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 expression through the Suppressor of Hairless [Su(H)] DNA-binding . 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 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 . 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.

KEY WORDS: Drosophila, Gene regulation, Myogenesis, Notch, Twist

INTRODUCTION co-regulators that confer such specificity on the Notch response is Signalling through the Notch receptor controls a broad spectrum of of primary importance for understanding the context-specific cell fates and developmental processes in organisms ranging from responses. sponges to human and is associated with various pathologies To date only a few cell type-specific transcription factors have including different types of cancer (Radtke and Raj, 2003; Lasky been shown to combine with the Notch pathway to regulate and Wu, 2005; van Es et al., 2005). This multitude of roles is induction of specific targets. These include GATA-like factors, associated with different outputs, depending upon the cellular which act in combination with Notch to regulate ref-1 in the C. context. For example, in certain contexts Notch activation promotes elegans endoderm (Neves et al., 2007), and NFB family proliferation and maintenance of progenitor populations [e.g. members, which co-regulate HES and deltex 1, which are both myogenesis (Kopan et al., 1994; Delfini et al., 2000; Hirsinger et Notch targets during B-lymphocyte development (Moran et al., al., 2001)], whereas in others it promotes cell type differentiation 2007). The best-characterised co-regulators are the proneural [e.g. T-cell formation (Robey, 1999) or neural fate (Gaiano and bHLH transcription factors that combine with Notch to activate Fishell, 2002)]. Transduction of the Notch pathway is relatively targets of the E(spl)/HES gene family during neurogenesis in a simple: activation of Notch elicits proteolytic cleavage, releasing wide range of species (Nakao and Campos-Ortega, 1996; Parras et the Notch intracellular domain (NICD), which enters the nucleus al., 1996; Culi and Modolell, 1998). Proneural co-regulation is and collaborates directly with DNA-binding of the CSL proposed to depend on a particular binding site architecture, family {CBF1 (RBPJ) in mammals, Suppressor of Hairless [Su(H)] consisting of paired CSL binding sites and a proneural protein in Drosophila and LAG-1 in worms (Bray, 2006; Kopan and binding site (Cave et al., 2005). As this code has been derived from Ilagan, 2009)}. Effects on are thus a primary the evolutionarily related genes of the E(spl) family it is not clear consequence of activating the pathway, implying that the different whether such specific binding site architecture extends more outputs of Notch activation require a selective transcriptional broadly to other Notch targets, nor whether other co-regulators response. This is highlighted by the observations that the sites of would involve similar binding site organisation at their targets. Su(H) occupancy after Notch activation differ according to cell Furthermore, in none of the partnerships analysed, including that type (Krejci and Bray, 2007) and that only a fraction (slightly more involving proneural proteins, is it clear whether the regulatory than 10%) of the potential Notch-regulated targets are shared relationship is specific for the targets analysed or whether it extends between two cell types (Krejci et al., 2009). The identification of 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 1 Department of Physiology, Development and Neuroscience, University of to identify direct Notch targets in different Drosophila cell lines, Cambridge, Downing Street, Cambridge CB2 3DY, UK. 2Cambridge Systems Biology Centre and Department of Genetics, University of Cambridge, Tennis Court Road, including DmD8 cells, related to adult muscle progenitors (AMPs) Cambridge CB2 1QR, UK. (Krejci et al., 2009). In this context, Notch is required to maintain the progenitors and prevent their differentiation, a function that appears *Author for correspondence ([email protected]) similar to the role of Notch in vertebrate myogenesis, in which

