BACKGROUND
the mitogen-activated protein kinase  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 .
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  are mainly activated by mitogenic stimuli such as growth factors and hormones, whereas c-jun n-terminal kinases  and p <dig> 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 mp <dig> forms a signaling complex with mek and erk thus facilitating erk activation  <cit> .
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 .
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, p <dig> is recruited to the promoters of myogenic genes together with the muscle-regulatory factors myod and mef2c  <cit> .
in pancreatic β-cells, in response to increased glucose concentration, erk1/ <dig> mapks are bound to the insulin gene promoter in the same complex as their transcription factor substrates mafa, beta <dig> and pdx- <dig>  <cit> .
in yeast, the p38-related hog <dig> kinase coordinates the transcriptional program required for cell survival upon osmostress: active hog <dig> interacts with the transcription factor hot <dig>  inducing recruitment of hog <dig> to osmostress-responsive promoters  <cit> .
anchoring of hog <dig> to chromatin was shown to be important to stimulate the recruitment and activation of rna pol ii  <cit> .
in addition to this role in transcriptional initiation, hog <dig> also behaves as a transcriptional elongation factor  <cit> .
genome-wide chromatin immunoprecipitation coupled with microarrays  experiments have revealed that two other yeast mapks involved in pheromone response, fus <dig> and kss <dig>  are bound to several genes that are expressed upon pheromone pathway activation  <cit> .
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 hog <dig> facilitates recruitment of either the histone deacetylase rpd3-sin <dig> complex  <cit> , the saga complex which contains both histone acetylation and de-ubiquitylation activities  <cit> , or the swi-snf chromatin remodeling complex  <cit> .
during mammalian myoblast differentiation, p <dig> targets the swi-snf complex  <cit>  as well as the ash2l trithorax complex, that contains a histone methyl-transferase, to muscle-specific genes  <cit> .
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 mapkap <dig> kinase, once activated by phosphorylation in response to mitogenic or stress signals, phosphorylates some members of the chromatin polycomb repressive complex  <dig> .
this results in dissociation of prc <dig> from chromatin and subsequent de-repression of target genes  <cit> .
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  and trithorax-group  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 .
a third class of proteins, the enhancers of trithorax and polycomb , is involved in pcg- as well as trxg-mediated gene regulation .
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  and trithorax-group  mutations  <cit> .
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  <cit> .
the gaga factor encoded by trithorax-like  was first described as an activator of hox gene expression and later shown to play a role in recruitment of pcg complexes  <cit> .
the hmgb protein dsp <dig> behaves as an etp since a dsp <dig> null allele exhibits a pcg phenotype but enhances at the same time the phenotype of several trxg mutants  <cit> .
furthermore, dsp <dig> is required for pcg complex recruitment to chromatin  <cit> .
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  <cit> .
the corto protein interacts not only with pcg and trxg proteins, but also with other etps such as gaga and dsp <dig>  which suggests that different combinations of etps could favor the recruitment of either pcg or trxg complexes on chromatin  <cit> .
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  that encodes the mapk erk  <cit> .
in drosophila, specification and differentiation of wing tissues  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 .
signaling mediated by the drosophila egf receptor  is crucial for early specification of the longitudinal vein primordia called proveins, as well as for differentiation of vein and intervein cells .
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  <cit> .
in provein territories, erk maintains expression of rhomboïd , which is required to direct provein cells to differentiate as vein cells  <cit> .
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  <cit> .
on the other hand, erk signaling represses blistered  expression.
bs, that encodes a homolog of the mammalian serum response factor , is expressed in the future intervein cells and controls the specification of intervein tissue  <cit> .
later during development, at the pupal stage, erk signaling is also required to promote intervein cell differentiation  <cit> .
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 dmp <dig> also participates in erk signaling during vein and intervein differentiation  <cit> .
the wing phenotype of corto mutants, but also the fact that we isolated dmp <dig> 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, dmp <dig>  bs and rho show that corto and dmp <dig> 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 dmp <dig> 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.
