- Research article
- Open Access
The MAP kinase ERK and its scaffold protein MP1 interact with the chromatin regulator Corto during Drosophilawing tissue development
© Mouchel-Vielh et al; licensee BioMed Central Ltd. 2011
- Received: 26 August 2010
- Accepted: 14 March 2011
- Published: 14 March 2011
Mitogen-activated protein kinase (MAPK) cascades (p38, JNK, ERK pathways) are involved in cell fate acquisition during development. These kinase modules are associated with scaffold proteins that control their activity. In Drosophila, dMP1, that encodes an ERK scaffold protein, regulates ERK signaling during wing development and contributes to intervein and vein cell differentiation. Functional relationships during wing development between a chromatin regulator, the Enhancer of Trithorax and Polycomb Corto, ERK and its scaffold protein dMP1, are examined here.
Genetic interactions show that corto and dMP1 act together to antagonize rolled (which encodes ERK) in the future intervein cells, thus promoting intervein fate. Although Corto, ERK and dMP1 are present in both cytoplasmic and nucleus compartments, they interact exclusively in nucleus extracts. Furthermore, Corto, ERK and dMP1 co-localize on several sites on polytene chromosomes, suggesting that they regulate gene expression directly on chromatin. Finally, Corto is phosphorylated. Interestingly, its phosphorylation pattern differs between cytoplasm and nucleus and changes upon ERK activation.
Our data therefore suggest that the Enhancer of Trithorax and Polycomb Corto could participate in regulating vein and intervein genes during wing tissue development in response to ERK signaling.
- Extracellular Regulate Kinase Signaling
- Polytene Chromosome
- Extracellular Regulate Kinase
- Gal4 Driver
- Extracellular Regulate Kinase Signaling Pathway
The mitogen-activated protein kinase (MAPK) pathways are evolutionary conserved signaling pathways used by eukaryotic cells to regulate gene expression during diverse processes such as proliferation, differentiation, apoptosis, adaptation to changes in their environment, and so on (for a review, see ). MAPK proteins are serine-threonine kinases that can phoshorylate targets in the cytoplasm or the nucleus in response to stimuli such as mitogenic or stress signals. MAPKs can be grouped into three classes depending on the stimuli they respond to. Extracellular regulated kinases (ERK) are mainly activated by mitogenic stimuli such as growth factors and hormones, whereas c-Jun N-terminal kinases (JNK) and p38 kinases respond predominantly to stress stimuli. Kinases associate with scaffold proteins that regulate signaling by providing critical spatial and temporal specificities. Notably, the scaffold protein MP1 forms a signaling complex with MEK and ERK thus facilitating ERK activation [2, 3]. One of the best characterized mechanisms by which MAPKs regulate gene expression involves phosphorylation of transcription factors, which consequently modifies their activity, regulating either their intracellular location, their stability, their binding to DNA, or their interactions with regulatory proteins (for a review, see ).
Although the traditional view has been that most phosphorylation events do not occur directly at promoters of genes that are ultimately controlled by MAPK pathways, recent reports have highlighted some cases where MAPKs are integral components of transcriptional activation complexes. For example, during mammalian myoblast differentiation, p38 is recruited to the promoters of myogenic genes together with the muscle-regulatory factors MyoD and MEF2C . In pancreatic β-cells, in response to increased glucose concentration, ERK1/2 MAPKs are bound to the insulin gene promoter in the same complex as their transcription factor substrates MafA, Beta2 and PDX-1 . In yeast, the p38-related Hog1 kinase coordinates the transcriptional program required for cell survival upon osmostress: active Hog1 interacts with the transcription factor Hot1, inducing recruitment of Hog1 to osmostress-responsive promoters . Anchoring of Hog1 to chromatin was shown to be important to stimulate the recruitment and activation of RNA Pol II . In addition to this role in transcriptional initiation, Hog1 also behaves as a transcriptional elongation factor . Genome-wide chromatin immunoprecipitation coupled with microarrays (ChIP-Chip) experiments have revealed that two other yeast MAPKs involved in pheromone response, Fus3 and Kss1, are bound to several genes that are expressed upon pheromone pathway activation .
