anterior open/yan

Promoter

Receptor tyrosine kinase (RTK) signaling plays an instructive role in cell fate decisions, whereas Notch signaling is often involved in restricting cellular competence for differentiation. Genetic interactions between these two evolutionarily conserved pathways have been extensively documented. The underlying molecular mechanisms, however, are not well understood. Yan, an Ets transcriptional repressor that blocks cellular potential for specification and differentiation, is a target of Notch signaling during Drosophila eye development. The Suppressor of Hairless (Su[H]) protein of the Notch pathway is required for activating yan expression, and Su(H) binds directly to an eye-specific yan enhancer in vitro. In contrast, yan expression is repressed by Pointed (Pnt), which is a key component of the RTK pathway. Pnt binds specifically to the yan enhancer and competes with Su(H) for DNA binding. This competition illustrates a potential mechanism for RTK and Notch signals to oppose one another. Thus, yan serves as a common target of Notch/Su(H) and RTK/Pointed signaling pathways during cell fate specification (Rohrbaugh, 2002).

To investigate how yan expression is regulated, DNA fragments comprising a 20-kb genomic sequence surrounding the first exon of yan were tested for regulatory potential in corresponding transgenic flies. Through this approach, a 122-bp eye-specific enhancer located approximately 3.5 kb upstream of the first exon was identified. In eight out of nine transgenic lines, this enhancer activated expression of a bacterial lacZ reporter gene within posterior undifferentiated cells of eye discs. This recapitulates the endogenous yan gene expression in eye discs with the exception of the MF region. Three putative Su(H) binding sites were found in the yan enhancer. When tested through an in vitro electrophoretic mobility shift assay (EMSA), the Su(H) protein was shown to specifically bind to these sequences. Further, the yan enhancer became inactive in most of the posterior undifferentiated retinal cells when the Su(H) function was removed. All together, these loss-of-function and DNA binding analyses support the notion that Su(H) is required to promote yan transcription and that yan is a target gene of Su(H) in the eye (Rohrbaugh, 2002).

E(spl) proteins are basic helix-loop-helix (bHLH) repressors, and most of them (m7, m8, mß, mdelta, and mgamma) are expressed in the posterior undifferentiated cells in eye discs. When E(spl) proteins (e.g., m7 and m8) are overproduced in eye discs, the yan enhancer activity is strongly reduced. Similarly, the level of Yan protein is also reduced. These results show that yan expression can be negatively regulated by E(spl) proteins. E(spl) proteins might act through an N box (5'-CACAAG-3') in the enhancer. Interestingly, mutations of the N box didn't cause upregulation of the reporter gene, but, instead, the reporter expression was abolished in all three transgenic lines. One explanation for this result is that the N box sequence might be shared by an activation element located in the region. Indeed, a Runt domain binding site (RBS) (5'-RACCRCA-3', R = purine) overlaps with the N box, which could mediate an effect by the Runt domain protein Lozenge (Lz), which has previously been shown to act as a transcriptional activator in the developing eye. Supporting this idea, the yan enhancer was completely inactivated in lz^r15 mutant eye discs. However, the level of Yan protein was not apparently affected by the lz mutation. This result suggests that Lz is not essential for the expression of the endogenous yan gene and that the loss of lz function could be compensated by other molecules so that yan expression is unaffected in lz mutants (Rohrbaugh, 2002).

A candidate factor that may be involved in this compensation and could cooperate with N^intra/Su(H) proteins might be a DNA binding protein capable of interacting with a 5'-GAAACC/A-3' sequence. Two direct repeats of a 5'-GAAACC-3' sequence (hexamer, HEX) were found between S1 and S2. The second half of the S2 site might be considered as a third HEX, since there is only one variant base (as 5'-GAAACA-3'). When clustered point mutations were introduced into the first and second HEX, expression of the reporter gene was abolished in all six transgenic lines. Therefore, the HEX element is essential for the yan enhancer activity. Expression analysis of the HEX repeats has provided further evidence supporting the finding that the hexamer is an activation element. The reporter gene expression can be detected over the entire eye disc of all six lines when a six-copy concatomer of a 22-bp sequence containing three HEX repeats is used; however, one copy of this 22-bp oligonucleotide is not sufficient to induce gene expression in eye discs. It is proposed that a putative HEX binding protein functions together with Su(H) and N^intra to activate the yan enhancer. The nature of the HEX binding factor remains to be investigated (Rohrbaugh, 2002).