RESULTS
corto contributes to intervein tissue differentiation
we first investigated the ectopic vein phenotype of corto mutants using three different recessive lethal alleles: corto <dig>  corto07128b and cortol <dig>  corto <dig> is a deletion of the corto locus  <cit> , corto07128b a p-element insertion located  <dig>  kb upstream of corto 5'-utr  <cit> , and cortol <dig> an ems-induced mutation  <cit> .
as already described for corto <dig>  <cit> , heteroallelic combinations using corto07128b, cortol <dig> and a deficiency encompassing corto  are poorly viable, since we observed 0% to 10% escapers depending on combinations.
therefore, these three alleles are true loss-of-function alleles.
this was confirmed by quantitative rt-pcr analysis on wing discs from third instar larvae, that showed absence of corto transcripts in corto420/df6- <dig> and corto07128b/df6- <dig> larvae .
in contrast, cortol1/df6- <dig> larvae exhibited the same level of corto transcripts as wild-type flies, which suggests that the mutation in cortol <dig> rather affects the level or activity of corto protein.
as already described  <cit> , corto420/+ heterozygous flies exhibited very few ectopic veins .
this phenotype was more penetrant in cortol1/+  and corto07128b/+  heterozygous flies .
since both corto <dig> and corto07128b are devoid of corto transcripts, the discrepancy between these alleles may be a consequence of an interaction with the genetic background.
for all combinations, the few heteroallelic corto escapers displayed a stronger ectopic vein phenotype than corto heterozygous flies .
ectopic veins mainly arose close to longitudinal veins  <dig>   <dig>   <dig> and to the posterior cross-vein, which seems to be the case for most mutations that induce ectopic vein phenotypes  <cit> .
interestingly, over-expressing corto using a uas::corto construct and the wing specific beadex::gal <dig>  or scalloped::gal <dig>  driver also induced extra pieces of vein tissue in all flies .
since both corto over-expression and loss-of-function induced the same phenotype, one possibility is that corto may be required in stoechiometric amount to allow correct wing tissue differentiation.
this feature characterizes proteins that act through formation of complexes.
indeed, complexes are very sensitive to the relative amounts of their components, and can be disrupted either by an excess or a shortage of one of these  <cit> .
for all genotypes, the first allele was brought by the mother.
only female phenotypes are reported here, but similar results were obtained for males.
numbers of females with blistered wings in flies containing a corto mutation and the bsey <dig> or rhoep <dig> allele were compared to those of flies containing the bsey <dig> or rhoep <dig> allele only .
in order to assess the temporal requirement for corto function in wing tissue differentiation, we crossed the uas::corto line with the hs::gal <dig> driver strain allowing staged gal <dig> expression .
the highest percentage of ectopic vein phenotype was obtained when heat-shock was applied between  <dig> to  <dig> hours after egg laying, which corresponds to the mid to late third instar larval stage.
interestingly, it has been shown that, from late third instar larval stage to pupal stage, down-regulation of erk signaling is crucial for wing tissue formation: indeed, expression of a constitutively active form of the mapkk raf at the third instar larval stage induces vein loss, whereas expression of a dominant negative form of the receptor der at pupal stage leads to formation of ectopic veins  <cit> .
<dig> h- <dig> h ael
<dig> h- <dig> h ael
<dig> h- <dig> h ael
<dig> h- <dig> h ael
for all genotypes, the first allele was brought by the mother.
only female phenotypes are reported here, but similar results were obtained for males.
the number of females with ectopic veins when heat-shock was applied between  <dig> h- <dig> h ael was compared to the ones of females with ectopic veins obtained after heat-shock applied between  <dig> h- <dig> h ael or  <dig> h- <dig> h ael .
in conclusion, corto misregulation  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 ash <dig> mutants enhance the ectopic vein phenotype of corto <dig>  <cit> .
furthermore, several corto alleles enhance the ectopic vein phenotype of mutations in snr <dig> that encodes a component of the swi/snf complex  <cit> , a chromatin-remodeling complex also involved in wing tissue differentiation  <cit> .
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 , or the vein-promoting gene rhomboïd .
as expected for a bs loss-of-function allele  <cit> , wings of flies heterozygous for bsey <dig> exhibited a moderate ectopic vein phenotype, but none showed blisters in the wings .
corto07128b enhanced the ectopic vein phenotype induced by bsey <dig> .
in addition,  <dig> % of these trans-heterozygous flies had blisters in the wings .
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  <cit>  or when rho is over-expressed  <cit> .
this result therefore showed that bs and corto act synergistically to promote intervein cell fate.
ectopic over-expression of rho using the rhoep <dig> allele and the sd::gal <dig> driver induced ectopic veins for most of the flies and in a few cases  formation of blisters .
this phenotype was similar to that induced by over-expressing rho under control of a heat-inducible promoter  <cit> .
both corto <dig> and corto07128b alleles enhanced this phenotype since the number of flies with blisters in the wings significantly increased .