Once bound to chromatin, MAPKs do not only modulate transcription factor activity and RNA Pol II recruitment, but also regulate gene expression by inducing changes in chromatin organization and epigenetic histone modifications. Indeed, yeast Hog1 facilitates recruitment of either the histone deacetylase Rpd3-Sin3 complex , the SAGA complex which contains both histone acetylation and de-ubiquitylation activities , or the SWI-SNF chromatin remodeling complex . During mammalian myoblast differentiation, p38 targets the SWI-SNF complex  as well as the ASH2L Trithorax complex, that contains a histone methyl-transferase, to muscle-specific genes . It is tempting to speculate that binding of these complexes to chromatin relies on phosphorylation of some of their components by MAPKs. For example in mammals, the downstream MAPKAP3 kinase, once activated by phosphorylation in response to mitogenic or stress signals, phosphorylates some members of the chromatin Polycomb Repressive Complex 1 (PRC1). This results in dissociation of PRC1 from chromatin and subsequent de-repression of target genes . Altogether, these data show that chromatin reorganization mediated by nucleosome remodeling and epigenetic mark modifications is an important process to regulate gene expression in response to MAPK signaling. This process could involve a dynamic switch between the binding of either a silencing complex or an activating complex on a target gene.
In most of the examples mentioned above, these repressing and activating complexes are formed by proteins of the Polycomb-group (PcG) and Trithorax-group (TrxG) which combine into several heteromeric complexes that bind chromatin. PcG and TrxG complexes regulate gene expression by modulating chromatin structure, in particular by depositing specific post-translational histone modifications and by nucleosome remodeling. PcG complexes lead to compact, transcriptionally inactive chromatin, whereas TrxG complexes counteract PcG-mediated repression and maintain chromatin in an open conformation that facilitates transcription (for a review, see ). A third class of proteins, the Enhancers of Trithorax and Polycomb (ETP), is involved in PcG- as well as TrxG-mediated gene regulation (for a review, see ). Interestingly, ETPs allow the recruitment on chromatin of either PcG or TrxG complexes and could therefore participate in a switch between activation and repression. The first ETP mutants have been identified in Drosophila as enhancers of both Polycomb-group (PcG) and trithorax-group (trxG) mutations . Although not found in any of the PcG or TrxG core complexes purified so far, ETPs interact with these complexes and are required for both PcG-mediated silencing and TrxG-mediated activation. For example, mutants of the ETP gene Asx enhance homeotic phenotypes of both PcG and trxG mutations . The GAGA factor encoded by Trithorax-like (Trl) was first described as an activator of Hox gene expression and later shown to play a role in recruitment of PcG complexes [20, 21]. The HMGB protein DSP1 behaves as an ETP since a dsp1 null allele exhibits a PcG phenotype but enhances at the same time the phenotype of several trxG mutants . Furthermore, DSP1 is required for PcG complex recruitment to chromatin . Lastly, corto, which is ubiquitously expressed all along development, presents the characteristics of an ETP since loss-of-function mutants exhibit both PcG and trxG phenotypes and enhance the phenotype of some PcG as well as trxG mutants [24, 25]. The Corto protein interacts not only with PcG and TrxG proteins, but also with other ETPs such as GAGA and DSP1, which suggests that different combinations of ETPs could favor the recruitment of either PcG or TrxG complexes on chromatin [26, 27]. Nevertheless, the mechanism through which ETPs could exert this dual function remains to be investigated.