Accepted 27 May 2010 activation of Notch signalling prevents muscle differentiation both DEVELOPMENT 2634 RESEARCH ARTICLE Development 137 (16) in embryonic precursors and in satellite cells (Delfini et al., 2000; [1:500; gift of F. Martin and G. Morata (Herranz et al., 2008)], rabbit Hirsinger et al., 2001; Conboy and Rando, 2002; Kopan and Ilagan, anti-Roughest [1/50; gift of K. Fishbach (Schneider et al., 1995)]. 2009). Among the 235 direct Notch targets identified in DmD8 -galactosidase expression was detected histochemically. muscle progenitor-like cells, several have been specifically linked to Cell experiments and lacZ reporters roles in muscle development. However, many others are involved in E(spl)m3, NME (–ve control) and aosNRE luciferase reporters have been fundamental aspects of cell differentiation such as signalling and described (Krejci et al., 2009). A fragment encompassing the region –2098 morphogenesis (Krejci et al., 2009). In addressing how the genome to +37 of E(spl)m6 (Nellesen et al., 1999) was amplified and cloned into a integrates Notch activation differentially according to cell type, one luciferase vector containing a minimal from Hsp70 (pGL3::Min). question therefore is whether one or many transcription factors are Twi sites were mutated using oligonucleotides with three mismatches (introducing T at positions 3, 4 and 8) as described (Krejci et al., 2009). After required to specify this diverse cohort of targets and to confer an NRE output that is distinct for myogenic precursors. A second question is mutagenesis, aos fragments were cloned into either pGL3::Min or pBlueRabbit (which contains lacZ downstream of a minimal Hsp70 how these factors affect CSL binding. promoter). Cell culture conditions and transfections were as described Several key transcription factors are known to have important previously (Nagel et al., 2005; Narasimha et al., 2008). To activate Notch, roles in regulating myogenesis. These include the bHLH proteins cells were treated with EDTA as described (Krejci and Bray, 2007). For twi of the MyoD/ family and the MADS box proteins of the RNAi treatment of DmD8 cells, double-stranded RNA duplexes were family (Parker et al., 2003; Tajbakhsh, 2003). The latter are produced from a 750 bp fragment of the twi exon with T7 promoter- important in promoting muscle differentiation in all species (Black containing primers and subsequently transcribed using a T7 Transcription Kit and Olson, 1998). In Drosophila, the bHLH protein Twist (Twi) (Ambion). Cells were incubated with twi RNAi for 72 hours before Notch acts prior to Mef2 in both embryonic and adult myogenesis (Cripps activation. Three biological replicates were performed in all experiments. RNA isolation, quantitative PCR and ChIP experiments were performed et al., 1998; Baylies and Michelson, 2001; Castanon and Baylies, as described (Krejci and Bray, 2007). Antibodies for ChIP were goat anti- 2002). Initially, Twi is expressed broadly throughout the embryonic Su(H) (Santa Cruz Biotechnology, sc15813) and rabbit anti-Twi [from M. mesoderm and subsequently it regulates whether or not the cells Leptin and S. Roth (Roth et al., 1989); or from E. Furlong (Sandmann et are routed into somatic muscle fates. Its expression is then lost in al., 2007)]. larval muscles when they differentiate (Baylies and Bate, 1996), but persists in adult muscle precursors (Bate et al., 1991), only Bioinformatics and Twi genome-wide ChIP in DmD8 cells Data sets declining during pupation when the myoblasts fuse and ChIP-enriched regions in DmD8 cells were determined de novo using differentiate into the muscles of the adult (Anant et al., 1998). As Nimblegen HD2 tiling arrays following the procedures and post-hybridisation both the Notch pathway and Twi are important in maintaining analysis described previously for Su(H) (Krejci et al., 2009). The array AMPs in an undifferentiated state (Anant et al., 1998), Twi is a data have been deposited at NCBI Gene Expression Omnibus (GEO, potential candidate to co-operate with Notch in muscle precursors. http://www.ncbi.nlm.nih.gov/geo/) with