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 cortol1/df6- <dig> escapers: in these cells, bs is down-regulated  whereas rho is ectopically expressed .these cells could thus acquire a vein fate.
corto and dmp <dig> act together and participate in the control of wing tissue differentiation
since the wing phenotype of corto mutants resembles the one induced by hyperactivation of erk signaling pathway, we wondered whether corto was involved in the regulation of this pathway during wing development.
we first tested genetic interactions between corto and rolled using the uas::rolled strain which allows targeted erk over-expression when crossed with a gal <dig> driver.
all flies over-expressing rolled with the sd::gal <dig> driver at 25°c exhibited a mild ectopic vein phenotype .
expressivity of this phenotype was enhanced by the corto07128b allele .
this result suggests that the roles of corto and rolled in vein-promoting function are antagonistic.
we also used the uas::rolledsem  transgene that encodes a hyper-active form of erk  <cit> .
at 18°c, flies that over-expressed the uas::rlsem transgene under control of the sd::gal <dig> driver exhibited ectopic veins.
this phenotype was much stronger than the one induced by rolled over-expression .
for 88% of these flies, this phenotype was very strong since one or the two wings showed blisters .
surprisingly, penetrance and expressivity of the rlsem over-expression phenotype were lowered by corto <dig> and corto07128b alleles, as only  <dig> % of corto <dig> flies and  <dig> % of corto07128b ones exhibited blisters in one or both wings  and blisters were smaller .
this result confirmed that corto and rl interact during wing tissue formation.
however, the observation that corto mutation enhanced a mild-activation of erk pathway  whereas slowing-down a hyper-activation  is paradoxical and requires further experiments to be fully understood.
for all genotypes, the first allele was brought by the mother.
only female phenotypes are reported here, but similar results were obtained for males.
numbers of females with ectopic veins only or with two blistered wings among flies bearing a corto allele and over-expressing rlsem were compared to the one of flies over-expressing rlsem only.
numbers of females with ectopic veins among flies containing the uas::dsmp <dig> transgene, the sd::gal <dig> transgene and a corto mutation were compared to that of flies containing the uas::dsmp <dig> and the sd::gal <dig> transgenes only .
we have recently shown that the drosophila ortholog of mp <dig>  dmp <dig>  antagonizes rl vein-promoting function in the future intervein cells of the wing  <cit> .
furthermore, we isolated dmp <dig> in a two-hybrid screen using corto as bait .
we thus tested the genetic interactions between corto and dmp <dig>  as already described  <cit> , down-regulation of dmp <dig> by rna interference using the sd::gal <dig> driver induced ectopic veins in  <dig> % of flies .
this percentage increased to  <dig> % and 100% in combination with corto <dig> or corto07128b, respectively .
with corto07128b, the expressivity of the ectopic vein phenotype was also enhanced .
therefore, these results showed that corto and dmp <dig> act synergistically and participate in intervein tissue differentiation in response to erk signaling.
corto interacts in vitro directly with erk and indirectly with dmp1
we have previously shown that dmp <dig> forms a complex with erk, which is required for the proper development of intervein cells  <cit> .
to understand the molecular bases of the relationship between corto, dmp <dig> 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  <dig> amino-acid protein contains three globular domains that might correspond to functional domains .
the first one is located at position 127- <dig> and exhibits strong structural similarities with chromodomains, that are chromatin targeting modules found in some regulators of chromatin structure .
the two others, located at positions 418- <dig> and 480- <dig>  present no obvious similarities with known protein domains.
in vitro translated erk protein was retained on gst-c1/ <dig> and gst-c325/ <dig> beads containing the nh2-terminal half and the cooh-terminal half of corto, respectively .
in contrast, erk was not retained on gst-c127/ <dig> beads containing the corto chromodomain, or on gst-c418/ <dig> beads containing part of the two cooh-terminal globular domains.
the lack of interaction with gst-c127/ <dig> and gst-c418/ <dig> 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 dmp <dig> in a two-hybrid screen using the nh2-terminal part of corto as a bait , we next questioned the physical interaction between corto and dmp <dig>  we performed gst pull-down assays using gst-dmp <dig> 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  <cit> .
as shown in the middle panel of figure 4c, the same result was obtained using gst or gst-dmp <dig> beads indicating that there was no specific direct interaction between corto and dmp <dig>  however, by incubating gst-dmp <dig> beads with total embryonic protein extract, we observed after blotting with anti-corto antibodies that corto was specifically retained on gst-dmp <dig> beads .
therefore, we concluded that corto and dmp <dig> interact via additional factors.
one potential candidate could be erk, since it directly interacts with corto  and with dmp <dig>  <cit> .
corto is located both in the cytoplasm and the nucleus and is phosphorylated
upon activation of the mapk cascade, erk is phosphorylated in the cytoplasm.
di-phosphorylated erk  phosphorylates in turn a large number of targets with diverse functions and different subcellular localizations.