An Ets domain binding site (EBS, 5'-GGAA/T-3') was found within the S2 site. Since Yan is an Ets domain protein and a transcriptional repressor, whether Yan could be involved in autoregulation was examined. When a constitutively activated Yan (Yan^Act) was overproduced in eye discs, the reporter gene expression was strongly reduced. Since Yan is capable of negatively regulating yan transcription, this autoinhibitory mechanism might be used to prevent overproduction of Yan in undifferentiated cells. DNA binding data suggests that Yan can be directly involved in this negative regulation. However, this Yan-mediated autoinhibitory feedback appears to play a minor role in regulating yan expression, because the yan enhancer activity is apparently not affected in yan mutant clones produced in eye discs (Rohrbaugh, 2002).

A role for RTK signaling in regulating yan transcription was investigated. When the RTK pathway is constitutively activated by tor^D-DER or Ras1^V12, the yan enhancer activity is greatly reduced. Thus, RTK signaling appears to negatively regulate yan transcription, in addition to its effect on Yan protein stability. Evidence supports a view that the inhibitory effect of RTK/Ras1 signaling on yan expression is mediated through the pointed (pnt) gene. Taken together, the results demonstrated that Pnt negatively regulates yan expression, and it is likely that Pnt is directly involved in repressing yan transcription. Although a role for Pnt as a transcriptional repressor has not been extensively investigated, pnt has been shown to negatively regulate hid transcription in embryos. Interestingly, a P-DLS motif is present in the Pnt protein (amino acids 356–360 in PntP1), which might mediate interaction with the transcriptional corepressor dCtBP. At this point, the data does not exclude the possibility that Pnt might also activate expression of a repressor, which in turn switches off yan transcription (Rohrbaugh, 2002).

The nesting of an Ets binding site within the S2 site suggests a possible mechanism whereby the binding of Pnt could interfere with Su(H)'s DNA binding activity. Indeed, increasing the amount of Pnt effectively prevents Su(H) from DNA binding. Such competition provides a mechanism by which RTK/Pnt signaling directly antagonizes Notch-mediated lateral inhibition at the transcriptional level. Since Ets binding sites are nested in many Su(H) binding sites, competitive occupancy of the common sequence could be a general mechanism for regulating expression of genes targeted by both Notch and RTK pathways (Rohrbaugh, 2002).

It is proposed that spatially restricted yan expression in the developing eye is coordinated by actions of multiple regulatory factors that include Su(H) and Pnt. Consequently, the yan enhancer provides an interface for Notch and RTK signals to oppose one another. The DNA binding analysis and mutagenesis of yan Su(H) binding sites provide evidence that supports a cell-autonomous role of Notch and RTK signaling in the regulation of yan expression. Interestingly, Yan expression is reduced not only in Su(H)^D47 clones but also in some Su(H)⁺ cells that surround the mutant clones in eye discs. This result implies that loss-of-Su(H) function might also cause a cell-nonautonomous effect on yan expression, possibly due to upregulation of RTK signaling in those Su(H)⁺ cells. This upregulation may occur via an increase of a diffusible activator of the RTK pathway due to the loss of Su(H). The model presented here illustrates a mechanism that should help explain how progenitor cells are maintained in an undifferentiated state by Notch-mediated inhibitory signals and how they can be effectively induced for cellular differentiation by RTK-mediated inductive signals (Rohrbaugh, 2002).

EGF receptor signaling regulates pulses of cell delamination from the Drosophila ectoderm: Activation of Yan

Many different intercellular signaling pathways are known but, for most, it is unclear whether they can generate oscillating cell behaviors. Time-lapse analysis of Drosophila embryogenesis has been used to show that oenocytes delaminate from the ectoderm in discrete bursts of three. This pulsatile process has a 1 hour period, occurs without cell division, and requires a localized EGF receptor (EGFR) response. High-threshold EGFR targets are sequentially activated in rings of three cells, prefiguring the temporal pattern of delamination. Surprisingly, widespread misexpression of the relevant activating ligand, Spitz, is compatible with robust delamination pulses. A single chordotonal organ precursor (called C1) and its progeny provide the source of secreted Spi relevant for oenocyte induction. Although Spitz ligand becomes limiting after only two pulses, artificially prolonging its secretion generates up to six additional cycles, revealing a rhythmic underlying mechanism. These findings illustrate how intercellular signaling and cell movements can generate multiple cycles of a cell behavior, despite individual cells experiencing only one cycle of receptor activation (Brodu, 2004).