accession number GSE22650. Peaks Taking advantage of the Notch targets identified in DmD8 cells, were defined using Tamalpais (Bieda et al., 2006), with P<0.0001. ChIP- we have investigated how specificity is conferred on the Notch enriched regions for Su(H) (292), Twi (2512), Mef2 (1464) or Biniou (292) response in the myogenic cell type. Focussing initially on three in embryos were taken from previously published experiments (Sandmann et al., 2006; Jakobsen et al., 2007; Sandmann et al., 2007; Krejci et al., 2009; targets, we find that their expression in vivo is dependent on Twi Zinzen et al., 2009). In the case of Biniou, Twi and Mef2, for which analysis acting in combination with Notch. Twi binding to target enhancers of binding had been performed at multiple time-points (Sandmann et al., precedes Su(H) recruitment, which is detected only after activation. 2006; Jakobsen et al., 2007; Sandmann et al., 2007; Zinzen et al., 2009), a To assess whether Twi is likely to confer specificity on a broader non-redundant data set was generated with all the regions that were occupied range of targets, we analysed whether Twi binding regions in at least one stage. Where necessary, the UCSC Genome Browser (Kuhn et identified genome-wide in the DmD8 myogenic precursor-related al., 2009) Liftover tool was used to transpose all genomic positions to cells are found in proximity to the Su(H) binding regions identified coordinates of release 5 of the D. melanogaster genome. in the same cells. This revealed a significant association and Overlap identified more than 170 genes that are potentially co-regulated by Custom-written Perl scripts were used to determine the overlap between both factors. These results demonstrate that one co-factor, Twi, is ChIP-enriched regions and their statistical significance. The significance likely to regulate on a large scale the output from the Notch of overlap was determined by 1000 rounds of random simulation. In each pathway in muscle progenitor cells. round, every Su(H) site was allowed to deviate around its original position (Fig. 1A and see Table S2 in the supplementary material; uniform MATERIALS AND METHODS distribution within a range around the original site). The overlap between Drosophila experiments random Su(H) and Twi sites was determined in each round and recorded All alleles and stocks are described in FlyBase unless indicated otherwise. to derive a Z-score [(actual overlap – average random overlap) / standard The following reporters were used: m6-GFP (Lai et al., 2000); 4.0Him- deviation of random overlap] and empirical P-value (0.001 increment for GFP (Rebeiz et al., 2002); aosNRE-lacZ, aosNREmutSu(H)-lacZ, edlNRE-lacZ each random overlap ≥ actual overlap). (Krejci et al., 2009); and m8-lacZ (Kramatschek and Campos-Ortega, Position weight matrix (PWM) matches 1994). In overexpression and RNAi experiments, Gal4 driver stocks Alignment matrices for Su(H) (Krejci et al., 2009), Twi (Down et al., 2007) [1151:Gal4 (Anant et al., 1998), Ptc:Gal4 Tub:Gal80ts, sca:Gal4] were and Mef2 were built based on a compilation of previously published binding combined with UAS lines [UAS:twi (Baylies and Bate, 1996), UAS-Ni79.2] sites (see Fig. S3 in the supplementary material). The motif scanner nmscan and/or RNAi lines [UAS-Notch-RNAi (BL7078), UAS-twi-RNAi from the NestedMICA package (Down and Hubbard, 2005) was used for (GD37092), UAS-da-RNAi (GD51297) (Dietzl et al., 2007), UAS-twi- genome-wide motif matching, with empirically defined cut-off values for RNAi2x (Wong et al., 2008)] and larvae were shifted to 30°C 48 hours each (roughly yielding the same number of genomic before dissections. Immunofluorescence was performed as described binding sites). For sites shown in Fig. 1, matches to PWMs were computed previously (Cooper and Bray, 1999) with the following antibodies: mouse using Patser (Hertz and Stormo, 1999) with a threshold of 5.5 (the anti-NICD (DSHB, 1/20), mouse anti-Cut (DSHB, 1/20), mouse anti--gal approximate cut-off used above is indicated on the resulting graph by a