corto loss-of-function mutants exhibit several phenotypes, among them ectopic veins on wings which recall the phenotype induced by a gain-of-function mutation of rolled (rl) that encodes the MAPK ERK [25, 28]. In Drosophila, specification and differentiation of wing tissues (i.e. vein and intervein) occur in wing imaginal discs during the third larval and pupal stages and rely on several developmental signals including those mediated by EGF, BMP, Hedgehog and Wnt (for a review, see ). Signaling mediated by the Drosophila EGF Receptor (DER) is crucial for early specification of the longitudinal vein primordia called proveins, as well as for differentiation of vein and intervein cells (for a review, see ). Once activated by one of its ligands, DER activates a phosphorylation cascade leading to ERK signaling. Early ERK signaling in wing discs of third instar larvae specifies provein . In provein territories, ERK maintains expression of rhomboïd (rho), which is required to direct provein cells to differentiate as vein cells . rho encodes a serine-threonine protease which is required to process EGFR ligands and thus participates in a positive feed-back loop that maintains high levels of ERK activity [33, 34]. On the other hand, ERK signaling represses blistered (bs) expression. bs, that encodes a homolog of the mammalian Serum Response Factor (SRF), is expressed in the future intervein cells and controls the specification of intervein tissue [35, 36]. Later during development, at the pupal stage, ERK signaling is also required to promote intervein cell differentiation . The formation of vein and intervein tissues thus depends on the outcome of a fine-tuned balance between rho and bs expression patterns, which are both regulated by ERK signaling. Furthermore, the scaffold protein dMP1 also participates in ERK signaling during vein and intervein differentiation . The wing phenotype of corto mutants, but also the fact that we isolated dMP1 in a two-hybrid screen using Corto as bait, prompted us to address the potential role of this ETP in relation to ERK signaling during wing vein and intervein differentiation. Our genetic interactions between corto and genes encoding some actors of the ERK signaling pathway, i.e. rl itself, dMP1, bs and rho show that corto and dMP1 contribute to antagonize rl vein-promoting function in future intervein cells. Biochemical analyzes show that Corto interacts directly with ERK. Furthermore, Corto is phosphorylated and its phosphorylation increases upon ERK activation. Surprisingly, ERK and dMP1 associate with Corto exclusively in the nucleus. As suggested by immunolocalizations on polytene chromosomes, a dMP1/ERK/Corto complex might be targeted to chromatin to directly regulate gene expression, thus allowing proper wing tissue differentiation.
cortocontributes to intervein tissue differentiation
corto mutants induce ectopic vein phenotypes and interact with blistered (bs) and rhomboïd (rho) during wing tissue differentiation
Total females observed
% females with ectopic veins only
% females with blistered wings
corto 420 /+
corto L1 /+
corto 07128b /+
+/bs EY23316 ; corto 07128b/+
sd::Gal4/+; +/rho EP3704
sd::Gal4/+; corto 420 /rho EP3704
sd::Gal4/+; corto 07128b /rho EP3704
Staged corto over-expression induced ectopic vein phenotypes mostly during late larval development
Time of heat-shock (hours After Egg Laying AEL)
Total females observed
% females with ectopic veins
Total females observed
% females with ectopic veins
48 h-72 h AEL
72 h-96 h AEL
(early to mid L3 larvae)
96 h-120 h AEL
(mid to late L3 larvae)
120 h-136 h AEL
In conclusion, corto misregulation (either loss-of-function or over-expression) induced ectopic veins that formed within intervein tissue and never truncated veins. This observation suggested that Corto contributes to intervein tissue differentiation, whereas it does not seem to be involved in vein formation. We have previously shown that corto interacts with some TrxG genes during wing tissue formation. Indeed, moira, kismet and ash1 mutants enhance the ectopic vein phenotype of corto 420 . Furthermore, several corto alleles enhance the ectopic vein phenotype of mutations in snr1 that encodes a component of the SWI/SNF complex [38, 40], a chromatin-remodeling complex also involved in wing tissue differentiation [41–43]. One hypothesis is that Corto, as an ETP, could participate in the recruitment of TrxG complexes to regulate expression of genes involved in wing tissue differentiation.