The induction of larval oenocytes in Drosophila has been used as a simple model system for investigating the developmental regulation of EGFR signaling. Oenocytes are induced from the dorsal ectoderm of abdominal segments by a fixed and highly restricted source of Spi. This triggers a local EGFR response within a ring of overlying dorsal ectodermal cells, termed a whorl, leading to the upregulation of numerous oenocyte-specification genes and subsequent cell delamination. The simple cell geometry of the oenocyte whorl, together with time-lapse microscopy, was used to explore the timing of Spi secretion, EGFR-target activation, early cell induction, and later cell delamination. These studies reveal that oenocytes delaminate in bursts of three and identify the cell-counting mechanism as an EGFR-dependent pulse generator converting the time window of Spi secretion into final oenocyte number. This represents the first example of a rhythmic cell behavior other than the cell cycle to be reported in the Drosophila embryo (Brodu, 2004).
Rather than delaminating from the ectoderm in a continuous stream, oenocyte precursors segregate in discrete well-separated bursts of three cells. Genetic backgrounds affecting the pattern of cell segregation but not early fate specification were used to show how these pulses are regulated by EGFR signaling. The signaling parameters regulating the time of onset, time of cessation, and in particular, the cyclical nature of cell delamination of oenocytes are discussed (Brodu, 2004).

Using a panel of markers for double- and single-ring stages, it was possible to place gene expression 'snapshots' in temporal order with the cell movements recorded in movies. Three generic EGFR targets (activated Rolled/ERK, Yan, and argos) and three oenocyte-specific EGFR targets (Sal, svp^lacZ, and svp^lacZΔ18) were analyzed. In wild-type embryos, the high-threshold EGFR outputs of argos and svp^lacZ expression, detectable Rolled activation, and strong Yan downregulation are all inner ring specific, whereas lower-threshold outputs such as Sal upregulation and svp^lacZΔ18 expression are present in both precursor rings. Delamination itself also appears to be a high-threshold EGFR response and is thus confined to the inner ring (Brodu, 2004).

Molecules involved in cell adhesion can regulate both early signal transduction events, triggered by soluble factors, and downstream events involved in cell cycle progression. Correct integration of these signals allows appropriate cellular growth, differentiation and ultimately tissue morphogenesis, but incorrect interpretation contributes to pathologies such as tumor growth. The Fat cadherin is a tumor suppressor protein required in Drosophila for epithelial morphogenesis, proliferation control and epithelial planar polarization, and its loss results in a hyperplastic growth of imaginal tissues. While several molecular events have been characterized through which fat participates in the establishment of the epithelial planar polarity, little is known about mechanisms underlying fat-mediated control of cell proliferation. Evidence is provided that fat specifically cooperates with the epidermal growth factor receptor (EGFR) pathway in controlling cell proliferation in developing imaginal epithelia. Hyperplastic larval and adult fat structures indeed undergo an amazing, synergistic enlargement following to EGFR oversignalling. Such a strong functional interaction occurs downstream of MAPK activation through the transcriptional regulation of genes involved in the EGFR nuclear signalling. Considering that fat mutation shows di per se a hyperplastic phenotype, a model is suggested in which fat acts in parallel to EGFR pathway in transducing different cell communication signals; furthermore its function is requested downstream of MAPK for a correct rendering of the growth signals converging to the epidermal growth factor receptor (Garoia, 2005).

The results shown in this paper suggest that the interaction between ft and EGFR takes place at the proliferation level, while differentiation signals controlled by the EGFR pathway appear unaffected. With the aim to find some mechanisms that could explain the synergic phenotype of ft and EGFR mutations, the transcriptional levels of yan, dmyc and pnt, genes involved in proliferation control whose function is regulated by the EGFR cascade, were studied in ft and wild-type imaginal tissues. The results of semi-quantitative RT-PCR trials showed in ft tissues an increase of the transcription levels of yan and dmyc, whereas pnt was unaffected. The Dmyc transcription factor, the unique Drosophila homologue of the Myc family of proto-oncogenes, plays a central role in the control of cell growth in Drosophila. Overexpression of ras is capable to increase post-transcriptionally the Dmyc protein levels, promoting the G1-S transition via the increase of CycE translation. The increase in the Dmyc levels, however, affects growth rate but not proliferation, since the shortening of the G1 phase is balanced by the compensatory lengthening of G2, resulting in an increase in cell size but not in cell number. ft mutation otherwise induces an increase of cell proliferation without altering the cell size. Taken together, these results indicate that ft mutation affects not only the G1-S transition via Dmyc but also the G2-M transition, since the coordinated stimulation of the two cell-cycle checkpoints is necessary to increase the proliferation rate in Drosophila imaginal discs. Interestingly, the transcription level of pnt was unaffected in ft mutant discs. pnt is an ETS transcriptional activator that plays a central role in the mitosis control mediated by the EGFR signalling cascade; several studies however suggest the presence of additional Pnt-independent effectors in EGFR-mediated mitosis control. The ft control of the G2–M transition may involve EGFR effectors other than pnt, or molecules functioning through different signalling pathways. The yan gene is another component of the ETS transcriptional regulator family involved in the EGFR signalling. Phosphorylation by MAPK affects stability and subcellular localization of Yan, resulting in a rapid down-regulation of its activity. Yan functions as a fairly general inhibitor of differentiation, allowing both neuronal and non-neuronal cell types to choose between cell division and differentiation in multiple developmental contexts and recent studies indicate that the mammalian homologue of the Drosophila yan, TEL, is overexpressed in tumors. In the Drosophila developing eye yan is expressed in all undifferentiated cells and is down regulated as cells differentiate, so a high yan activity in ft mutant discs is correlatable with the observed proliferative advantage of ft cells (Garoia, 2005).