in particular, part of dp-erk is translocated into the nucleus where it phosphorylates some transcription factors .
mammalian mp <dig> is present in the cytoplasm in association with erk but its possible nuclear localization has not been reported  <cit> .
nevertheless, we have recently shown that dmp <dig> is present both in the cytoplasm and the nucleus  <cit> .
we thus asked whether corto was present in the same compartments as erk and dmp <dig>  corto was detected in nuclear and cytoplasmic extracts from embryos , third instar larvae  and schneider s <dig> cells .
a similar nuclear and cytoplasmic distribution was observed for a corto-flag fusion protein expressed in s <dig> cells .
both corto and corto-flag exhibited several isoforms very close to each other in size, with the lowest isoforms being more abundant in the cytoplasm than in the nucleus .
corto is very rich in serine , threonine , tyrosine , and presents many predicted phosphorylation sites for several kinases distributed all along the sequence .
hence we checked whether the isoforms we observed could indeed correspond to differentially phosphorylated molecules.
as shown in figure 5d, the upper bands of corto-flag disappeared after lambda phosphatase treatment.
altogether, these results showed that corto is a phosphorylated protein present both in the cytoplasm and in the nucleus.
in addition, the corto phosphorylation pattern seemed to be different between the two cellular compartments, with enrichment in highly phosphorylated forms in the nucleus.
phosphorylation of m <dig>  a chromatin regulator, has been shown to regulate its nuclear translocation  <cit> .
further experiments are required to determine if corto localization is regulated through a similar mechanism.
the phosphorylation pattern of corto is controlled at least partially by erk pathway
since corto is phosphorylated and interacts in vitro with erk, it is tempting to speculate that corto could be phosphorylated in response to activation of the corresponding map kinase cascade.
to answer this question, we transfected s <dig> cells in absence of serum with a plasmid allowing corto-flag expression.
after two days, we transiently activated the map kinase cascade by a short  treatment either with serum, with serum plus insulin or with serum plus pma.
in all three conditions, faint upper corto isoforms appeared , showing that corto phosphorylation was induced very rapidly upon erk activation.
to confirm the existence of these faint upper isoforms, we immunoprecipitated corto from these extracts using anti-flag antibodies and blotted the immunoprecipitates with antibodies directed against phosphoproteins .
this experiment revealed first that corto is constitutively phosphorylated even without map kinase activation.
second, erk activation induced an hyperphosphorylation of corto since an upper smear containing several bands very close in size appeared in all three conditions of activation tested.
as another way to activate the erk pathway, we co-expressed corto-flag and tagged forms of either erk, erksem or rasv <dig> in s <dig> cells.
similar to erk and erksem over-expression, over-expression in flies of a constitutively active form of ras, rasv <dig>  induces ectopic vein cells  <cit> .
surprisingly, smaller corto isoforms appeared when constitutively activating the erk pathway with either erk, erksem or rasv <dig> .
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  <dig> sp sites at positions  <dig>   <dig> and  <dig> that correlate with theoretical erk1/erk <dig> phosphorylation sites  <cit> .
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 dmp <dig> or corto takes place in the nucleus only
we next performed co-immunoprecipitation experiments to see whether corto interacts with erk and dmp <dig> in vivo.
we have previously shown that erk and dmp <dig> co-immunoprecipitate in a total protein extract  <cit> .
in order to determine the subcellular localization of this dmp1/erk complex, we carried out co-immunoprecipitation experiments using cytoplasmic or nuclear extracts of s <dig> cells expressing dmp <dig> and erk tagged proteins.
surprisingly, dmp1-myc co-immunoprecipitated with erk-flag in the nuclear extract only .
corto-myc and erk-flag also co-immunoprecipitated in the nuclear extract only .
in this last experiment, all corto isoforms were co-immunoprecipitated with erk.
in both cases, the lack of co-immunoprecipitation in cytoplasmic extracts was confirmed by using two different protocols to prepare nuclear and cytoplasmic extracts .
the co-immunoprecipitation observed between corto and erk fusion proteins was confirmed with endogenous proteins from a total embryonic extract using anti-dp-erk antibody .
furthermore, this experiment showed that corto was able to interact with dp-erk.
lastly, we observed no co-immunoprecipitation between corto-flag and dmp1-myc whether in total, cytoplasmic or nuclear extracts .
labile protein interactions can be stabilized by cross-linking before performing cell extracts, although such treatment does not allow separating cytoplasmic and nuclear extracts.
when using a total extract from cross-linked cells, we could detect co-immunoprecipitation between corto-flag and dmp1-myc .
this observation was consistent with our gst pull-down and peptide pull-down assays showing that the interaction between dmp <dig> and corto was not direct but was probably mediated by other proteins.