To clarify the role of corto in the formation of intervein tissue, we performed genetic interaction assays between corto and the intervein-promoting gene blistered (bs), or the vein-promoting gene rhomboïd (rho). As expected for a bs loss-of-function allele [35, 36], wings of flies heterozygous for bs EY23316 exhibited a moderate ectopic vein phenotype, but none showed blisters in the wings (Table 1 and Figure 2F). corto 07128b enhanced the ectopic vein phenotype induced by bs EY23316 (compare Figure 2G to Figure 2F). In addition, 32.5% of these trans-heterozygous flies had blisters in the wings (Table 1 and Figure 2H). These blisters, which result from impaired adhesion between the ventral and dorsal wing surfaces, could be caused by formation of many vein cells within intervein tissue. They are frequently observed in bs mutants  or when rho is over-expressed . This result therefore showed that bs and corto act synergistically to promote intervein cell fate. Ectopic over-expression of rho using the rho EP3704 allele and the sd::Gal4 driver induced ectopic veins for most of the flies and in a few cases (9.5%) formation of blisters (Figure 2I and Table 1). This phenotype was similar to that induced by over-expressing rho under control of a heat-inducible promoter . Both corto 420 and corto 07128b alleles enhanced this phenotype since the number of flies with blisters in the wings significantly increased (Table 1 and Figure 2J). This observation showed that corto antagonizes rho in vein formation.
Taken together, these results suggest that corto might antagonize rl vein-promoting function in future intervein cells. corto misregulation could therefore lead to deregulation of certain vein and intervein-promoting genes. Indeed, we observed deregulation of bs and rho in some intervein cells of pupal wings from corto L1 /Df(3R)6-7 escapers: in these cells, bs is down-regulated (Figure 2K) whereas rho is ectopically expressed (Figure 2L).These cells could thus acquire a vein fate.
Corto and dMP1 act together and participate in the control of wing tissue differentiation
corto interacts with rolled (rl) and dMP1 during wing tissue differentiation
Total females observed
% females with ectopic veins only
% females with one blistered wing
% females with two blistered wings
sd::Gal4/+;corto 420 /UAS::rl Sem
sd::Gal4/+;corto 07128b /UAS::rl Sem
+/sd::Gal4; UAS::dsMP1/+; +/corto 420
+/sd::Gal4; UAS::dsMP1/+; +/corto 07128b
Corto interacts in vitrodirectly with ERK and indirectly with dMP1
We have previously shown that dMP1 forms a complex with ERK, which is required for the proper development of intervein cells . To understand the molecular bases of the relationship between Corto, dMP1 and ERK, we first questioned the physical interaction between Corto and ERK. We carried out GST pull-down assays using in vitro translated ERK and GST-Corto fusion proteins. Structural analysis of Corto has shown that this 550 amino-acid protein contains three globular domains that might correspond to functional domains ( and Figure 4A). The first one is located at position 127-203 and exhibits strong structural similarities with chromodomains, that are chromatin targeting modules found in some regulators of chromatin structure (for a review, see ). The two others, located at positions 418-455 and 480-550, present no obvious similarities with known protein domains. In vitro translated ERK protein was retained on GST-C1/324 and GST-C325/550 beads containing the NH2-terminal half and the COOH-terminal half of Corto, respectively (Figure 4A). In contrast, ERK was not retained on GST-C127/207 beads containing the Corto chromodomain, or on GST-C418/503 beads containing part of the two COOH-terminal globular domains. The lack of interaction with GST-C127/207 and GST-C418/503 suggested that none of these domains was sufficient to mediate Corto-ERK interaction, either because of inappropriate folding of these short domains in the GST fusion proteins, or because none of these two fragments contains the sequences that mediate ERK binding. Taken together, these results showed that Corto interacts directly with ERK in vitro. Further experiments are needed to determine the precise domains or residues that mediate the interaction between Corto and ERK.
