells. Furthermore, the enhancers tested in our ectopic assay were
only strongly induced when Twi was present in combination with
Notch activation. Indeed, an enhancer in which the Su(H) sites were
mutated was no longer able to respond to Twi, and vice versa,
demonstrating that their combined activities are necessary.
To determine whether the Twi co-regulation could be
extrapolated to a broad spectrum of Notch targets in muscle
progenitors, we assessed whether there was a significant
association between Su(H) and Twi binding in the muscle
progenitor-related DmD8 cells. Comparison of the binding regions
genome-wide revealed a strong association of Twi and Su(H)
among these targets: 71% of Su(H) peaks directly overlapped with
Twi peaks. This association was highly significant based on
random models that constrained the positions of peaks across the
genome to take account of the non-random distribution of
transcription factor binding sites and in comparison to several other
ChIP data sets. Expression of putative Notch-Twi targets in
myogenic precursors was dependent on Twi, as predicted by the
association. Together, these data indicate that one transcription
factor, Twi, has the potential to co-ordinate the expression of a
broad cross-section of Notch targets in muscle progenitors (84% of
previously assigned Notch targets are associated with a Twi peak)
and thus to confer a specific context on the Notch response.
Further evidence in support of the instructive role of Twi comes
from its ability to confer Notch responsiveness on some muscle
precursor targets when expressed in a heterologous cell line. Twi
itself was found to occupy sites on the target enhancers prior to
Notch activation, and in the heterologous cells it was accompanied
by increased Su(H) binding after Notch activation. This suggests that
Twi binding precedes Su(H) recruitment. However, the co-regulation
does not appear to require the Twi partner Da [the E47 (TCF3)
homologue], which was previously reported to contact Notch.
Furthermore, there does not appear to be any specific organisation or
spacing of Twi and Su(H) motifs among the co-regulated targets, in
contrast to the conserved motif, consisting of paired Su(H) sites
closely linked to an A-class bHLH binding site, that is thought to
underlie Notch-proneural bHLH synergy (Cave et al., 2005).
Nevertheless, in most of the co-regulated targets, the Su(H) and Twi
mid-peaks are separated by less than 500 bp, suggesting that the
interaction operates over a limited range. The Twi-Su(H) coregulation appears, therefore, more in keeping with models in which
binding sites for transcription factors are flexibly disposed and act
independently with targets in the basal transcriptional machinery [the
so-called ‘billboard’ model (Arnosti and Kulkarni, 2005)]. However,
mutation of a single Twi binding motif in the aos enhancer was
sufficient to compromise activity, despite the fact that there are
several matches to the Twi consensus site, suggesting that only a
subset of the possible binding motifs are crucial. In addition, as the
characterised Notch-Twi-dependent enhancers do not have identical
patterns of expression, it is likely that their activity is further
constrained by others factors. This is most evident for E(spl)m6,
which is only expressed in a small patch of the AMPs but
nevertheless responds very robustly to Twi and NICD.
genes regulated by the combination of Twi and Notch might
therefore be important for maintaining these cells as progenitors
with myogenic potential. Normally, Twi expression declines as the
muscles differentiate. Interfering with this regulation by persistent
expression of Twi or Notch inhibits the development of mature
fibres (Anant et al., 1998). Conversely, ablating Notch results in
premature differentiation (Krejci et al., 2009). The genes regulated
by the combination of Twi and Notch include those with proven
roles in myogenic regulation that are relevant to the maintenance
of muscle progenitors. These include twi itself, Him [an inhibitor
of Mef2 (Liotta et al., 2007)] and zfh1 [a repressor of myogenesis
(Postigo et al., 1999)]. As Notch signalling and Twi homologues
also inhibit vertebrate myogenic differentiation (Kopan et al., 1994;
Rohwedel et al., 1995; Spicer et al., 1996; Delfini et al., 2000;
Hirsinger et al., 2001; Schuster-Gossler et al., 2007; Vasyutina et
al., 2007), and overexpression of Twi in terminally differentiated
myotubes can induce reversal of cell differentiation (Hjiantoniou
et al., 2008), it will be interesting to test whether homologues of
the identified co-regulated targets of Notch and Twi are similarly
regulated.
The Notch-Twi combination might also be required to confer
properties on the adult myogenic precursors, such as differential
adhesion, migration and proliferation. Besides the genes with
proven roles in myogenic regulation, many of the Notch-Twi coregulated genes are implicated in morphogenesis. These include the
Ig-domain proteins Roughest, Kirre and Dscam (Bao and Cagan,
2005; Schmucker and Chen, 2009; Zhuang et al., 2009), the netrin
receptor Unc-5 and leucine repeat protein Capricious. Notch and
Twi have both been found to contribute to the regulation of the
epithelial-mesenchymal transition (EMT), and Twi is proposed to
affect malignant progression by inducing EMT and suppressing the
senescence response (Ansieau et al., 2008). Therefore, it is possible
that the co-regulated genes might also confer specialised
behaviours that are required in the adult precursors and in cells
undergoing EMT (Huber et al., 2005).
In conclusion, we find that Notch and Twi potentially coregulate a broad spectrum of genes required for the maintenance of
muscle progenitors. This suggests that a single co-regulatory
relationship can account for a significant component (>170 genes)
of the Notch output in one cell type.
What are the likely characteristics conferred on
cells by the Twi-Notch combination?
The AMPs have the capacity for self-renewal and are not
committed to a particular muscle lineage, characteristics similar to
those of mammalian muscle satellite cells (Figeac et al., 2007). The
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Acknowledgements
We are particularly grateful to Maria Leptin for the Twi antibody used for the
genome-wide ChIP. We also thank Mary Baylies, Karl Fishbach, Eileen Furlong,
Alexis Lalouette, Gines Morata and Joel Silber for generously providing fly
stocks or antibodies and Rob White and members of our groups for helpful
discussions. This work was supported by programme and project grants to S.B.
from the Medical Research Council and by a grant from the Association for
International Cancer Research. A.K. and F.B. were European Molecular Biology
Organization long-term fellows. B.A. is a Royal Society University Research
Fellow and acknowledges the Wellcome Trust for supporting the development
of computational methods used in this study. Deposited in PMC for release
after 6 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.053181/-/DC1
DEVELOPMENT
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