altogether, our co-immunoprecipitation results suggest that a complex containing corto, erk and dmp <dig> might exist in the nucleus only.
the core protein of this complex should be erk, since it interacts directly with both corto and dmp <dig>
erk and dmp <dig> bind polytene chromosomes where they partially co-localize with corto
to further analyze the relationship between corto, dmp <dig> and erk, and since corto has been shown to bind polytene chromosomes  <cit> , we analyzed the binding of erk and dmp <dig> onto polytene chromosomes.
we observed that erk and dmp <dig> bound polytene chromosomes on many sites where they completely co-localized .
furthermore, corto and dmp <dig> co-localized on several sites .
these results suggest that a dmp1/erk/corto complex might regulate targets directly on chromatin.
since the co-localization of corto with dmp <dig> is not complete, corto appears to be an optional partner of dmp1/erk on chromatin.
corto association may require additional factors or may be controlled by signaling events.
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  <cit> .
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  <cit> .
the scaffold protein dmp <dig> 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  <cit> .
it would therefore be interesting to know if erk is monomeric or dimeric when bound to chromatin.
CONCLUSIONS
we show here that the etp corto, rl and dmp <dig> interact during wing tissue differentiation in drosophila.
corto, erk and dmp <dig> form a complex exclusively in the nucleus.
in addition, these proteins bind polytene chromosomes where they partially co-localize, suggesting that the corto-erk-dmp <dig> 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.

 the mitogen-activated protein kinase 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. 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 are mainly activated by mitogenic stimuli such as growth factors and hormones, whereas c-jun n-terminal kinases and p 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 mp forms a signaling complex with mek and erk thus facilitating erk activation. 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. 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, p 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/ mapks are bound to the insulin gene promoter in the same complex as their transcription factor substrates mafa, beta and pdx-. in yeast, the p38-related hog kinase coordinates the transcriptional program required for cell survival upon osmostress: active hog interacts with the transcription factor hot inducing recruitment of hog to osmostress-responsive promoters. anchoring of hog 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, hog also behaves as a transcriptional elongation factor. genome-wide chromatin immunoprecipitation coupled with microarrays experiments have revealed that two other yeast mapks involved in pheromone response, fus and kss 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 hog facilitates recruitment of either the histone deacetylase rpd3-sin complex, the saga complex which contains both histone acetylation and de-ubiquitylation activities, or the swi-snf chromatin remodeling complex. during mammalian myoblast differentiation, p 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 mapkap kinase, once activated by phosphorylation in response to mitogenic or stress signals, phosphorylates some members of the chromatin polycomb repressive complex. this results in dissociation of prc 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 and trithorax-group 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. a third class of proteins, the enhancers of trithorax and polycomb, is involved in pcg- as well as trxg-mediated gene regulation. 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 and trithorax-group 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 was first described as an activator of hox gene expression and later shown to play a role in recruitment of pcg complexes. the hmgb protein dsp behaves as an etp since a dsp null allele exhibits a pcg phenotype but enhances at the same time the phenotype of several trxg mutants. furthermore, dsp 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. the corto protein interacts not only with pcg and trxg proteins, but also with other etps such as gaga and dsp which suggests that different combinations of etps could favor the recruitment of either pcg or trxg complexes on chromatin. 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 that encodes the mapk erk. in drosophila, specification and differentiation of wing tissues 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. signaling mediated by the drosophila egf receptor is crucial for early specification of the longitudinal vein primordia called proveins, as well as for differentiation of vein and intervein cells. 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, 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. on the other hand, erk signaling represses blistered expression. bs, that encodes a homolog of the mammalian serum response factor, is expressed in the future intervein cells and controls the specification of intervein tissue. 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 dmp also participates in erk signaling during vein and intervein differentiation. the wing phenotype of corto mutants, but also the fact that we isolated dmp 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, dmp bs and rho show that corto and dmp 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 dmp 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.  