Since we isolated dMP1 in a two-hybrid screen using the NH2-terminal part of Corto as a bait (Figure 4B), we next questioned the physical interaction between Corto and dMP1. We performed GST pull-down assays using GST-dMP1 fusion protein and in vitro translated Corto to see whether their interaction was direct or indirect. Indeed, indirect interactions via yeast proteins have already been observed in two-hybrid experiments . As shown in the middle panel of Figure 4C, the same result was obtained using GST or GST-dMP1 beads indicating that there was no specific direct interaction between Corto and dMP1. However, by incubating GST-dMP1 beads with total embryonic protein extract, we observed after blotting with anti-Corto antibodies that Corto was specifically retained on GST-dMP1 beads (bottom panel of Figure 4C). Therefore, we concluded that Corto and dMP1 interact via additional factors. One potential candidate could be ERK, since it directly interacts with Corto (as shown above) and with dMP1 .
Corto is located both in the cytoplasm and the nucleus and is phosphorylated
The phosphorylation pattern of Corto is controlled at least partially by ERK pathway
As another way to activate the ERK pathway, we co-expressed Corto-FLAG and tagged forms of either ERK, ERKSem or RasV12 in S2 cells. Similar to ERK and ERKSem over-expression, over-expression in flies of a constitutively active form of Ras, RasV12, induces ectopic vein cells . Surprisingly, smaller Corto isoforms appeared when constitutively activating the ERK pathway with either ERK, ERKSem or RasV12 (Figure 6B). One possibility is that these smaller isoforms could correspond to partially dephosphorylated Corto molecules. Taken together, these experiments demonstrate that Corto presents a complex phosphorylation pattern that depends at least on ERK signaling. It is tempting to speculate that some phosphorylations are performed directly by ERK. Indeed, Corto contains 3 SP sites at positions 139, 190 and 428 that correlate with theoretical ERK1/ERK2 phosphorylation sites . Identification of Corto phosphorylation sites as well as phospho-mutant analysis and determination of Corto phosphorylation status when bound to chromatin would help to better understand the role of these phosphorylation events.
Interaction between ERK and dMP1 or Corto takes place in the nucleus only
ERK and dMP1 bind polytene chromosomes where they partially co-localize with Corto
Previously, other scaffold proteins have been reported to bind chromatin. This is the case of the scaffold protein Ste5p in the pheromone pathway of yeast which interacts with the MAPKs Fus3p and Kss1p and occupies the same mating-type genes. Ste5p has then been suggested to function as an adaptor for protein-protein interactions both at the plasma membrane and in the nucleus . In mammals, the scaffold protein β-arrestin, localized both in the cytoplasm and in the nucleus, is also recruited to target promoters under opioid receptor stimulation thus enhancing gene transcription . The scaffold protein dMP1 could serve as an adaptor to connect ERK with other partners directly on chromatin. It could also allow ERK to form dimers, as mammalian scaffold proteins have been shown to be essential to connect ERK dimers to cytoplasmic targets . It would therefore be interesting to know if ERK is monomeric or dimeric when bound to chromatin.
We show here that the ETP corto, rl and dMP1 interact during wing tissue differentiation in Drosophila. Corto, ERK and dMP1 form a complex exclusively in the nucleus. In addition, these proteins bind polytene chromosomes where they partially co-localize, suggesting that the Corto-ERK-dMP1 complex might regulate vein and/or intervein gene expression directly on chromatin. Future experiments will be needed to test whether this complex, via the ETP Corto, participates in the recruitment of TrxG complexes on target genes in response to ERK signaling.