(DSHB, 1/20), rabbit anti-GFP (Invitrogen, 1/1000), guinea-pig anti- dashed line). DEVELOPMENT Twist confers specificity on Notch RESEARCH ARTICLE 2635

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.

RESULTS that we have tested (Krejci and Bray, 2007). The resulting reporter Functional relevance of Twist for Notch target genes were co-transfected into DmD8 muscle progenitor type cells gene expression and into S2 blood-related cells. E(spl)m6 and aos enhancer We first focussed our investigations on a selection of DmD8 Notch fragments both recapitulated the cell specificity, responding to targets with distinct cellular functions: E(spl)m6, an immediate NICD much more robustly in DmD8 cells (Fig. 1D). The early Notch target involved in regulating Notch pathway activity magnitude of the response was larger for aosS than for the longer (Bardin and Schweisguth, 2006); argos (aos), an inhibitor of EGF aosL, suggesting that the latter can recruit factors that antagonise receptor signalling (Howes et al., 1998); and Him, a repressor that NICD or that affect accessibility. Nevertheless, the results suggest inhibits Mef2 (Liotta et al., 2007). The first question we addressed that the enhancer fragments are sufficient to confer a context- was whether the cell-context specificity was retained in enhancer specific Notch response that is dependent on factors present in fragments isolated from two of these target genes, by testing DmD8 cells. whether the enhancers responded differently to NICD according to Transcription factors that might contribute to the specificity of cell type. Fragments encompassing Su(H) ChIP peak regions from Notch in DmD8 cells and myogenic precursors include Twi and E(spl)m6 and aos (Krejci et al., 2009) (see Fig. 1A,B) were tested Mef2 (Castanon and Baylies, 2002; Cripps et al., 2004; Lovato et

in comparison to E(spl)m3, which is inducible by Notch in all cells al., 2005). RNA sequencing profiles have shown that DmD8 cells DEVELOPMENT 2636 RESEARCH ARTICLE Development 137 (16) 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 Twi- dependent 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 Fig 2. Twist regulation of Notch target enhancers in muscle expression of da RNAi in proneural clusters with sca-Gal4 did progenitors. (A-C)Effect of two independent twi RNAi constructs alter the expression of E(spl)m8 as expected] (see Fig. S1 in the (GD37092, B; twi-RNAi2x, C) on E(spl)m6-GFP expression (green in supplementary material). Likewise, no change in E(spl)m6-GFP A,B,C; white in AЈ). A patch of E(spl)m6-GFP is detectable in wild type expression was seen with RNAi targeting nine other bHLH genes in Cut-expressing adult muscle progenitors (AMPs) (A, arrow; inset) but (see Table S1 in the supplementary material). Thus, the conditional is severely reduced (B, arrow) or abolished (C, arrow) in the presence of knockdown of twi, but not da or other bHLH transcription factors, twi RNAi. Cut expression (which marks all muscle progenitors; purple, is sufficient to prevent expression from E(spl)m6, aos and Him 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 enhancers. (E,G). Expression was monitored using the indicated reporters, detected by antibodies (A-E) or histochemistry (F,G), and colocalisation with Twist and Notch co-operate to activate target AMPs was verified by co-staining with Cut (A-C) or by interference genes microscopy (F,G). 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 boundary (e.g. Fig. 3B,G), neither E(spl)m6-GFP, 4.0Him-GFP nor whether it requires collaboration with Notch. To address this aosNRE-lacZ exhibited any ectopic activation (Fig. 3B,G,L). By question, we assessed the ability of Twi to confer appropriate Notch contrast, expression of Twi using the same driver elicited ectopic responsiveness when expressed in a different cell type in the expression from all three enhancers (Fig. 3C,H,M), giving strong presence and absence of Notch. Genes such as E(spl)m6 or Him are induction of 4.0Him-GFP and weak induction of E(spl)m6-GFP and normally only expressed in AMPs, not elsewhere in the wing disc, aosNRE-lacZ. Their ectopic induction indicates that the enhancers are despite Notch activity at other locations. Similarly, expression from capable of responding to Twi. This response is highly selective, as enhancers identified by Su(H) binding in DmD8 cells is also increased or ectopic expression of 20 other bHLH proteins, restricted to muscle progenitors (e.g. aos). We therefore investigated including proneural proteins, failed to elicit any change in E(spl)m6 whether Notch and/or Twi were able to activate these enhancers expression (see Table S1 in the supplementary material). when expressed ectopically in the wing pouch using ptc-Gal4 (Fig. Combining expression of Twi with NICD yielded much stronger 3T) in combination with thermosensitive Gal80, so that expression expression from all three enhancers in the wing pouch than was limited to a defined period at the end of larval development. expression of Twi alone (Fig. 3D,I,N). These results suggest that First we assessed whether simply increasing Notch activity in the Notch and Twi co-operate to induce expression of these genes. To wing pouch domain would activate the muscle progenitor further confirm whether this co-operation was essential, we also enhancers. Although this was sufficient to produce ectopic tested the consequences of ablating Notch activity when Twi was

expression of genes that are direct Notch targets at the dorsal-ventral ectopically expressed. All three enhancers showed substantially DEVELOPMENT 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).