corto contributes to intervein tissue differentiation we first investigated the ectopic vein phenotype of corto mutants using three different recessive lethal alleles: corto corto07128b and cortol corto is a deletion of the corto locus, corto07128b a p-element insertion located kb upstream of corto 5'-utr, and cortol an ems-induced mutation. as already described for corto, heteroallelic combinations using corto07128b, cortol and a deficiency encompassing corto are poorly viable, since we observed 0% to 10% escapers depending on combinations. therefore, these three alleles are true loss-of-function alleles. this was confirmed by quantitative rt-pcr analysis on wing discs from third instar larvae, that showed absence of corto transcripts in corto420/df6- and corto07128b/df6- larvae. in contrast, cortol1/df6- larvae exhibited the same level of corto transcripts as wild-type flies, which suggests that the mutation in cortol rather affects the level or activity of corto protein. as already described, corto420/+ heterozygous flies exhibited very few ectopic veins. this phenotype was more penetrant in cortol1/+ and corto07128b/+ heterozygous flies. since both corto and corto07128b are devoid of corto transcripts, the discrepancy between these alleles may be a consequence of an interaction with the genetic background. for all combinations, the few heteroallelic corto escapers displayed a stronger ectopic vein phenotype than corto heterozygous flies. ectopic veins mainly arose close to longitudinal veins and to the posterior cross-vein, which seems to be the case for most mutations that induce ectopic vein phenotypes. interestingly, over-expressing corto using a uas::corto construct and the wing specific beadex::gal or scalloped::gal driver also induced extra pieces of vein tissue in all flies. since both corto over-expression and loss-of-function induced the same phenotype, one possibility is that corto may be required in stoechiometric amount to allow correct wing tissue differentiation. this feature characterizes proteins that act through formation of complexes. indeed, complexes are very sensitive to the relative amounts of their components, and can be disrupted either by an excess or a shortage of one of these. for all genotypes, the first allele was brought by the mother. only female phenotypes are reported here, but similar results were obtained for males. numbers of females with blistered wings in flies containing a corto mutation and the bsey or rhoep allele were compared to those of flies containing the bsey or rhoep allele only. in order to assess the temporal requirement for corto function in wing tissue differentiation, we crossed the uas::corto line with the hs::gal driver strain allowing staged gal expression. the highest percentage of ectopic vein phenotype was obtained when heat-shock was applied between to hours after egg laying, which corresponds to the mid to late third instar larval stage. interestingly, it has been shown that, from late third instar larval stage to pupal stage, down-regulation of erk signaling is crucial for wing tissue formation: indeed, expression of a constitutively active form of the mapkk raf at the third instar larval stage induces vein loss, whereas expression of a dominant negative form of the receptor der at pupal stage leads to formation of ectopic veins. h- h ael h- h ael h- h ael h- h ael for all genotypes, the first allele was brought by the mother. only female phenotypes are reported here, but similar results were obtained for males. the number of females with ectopic veins when heat-shock was applied between h- h ael was compared to the ones of females with ectopic veins obtained after heat-shock applied between h- h ael or h- h ael. in conclusion, corto misregulation 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 ash mutants enhance the ectopic vein phenotype of corto. furthermore, several corto alleles enhance the ectopic vein phenotype of mutations in snr that encodes a component of the swi/snf complex, a chromatin-remodeling complex also involved in wing tissue differentiation. 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, or the vein-promoting gene rhomboïd. as expected for a bs loss-of-function allele, wings of flies heterozygous for bsey exhibited a moderate ectopic vein phenotype, but none showed blisters in the wings. corto07128b enhanced the ectopic vein phenotype induced by bsey. in addition, % of these trans-heterozygous flies had blisters in the wings. 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 rhoep allele and the sd::gal driver induced ectopic veins for most of the flies and in a few cases formation of blisters. this phenotype was similar to that induced by over-expressing rho under control of a heat-inducible promoter. both corto and corto07128b alleles enhanced this phenotype since the number of flies with blisters in the wings significantly increased. 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 cortol1/df6- escapers: in these cells, bs is down-regulated whereas rho is ectopically expressed.these cells could thus acquire a vein fate. corto and dmp act together and participate in the control of wing tissue differentiation since the wing phenotype of corto mutants resembles the one induced by hyperactivation of erk signaling pathway, we wondered whether corto was involved in the regulation of this pathway during wing development. we first tested genetic interactions between corto and rolled using the uas::rolled strain which allows targeted erk over-expression when crossed with a gal driver. all flies over-expressing rolled with the sd::gal driver at 25°c exhibited a mild ectopic vein phenotype. expressivity of this phenotype was enhanced by the corto07128b allele. this result suggests that the roles of corto and rolled in vein-promoting function are antagonistic. we also used the uas::rolledsem transgene that encodes a hyper-active form of erk. at 18°c, flies that over-expressed the uas::rlsem transgene under control of the sd::gal driver exhibited ectopic veins. this phenotype was much stronger than the one induced by rolled over-expression. for 88% of these flies, this phenotype was very strong since one or the two wings showed blisters. surprisingly, penetrance