Drosophilastrains and genetic crosses
Flies were raised on standard yeast-cornmeal medium at 25°C. w 1118 was used as control strain. The corto L1 (EMS-induced allele), corto 07128b , bs EY23316 , rho EP3704 (P-insertion alleles) lines were from the Bloomington Stock Center. The corto 420 line results from imprecise P-element excision [24, 25]. The transgenic lines UAS::corto  (transgene on the third chromosome), UAS::rolled (transgene on the X chromosome) and UAS::rl Sem  (transgene on the third chromosome) were gifts from Dr. R. Rosset (UAS::corto) and Dr. K. Moses (UAS::rl and UAS::rl Sem ). The UAS::dsMP1 line allowing dMP1 down-regulation by RNA interference (transgene on the second chromosome) was described previously . Lines containing a transgene with UAS sequences were crossed with the hs::Gal4, scalloped::Gal4 (sd::Gal4, ) and Beadex::Gal4 (Bx::Gal4, ) drivers. All crosses were performed at 25°C, except those of UAS::rl Sem with sd::Gal4 that were performed at 18°C to decrease Gal4 activity and therefore lower transgene expression. To perform staged corto expression with the hs::Gal4 driver, Gal4 was induced by 20 minute heat-shocks applied at various moments during larval and pupal development.
In situhybridization experiments on pupal wings
White pupae were collected and maintained for 30 h at 25°C. After puparium dissection, pupae were fixed in 8% formaldehyde for 12 h at 4°C. In situ hybridization with blistered (EST SD23611) and rhomboïd (EST RE59529) DIG-UTP labeled RNA probes was performed according to standard protocols .
Q-RT PCR experiments
Total RNA were extracted from 20 third instar larval discs of each genotype using the PureLink RNA Microkit (Invitrogen) according to the manufacturer's instructions. 1 μg of RNA was reverse-transcribed with the SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen). Q-RT PCR experiments were carried out on a Light Cycler 480 (Roche Diagnostics) with the Maxima SybrGreen mix (Fermentas). The primers used were: cortoF (5'-TGGCCACAGTTCCTAGCATT-3') and cortoR (5'-GCATGGGATTGGTGTCAGG-3'); rp49F (5'-CCGCTTCAAGGGACAGTATC-3') and rp49R (5'-GACAATCTCCTTGCGCTTC-3'); spt6F (5-'CGGAGGAGCTCTTCGATATG-3') and spt6R (5'-GACAGCTCTGGGAAGTCGTC-3'). A standard curve of amplification efficiency for each set of primers was generated with a serial dilution of cDNA. rp49 or spt6 levels were used for normalization according to the standard curve method. Three independent experiments were performed.
Plasmids and S2 cell transfection
The corto, dMP1, rl and rl Sem cDNAs were cloned into Gateway® Drosophila vectors allowing expression of the fusion proteins under control of the actin5C promoter, as previously described . The rl Sem sequence was obtained by in vitro mutagenesis using the QuickChange® Site Directed Mutagenesis kit (Stratagene) according to the manufacturer's instructions. This gain-of-function mutation is a G to A transition resulting in a D to N substitution at position 334 . pMT-RasV12 (a gift from Dr. A. Nagel) allowed transient expression of the constitutively active form of Ras, RasV12, under the control of the heavy metal inducible promoter metallothionein . RasV12 was induced by treating transfected cells for 24 h with CuSO4 0.5 mM. For transfection, S2 cells were cultivated at 25°C in Schneider medium with or without 10% fetal calf serum as indicated. 5.106 cells were transfected with 2 μg of DNA using Effecten® transfection reagent (Qiagen) according to the manufacturer's instructions (1/10 DNA-Effecten® ratio). Cells were collected 48 h (ERK pathway activation) or 72 h (immunoprecipitation) after transfection.