Fig. 3. Twist acts in combination with Notch to regulate myogenic target enhancers. (A-S)Expression from reporters mutated putative Twi binding sites in one of the responsive NRE E(spl)m6-GFP (green, A-E), 4.0Him-GFP (green, F-J), aosNRE-lacZ (grey, enhancers. aos contains three matches to a position weight K-O) or aosNRESu(H)mut-lacZ (grey, P-S) in wild type (A,F,K,P), and in discs matrix (PWM) for Twi binding sites (see Fig. 1A), with sites 2 and where the levels of Twi and Notch activity were manipulated in a stripe 3 having the best matches. We generated fragments with mutations (arrow, ptc-Gal4;Tub-Gal80ts; see T) by NICD expression (>NICD; in site 2 (aosNRE⌬Twi2) and with mutations in both site 2 and the B,G,L,Q), by Twi overexpression (>Twi; C,H,M,R), by combining NICD adjacent site 3 (aosNRE⌬Twi2,3). When tested in DmD8 cells, both and Twi overexpression (>Twi;NICD; D,I,N,S), or by ablating Notch (with enhancers had lost their ability to respond to Notch activation, Notch RNAi) in cells overexpressing Twi (>Twi;N-RNAi; E,J,O). (T)The suggesting that these sites are essential (Fig. 4A). Similarly, when expression domain of the Ptc:Gal4 driver, detected with UAS:lacZ. In tested in vivo, the mutated fragments had lost the ability to direct A-J, Cut expression (magenta) is used as an indicator of Notch activity at the dorsal-ventral boundary; in K-T, -galactosidase was detected expression in AMPs (Fig. 4B,C). Thus, Twi binding sites are  NRE histochemically. necessary for aos activity in DmD8 cells and in vivo in myogenic precursors. reduced expression when Twi was expressed in combination with Genome-wide association of Twist and Suppressor Notch RNAi (Fig. 3E,J,O), as compared with Twi alone, and two of Hairless binding of the three had no residual ectopic expression. This indicates that As the tested enhancers all require Twi for their responsiveness to the expression induced by Twi alone is largely dependent on an Notch, this regulatory combination might be required for the underlying activity of Notch [sensitive Notch reporters reveal a low majority of Notch targets in DmD8 cells and muscle progenitors. level of Notch activity in most cells of the wing pouch (Furriols If this were the case, there should be a strong association of Su(H) and Bray, 2001)]. The dependence on Notch was further confirmed and Twi sites among the DmD8 target loci. To test this possibility, by analysing expression from an aos enhancer in which the Su(H) we first used data from previously published genome-wide ChIP binding sites had been mutated. This mutated aosNREmutSu(H) experiments performed in embryos (Sandmann et al., 2006; enhancer is no longer expressed in muscle progenitors (Krejci et Jakobsen et al., 2007; Sandmann et al., 2007; Zinzen et al., 2009). al., 2009) and was not able to respond to ectopic Twi expression or Using two approaches (Fig. 5A and see Fig. S2 in the to the combination of Twi and NICD (Fig. 3P-S). Together, these supplementary material), we found a significant association results show that Twi confers the ability on these target enhancers between Su(H) and Twi binding regions (based on a null model to respond to Notch activity, and vice versa. that constrained the position of the peaks to take account of their non-random distribution across the genome; see Materials and Twist binding sites are required for the activity of methods and Table S2 in the supplementary material). Furthermore, Notch target gene enhancers when the partner combinations between Su(H) and several other Our results point to a pivotal role for Twi in the context-specific transcription factors were calculated on the basis of the embryonic Notch response, but do not distinguish whether this effect is direct. data, there was a strong pair-wise correlation of Su(H) with Twi