and expressivity of the rlsem over-expression phenotype were lowered by corto and corto07128b alleles, as only % of corto flies and % of corto07128b ones exhibited blisters in one or both wings and blisters were smaller. this result confirmed that corto and rl interact during wing tissue formation. however, the observation that corto mutation enhanced a mild-activation of erk pathway whereas slowing-down a hyper-activation is paradoxical and requires further experiments to be fully understood. for all genotypes, the first allele was brought by the mother. only female phenotypes are reported here, but similar results were obtained for males. numbers of females with ectopic veins only or with two blistered wings among flies bearing a corto allele and over-expressing rlsem were compared to the one of flies over-expressing rlsem only. numbers of females with ectopic veins among flies containing the uas::dsmp transgene, the sd::gal transgene and a corto mutation were compared to that of flies containing the uas::dsmp and the sd::gal transgenes only. we have recently shown that the drosophila ortholog of mp dmp antagonizes rl vein-promoting function in the future intervein cells of the wing. furthermore, we isolated dmp in a two-hybrid screen using corto as bait. we thus tested the genetic interactions between corto and dmp as already described, down-regulation of dmp by rna interference using the sd::gal driver induced ectopic veins in % of flies. this percentage increased to % and 100% in combination with corto or corto07128b, respectively. with corto07128b, the expressivity of the ectopic vein phenotype was also enhanced. therefore, these results showed that corto and dmp act synergistically and participate in intervein tissue differentiation in response to erk signaling. corto interacts in vitro directly with erk and indirectly with dmp1 we have previously shown that dmp 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, dmp 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 amino-acid protein contains three globular domains that might correspond to functional domains. the first one is located at position 127- and exhibits strong structural similarities with chromodomains, that are chromatin targeting modules found in some regulators of chromatin structure. the two others, located at positions 418- and 480- present no obvious similarities with known protein domains. in vitro translated erk protein was retained on gst-c1/ and gst-c325/ beads containing the nh2-terminal half and the cooh-terminal half of corto, respectively. in contrast, erk was not retained on gst-c127/ beads containing the corto chromodomain, or on gst-c418/ beads containing part of the two cooh-terminal globular domains. the lack of interaction with gst-c127/ and gst-c418/ 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 dmp in a two-hybrid screen using the nh2-terminal part of corto as a bait, we next questioned the physical interaction between corto and dmp we performed gst pull-down assays using gst-dmp 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-dmp beads indicating that there was no specific direct interaction between corto and dmp however, by incubating gst-dmp beads with total embryonic protein extract, we observed after blotting with anti-corto antibodies that corto was specifically retained on gst-dmp beads. therefore, we concluded that corto and dmp interact via additional factors. one potential candidate could be erk, since it directly interacts with corto and with dmp. corto is located both in the cytoplasm and the nucleus and is phosphorylated upon activation of the mapk cascade, erk is phosphorylated in the cytoplasm. di-phosphorylated erk phosphorylates in turn a large number of targets with diverse functions and different subcellular localizations. in particular, part of dp-erk is translocated into the nucleus where it phosphorylates some transcription factors. mammalian mp is present in the cytoplasm in association with erk but its possible nuclear localization has not been reported. nevertheless, we have recently shown that dmp is present both in the cytoplasm and the nucleus. we thus asked whether corto was present in the same compartments as erk and dmp corto was detected in nuclear and cytoplasmic extracts from embryos, third instar larvae and schneider s cells. a similar nuclear and cytoplasmic distribution was observed for a corto-flag fusion protein expressed in s cells. both corto and corto-flag exhibited several isoforms very close to each other in size, with the lowest isoforms being more abundant in the cytoplasm than in the nucleus. corto is very rich in serine, threonine, tyrosine, and presents many predicted phosphorylation sites for several kinases distributed all along the sequence. hence we checked whether the isoforms we observed could indeed correspond to differentially phosphorylated molecules. as shown in figure 5d, the upper bands of corto-flag disappeared after lambda phosphatase treatment. altogether, these results showed that corto is a phosphorylated protein present both in the cytoplasm and in the nucleus. in addition, the corto phosphorylation pattern seemed to be different between the two cellular compartments, with enrichment in highly phosphorylated forms in the nucleus. phosphorylation of m a chromatin regulator, has been shown to regulate its nuclear translocation. further experiments are required to determine if corto localization is regulated through a similar mechanism. the phosphorylation pattern of corto is controlled at least partially by erk pathway since corto is phosphorylated and interacts in vitro with erk, it is tempting to speculate that corto could be phosphorylated in response to activation of the corresponding map kinase cascade. to answer this question, we transfected s cells in absence of serum with a plasmid allowing corto-flag expression. after two days, we transiently activated the map kinase