Protein extracts and phosphatase treatment
Embryos and S2 cell total extracts were prepared by sonication in RIPA buffer [50 mM Tris-HCl pH7.5, 150 mM NaCl, 25 mM NaVO4, 25 mM NaF, 0.1% SDS, 0.5% NP40, complete protease inhibitors (Roche)]. Cytoplasmic extracts were prepared either with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce) according to the manufacturer's instructions or by homogenization using a Dounce potter in low salt buffer as described in . When analyzed by immunoprecipitation, these two kinds of cytoplasmic extracts gave the same results. Nuclear extracts were obtained by sonication of the nuclear pellet in RIPA buffer. Phosphatase treatments of S2 cell extracts prepared without NaVO4 and NaF phosphatase inhibitors were performed using Lambda Protein Phosphatase (Upstate) for a 10 minute incubation time at 37°C.
Western blot analysis and antibodies
Cell lysates or immunoprecipitated proteins were resolved on 8% SDS-PAGE Anderson gels  when separating different isoforms of Corto, or on 12% or 15% classical SDS-PAGE depending on the molecular weight of proteins. Western blot experiments were performed according to standard protocols. Antibodies used were monoclonal anti-FLAG (F3165, Sigma) or anti-Myc (sc-40, Santa Cruz Biotechnology) antibodies for fusion proteins, anti-β- tubulin (E7) and anti-lamin (ADL67.10) antibodies (Developmental Studies Hybridoma Bank) for control of cytoplasmic and nuclear fractions, rat anti-Corto antibodies , monoclonal phosphoserine/threonine/tyrosine antibody (MA1-38450, Pierce), rabbit anti-ERK antibodies (C-16, Santa-Cruz Biotechnology) or monoclonal anti-dP-ERK E10 antibody (9106, Cell Signaling). Anti-HA antibody (H3663, Sigma) was used as a negative control in immunoprecipitation experiments.
For co-immunoprecipitation experiments, 500 μg of protein extracts (total, cytoplasmic or nuclear) were immunoprecipitated either with monoclonal anti-FLAG antibody, anti-Myc 9E10 antibody or anti-HA antibody using magnetic protein G-agarose beads (Ademtech). To co-immunoprecipitate Corto and dMP1, proteins were cross-linked before extraction by treating cells with 1% formaldehyde for 10 minutes followed by neutralization with 0.13 M glycine. To co-immunoprecipitate Corto and dP-ERK in embryonic extracts, monoclonal anti-dP-ERK E10 antibody was covalently bound onto protein G-agarose beads using standard protocols.
Two-hybrid experiments were performed as previously described , using leucine and X-Gal tests. Both tests gave the same result, and only the leucine test is shown. The full length dMP1 protein was fused with the B42 activation domain (B42AD). The NH2-terminal half of Corto was fused with the LexA DNA binding domain. B42AD and LexA were used as negative controls. B42/SV40 large T-antigen (B42ADT) and LexA/p53 (LexA53) fusion proteins were used as positive controls.
Immunolocalization on polytene chromosomes
Co-immunostaining of w 1118 polytene chromosomes was performed as previously described  using rabbit anti-ERK (1:20) (C-16; Santa-Cruz Biotechnology), guinea-pig anti-dMP1 (1:20)  and rabbit anti-Corto (1:20)  as primary antibodies. Secondary antibodies (Alexa Fluor® 594 goat anti-rabbit IgG and Alexa Fluor® 488 goat anti-guinea-pig IgG, Molecular Probes) were used at a 1:1000 dilution.
We thank Dr. Anja Nagel for the gift of the pMT-RasV12 plasmid, the Murphy lab for Drosophila Gateway vectors, Dr. Kevin Moses, Dr. Roland Rosset and the Bloomington Stock Center for flies, the Developmental Studies Hybridoma Bank for antibodies. We thank Valérie Ribeiro and Sarah Leridée for excellent technical assistance. Q-RT PCR experiments were performed at the IFR83 (UPMC). We thank Dr. Neel Randsholt, Dr. Willem Voncken, Dr. Sébastien Bloyer and Pr. Jean Deutsch for their comments on the manuscript. This work was supported by CNRS and UPMC, and by a scholarship from the Ministère de la Recherche to J.R.
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