In order to test whether direct binding by Twi is required, we (see Fig. S2B in the supplementary material, red box) and ChIP DEVELOPMENT 2638 RESEARCH ARTICLE Development 137 (16)

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. peaks of other muscle transcription factors exhibited less using a midpoint-centred method (Fig. 5B,C). This approach association with Su(H)-bound regions (Fig. 5A and see Fig. S2C in identified 181 (70%) Su(H) peaks with midpoints within 1 kb of the supplementary material), suggesting that the association with Twi peak midpoints (Fig. 5B) and 200 (76%) that were within 2 kb Twi is specific. of Twi peak midpoints (see Fig. S2 in the supplementary material). It is likely that Twi binding differs between cell types and Again, these are highly significant when compared with a similar developmental stages. Therefore, although the embryonic data are analysis using an Su(H) peak data set from another (Kc) cell type indicative of regions that have the potential to bind Twi, these (Fig. 5B; P<<10–16). Thus, our comparisons reveal a striking might not be identical to the occupied regions in Notch-activated association between Su(H) and Twi in DmD8 AMP-like cells and DmD8 cells. We therefore performed ChIP in the DmD8 cells and we note also that the majority of potential Twi sites (identified by hybridised the bound DNA to a whole-genome tiling array to motif-finding with PWM; see Fig. S3 in the supplementary determine the sites of Twi occupancy. This identified ~2500 Twi- material) in proximity to the Su(H) peaks are occupied by Twi in enriched regions (Twi peaks called at P<0.0001) in the DmD8 these cells (Fig. 5B). cells. Here, we focus on the relationship of the Twi peaks with Extrapolating the Twi peaks to the previously assigned direct Su(H)-bound regions to investigate Notch and Twi co-regulation Notch-regulated target genes in DmD8 cells (Krejci et al., 2009) and identify putative co-regulated genes. First, we found that revealed that 84% (197/235) of the assigned Notch targets had Twi 184/260 (71%) Su(H) peaks directly overlapped with Twi-bound peaks in their vicinity (see Table S3 in the supplementary material). regions (Z-score>15.35, based on the constrained random model; These included E(spl)m6, aos and Him, as well as other targets Fig. 5A and see Table S2 in the supplementary material). To avoid previously shown to be expressed in muscle progenitors in vivo bias due to differences in the average size of the peaks in the two (Fig. 5C) (Krejci et al., 2009). To further confirm that Twi was

data sets, we also analysed the proximity of Twi and Su(H) peaks required for normal expression of the associated Notch targets, we DEVELOPMENT Twist confers specificity on Notch 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. Fig 6. Depletion of Twi compromises target expression and Twi is (B)mRNA levels of the indicated genes. Colours indicate conditions as present at target enhancers prior to Notch activation. (A)Fold in A. 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 occupancy precedes recruitment of the Notch-Su(H) complex. target genes in wild type and in muscle progenitors expressing twi Similar results were obtained with an independent anti-Twi RNAi. (C,D)Enrichment of fragments from the indicated genes in anti- antibody (see Fig. S4 in the supplementary material). We therefore Twi ChIP from DmD8 cells before Notch activation. T, Twi binding/ asked whether Twi alters the recruitment of Su(H) to target enhancer region; ORF, open reading frame. Results are the average of enhancers. For this purpose, we created S2-Notch cells in which three independent experiments; error bars indicate s.e.m. 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). monitored the expression of four genes in twi RNAi-treated cells As previously, we detected increased binding of Su(H) close to (under conditions that reduced Twi by ~50%, as previously). All E(spl)m3 after Notch activation in S2 cells. By contrast, no binding showed a decrease in activation (e.g. Fig. 6A). We therefore was detected at the sites associated with E(spl)m6 or aos. Induction selected three further target genes and monitored the effects of twi of Twi alone had little effect on the Su(H) binding at these RNAi on their expression in vivo using available reporter genes or locations in the absence of Notch. However, when we activated antibodies (Fig. 6B). All three genes [edl (mae), roughest and Notch in cells in which Twi expression had been induced, we diminutive (myc)] were expressed in the muscle progenitors in detected a significant enrichment for Su(H) binding (Fig. 7A) and wild-type discs and had reduced expression in the presence of twi an induction of cognate mRNA expression (Fig. 7B). aos RNAi, supporting the model that they are co-regulated by Twi. expression was increased above basal levels by Twi alone, The genome-wide results suggest, therefore, that Twi has a supporting the observation that Twi can bind to target enhancers in widespread role in conferring the myogenic-specific response to the absence of Notch activation. As aos is associated with several Notch. We further queried whether the organisation of the Su(H) Twi peaks (Fig. 5C), its increased expression in the absence of and Twi sites in the target enhancers had any conserved features Notch might be explained by Twi recruitment at other regulatory such as spacing or topology, as seen for the proneural and Su(H) modules. As a further control, we examined E(spl)m8, which is a binding region in E(spl) genes (the A-SPS motif). However, we Notch target in the proneural clusters, where it is regulated by found no evidence for a consistent motif organisation within the proneural bHLH proteins (Achaete and Scute). E(spl)m8 was not identified regions. We note, however, that in the majority (71%) of activated in the Twi-expressing S2 cells and Su(H) binding was not the assigned co-regulated targets, the distance between the Su(H) enriched (Fig. 7A,B). These results suggest, therefore, that Twi and Twi peak midpoints is less than 500 bp (see Table S3 in the enhances Su(H) binding to myogenic Notch target enhancers under supplementary material). conditions in which Notch is also activated.