cascade by a short treatment either with serum, with serum plus insulin or with serum plus pma. in all three conditions, faint upper corto isoforms appeared, showing that corto phosphorylation was induced very rapidly upon erk activation. to confirm the existence of these faint upper isoforms, we immunoprecipitated corto from these extracts using anti-flag antibodies and blotted the immunoprecipitates with antibodies directed against phosphoproteins. this experiment revealed first that corto is constitutively phosphorylated even without map kinase activation. second, erk activation induced an hyperphosphorylation of corto since an upper smear containing several bands very close in size appeared in all three conditions of activation tested. as another way to activate the erk pathway, we co-expressed corto-flag and tagged forms of either erk, erksem or rasv in s cells. similar to erk and erksem over-expression, over-expression in flies of a constitutively active form of ras, rasv induces ectopic vein cells. surprisingly, smaller corto isoforms appeared when constitutively activating the erk pathway with either erk, erksem or rasv. 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 sp sites at positions and that correlate with theoretical erk1/erk 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 dmp or corto takes place in the nucleus only we next performed co-immunoprecipitation experiments to see whether corto interacts with erk and dmp in vivo. we have previously shown that erk and dmp co-immunoprecipitate in a total protein extract. in order to determine the subcellular localization of this dmp1/erk complex, we carried out co-immunoprecipitation experiments using cytoplasmic or nuclear extracts of s cells expressing dmp and erk tagged proteins. surprisingly, dmp1-myc co-immunoprecipitated with erk-flag in the nuclear extract only. corto-myc and erk-flag also co-immunoprecipitated in the nuclear extract only. in this last experiment, all corto isoforms were co-immunoprecipitated with erk. in both cases, the lack of co-immunoprecipitation in cytoplasmic extracts was confirmed by using two different protocols to prepare nuclear and cytoplasmic extracts. the co-immunoprecipitation observed between corto and erk fusion proteins was confirmed with endogenous proteins from a total embryonic extract using anti-dp-erk antibody. furthermore, this experiment showed that corto was able to interact with dp-erk. lastly, we observed no co-immunoprecipitation between corto-flag and dmp1-myc whether in total, cytoplasmic or nuclear extracts. labile protein interactions can be stabilized by cross-linking before performing cell extracts, although such treatment does not allow separating cytoplasmic and nuclear extracts. when using a total extract from cross-linked cells, we could detect co-immunoprecipitation between corto-flag and dmp1-myc. this observation was consistent with our gst pull-down and peptide pull-down assays showing that the interaction between dmp and corto was not direct but was probably mediated by other proteins. altogether, our co-immunoprecipitation results suggest that a complex containing corto, erk and dmp might exist in the nucleus only. the core protein of this complex should be erk, since it interacts directly with both corto and dmp erk and dmp bind polytene chromosomes where they partially co-localize with corto to further analyze the relationship between corto, dmp and erk, and since corto has been shown to bind polytene chromosomes, we analyzed the binding of erk and dmp onto polytene chromosomes. we observed that erk and dmp bound polytene chromosomes on many sites where they completely co-localized. furthermore, corto and dmp co-localized on several sites. these results suggest that a dmp1/erk/corto complex might regulate targets directly on chromatin. since the co-localization of corto with dmp is not complete, corto appears to be an optional partner of dmp1/erk on chromatin. corto association may require additional factors or may be controlled by signaling events. 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 dmp 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 dmp interact during wing tissue differentiation in drosophila. corto, erk and dmp form a complex exclusively in the nucleus. in addition, these proteins bind polytene chromosomes where they partially co-localize, suggesting that the corto-erk-dmp 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. 
interestingly, its phosphorylation pattern differs between cytoplasm and nucleus and changes upon erk activation. genetic interactions show that corto and dmp act together to antagonize rolled in the future intervein cells, thus promoting intervein fate. functional relationships during wing development between a chromatin regulator, the enhancer of trithorax and polycomb corto, erk and its scaffold protein dmp are examined here. in drosophila, dmp that encodes an erk scaffold protein, regulates erk signaling during wing development and contributes to intervein and vein cell differentiation. these kinase modules are associated with scaffold proteins that control their activity. furthermore, corto, erk and dmp co-localize on several sites on polytene chromosomes, suggesting that they regulate gene expression directly on chromatin.
 mitogen-activated protein kinase cascades are involved in cell fate acquisition during development. these kinase modules are associated with scaffold proteins that control their activity. in drosophila, dmp 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 dmp are examined here.  genetic interactions show that corto and dmp act together to antagonize rolled in the future intervein cells, thus promoting intervein fate. although corto, erk and dmp are present in both cytoplasmic and nucleus compartments, they interact exclusively in nucleus extracts. furthermore, corto, erk and dmp 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. 