Twist facilitates Suppressor of Hairless binding to DISCUSSION Notch target gene enhancers A major outcome of Notch activation resides in the transcriptional Twi has the characteristics to confer cell context on the Notch programme elicited in a given cell type. However, little is known response. We therefore first investigated whether it is present on about the factors that are required to confer such specificity. This is target enhancers prior to Notch activation. Fragments that primarily because most studies have focussed on a small number of encompass Twi sites in E(spl)m6, Him and aos were enriched, as HES/E(spl) gene targets. Here, we have taken advantage of novel compared with the cognate open reading frame, in a Twi ChIP from Notch targets identified in a genome-wide study of muscle

unactivated DmD8 cells (Fig. 6C,D), suggesting that Twi progenitor-related cells to uncover a key component in specifying the DEVELOPMENT 2640 RESEARCH ARTICLE Development 137 (16)

Notch response in this context: the bHLH transcription factor Twi. genes regulated by the combination of Twi and Notch might Starting initially with a diverse set of Notch targets that are expressed therefore be important for maintaining these cells as progenitors in AMPs, we found that Twi was crucial for their expression in these with myogenic potential. Normally, Twi expression declines as the cells. Furthermore, the enhancers tested in our ectopic assay were muscles differentiate. Interfering with this regulation by persistent only strongly induced when Twi was present in combination with expression of Twi or Notch inhibits the development of mature Notch activation. Indeed, an enhancer in which the Su(H) sites were fibres (Anant et al., 1998). Conversely, ablating Notch results in mutated was no longer able to respond to Twi, and vice versa, premature differentiation (Krejci et al., 2009). The genes regulated demonstrating that their combined activities are necessary. by the combination of Twi and Notch include those with proven To determine whether the Twi co-regulation could be roles in myogenic regulation that are relevant to the maintenance extrapolated to a broad spectrum of Notch targets in muscle of muscle progenitors. These include twi itself, Him [an inhibitor progenitors, we assessed whether there was a significant of Mef2 (Liotta et al., 2007)] and zfh1 [a repressor of myogenesis association between Su(H) and Twi binding in the muscle (Postigo et al., 1999)]. As Notch signalling and Twi homologues progenitor-related DmD8 cells. Comparison of the binding regions also inhibit vertebrate myogenic differentiation (Kopan et al., 1994; genome-wide revealed a strong association of Twi and Su(H) Rohwedel et al., 1995; Spicer et al., 1996; Delfini et al., 2000; among these targets: 71% of Su(H) peaks directly overlapped with Hirsinger et al., 2001; Schuster-Gossler et al., 2007; Vasyutina et Twi peaks. This association was highly significant based on al., 2007), and overexpression of Twi in terminally differentiated random models that constrained the positions of peaks across the myotubes can induce reversal of cell differentiation (Hjiantoniou genome to take account of the non-random distribution of et al., 2008), it will be interesting to test whether homologues of transcription factor binding sites and in comparison to several other the identified co-regulated targets of Notch and Twi are similarly ChIP data sets. Expression of putative Notch-Twi targets in regulated. myogenic precursors was dependent on Twi, as predicted by the The Notch-Twi combination might also be required to confer association. Together, these data indicate that one transcription properties on the adult myogenic precursors, such as differential factor, Twi, has the potential to co-ordinate the expression of a adhesion, migration and proliferation. Besides the genes with broad cross-section of Notch targets in muscle progenitors (84% of proven roles in myogenic regulation, many of the Notch-Twi co- previously assigned Notch targets are associated with a Twi peak) regulated genes are implicated in morphogenesis. These include the and thus to confer a specific context on the Notch response. Ig-domain proteins Roughest, Kirre and Dscam (Bao and Cagan, Further evidence in support of the instructive role of Twi comes 2005; Schmucker and Chen, 2009; Zhuang et al., 2009), the netrin from its ability to confer Notch responsiveness on some muscle receptor Unc-5 and leucine repeat protein Capricious. Notch and precursor targets when expressed in a heterologous cell line. Twi Twi have both been found to contribute to the regulation of the itself was found to occupy sites on the target enhancers prior to epithelial-mesenchymal transition (EMT), and Twi is proposed to Notch activation, and in the heterologous cells it was accompanied affect malignant progression by inducing EMT and suppressing the by increased Su(H) binding after Notch activation. This suggests that senescence response (Ansieau et al., 2008). Therefore, it is possible Twi binding precedes Su(H) recruitment. However, the co-regulation that the co-regulated genes might also confer specialised does not appear to require the Twi partner Da [the E47 (TCF3) behaviours that are required in the adult precursors and in cells homologue], which was previously reported to contact Notch. undergoing EMT (Huber et al., 2005). Furthermore, there does not appear to be any specific organisation or In conclusion, we find that Notch and Twi potentially co- spacing of Twi and Su(H) motifs among the co-regulated targets, in regulate a broad spectrum of genes required for the maintenance of contrast to the conserved motif, consisting of paired Su(H) sites muscle progenitors. This suggests that a single co-regulatory closely linked to an A-class bHLH binding site, that is thought to relationship can account for a significant component (>170 genes) underlie Notch-proneural bHLH synergy (Cave et al., 2005). of the Notch output in one cell type. 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 Acknowledgements We are particularly grateful to Maria Leptin for the Twi antibody used for the interaction operates over a limited range. The Twi-Su(H) co- genome-wide ChIP. We also thank Mary Baylies, Karl Fishbach, Eileen Furlong, regulation appears, therefore, more in keeping with models in which Alexis Lalouette, Gines Morata and Joel Silber for generously providing fly binding sites for transcription factors are flexibly disposed and act stocks or antibodies and Rob White and members of our groups for helpful independently with targets in the basal transcriptional machinery [the 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 so-called ‘billboard’ model (Arnosti and Kulkarni, 2005)]. However, International Cancer Research. A.K. and F.B. were European Molecular Biology mutation of a single Twi binding motif in the aos enhancer was Organization long-term fellows. B.A. is a Royal Society University Research sufficient to compromise activity, despite the fact that there are Fellow and acknowledges the Wellcome Trust for supporting the development several matches to the Twi consensus site, suggesting that only a of computational methods used in this study. Deposited in PMC for release subset of the possible binding motifs are crucial. In addition, as the after 6 months. characterised Notch-Twi-dependent enhancers do not have identical Competing interests statement patterns of expression, it is likely that their activity is further The authors declare no competing financial interests. constrained by others factors. This is most evident for E(spl)m6, which is only expressed in a small patch of the AMPs but Supplementary material Supplementary material for this article is available at nevertheless responds very robustly to Twi and NICD. http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.053181/-/DC1

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