There are several indications that EGFR signalling can trigger different responses by different activity levels: in the Drosophila eye disc, differentiation requires high signalling levels, whereas lesser EGFR activity promotes mitosis and protects against cell death. These findings indicate that EGFR signalling may coordinate partially independent processes, transferring graded activity to the nucleus, rather than triggering 'all or none' responses. The simultaneous increase of activity in both growth promoters (dmyc) and differentiation repressors (yan) in ft mutant imaginal discs suggests the presence of a mechanism that shifts the EGFR nuclear equilibrium towards a level insufficient to induce differentiation but adequate for promoting cell growth and proliferation (Garoia, 2005).

The retinal determination gene eyes absent is regulated by the EGF receptor pathway throughout development in Drosophila

Members of the Eyes absent (Eya) protein family play important roles in tissue specification and patterning by serving as both transcriptional activators and protein tyrosine phosphatases. These activities are often carried out in the context of complexes containing members of the Six and/or Dach families of DNA binding proteins. eyes absent, the founding member of the Eya family is expressed dynamically within several embryonic, larval, and adult tissues of Drosophila. Loss-of-function mutations are known to result in disruptions of the embryonic head and central nervous system as well as the adult brain and visual system, including the compound eyes. In an effort to understand how eya is regulated during development, a genetic screen was carried out designed to identify genes that lie upstream of eya and govern its expression. This study identified a large number of putative regulators, including members of several signaling pathways. Of particular interest is the identification of both yan/anterior open and pointed, two members of the EGF Receptor (EGFR) signaling cascade. The EGFR pathway is known to regulate the activity of Eya through phosphorylation via MAPK. These findings suggest that this pathway is also used to influence eya transcriptional levels. Together these mechanisms provide a route for greater precision in regulating a factor that is critical for the formation of a wide range of diverse tissues (Salzer, 2010).

This report describes a genetic screen that identified factors that direct the expression of the retinal determination gene eyes absent to the developing embryonic head and eye imaginal disc. Putative regulators were identified by the loss or expansion of Eya protein distribution within the embryonic head of stage 9 loss-of-function mutants. The findings indicate multiple signaling cascades including Notch, Hedgehog, TGFβ, and the EGFR regulate eya expression. These results are consistent with previous studies identifying Hedgehog, Ras, and TGFβ as regulators of eya function in eye development. No mutations were recovered in any of known Wingless pathway members. This was slightly unexpected as Wnt signaling and eya are known to reciprocally regulate each other. This result could imply, however, that eya is regulated differently in diverse tissues (Salzer, 2010).

A screen similar to the one described in this study successfully identified the TGFβ pathway as an important upstream regulator of another retinal determination gene, dachshund. Of interest is the observation that the loss of TGFβ signaling has differential effects on eya and dac expression. In TGFβ mutant embryos ectopic dac expression was observed in cells of the visual primordium. However, eya expression remains unaffected in this tissue and is instead lost in the subsets of cells that give rise to the protocerebrum. These differential effects are interesting as eya and dac interact genetically within the retinal determination network. Therefore it seems that these regulatory relationships vary among different tissues. It also appears that the number of distinct signaling pathways that regulate eya expression outnumbers that of dac. This is unsurprising as the expression pattern of eya, when compared to dac, is considerably more dynamic, at least within the embryonic head (Salzer, 2010).

It was of particular interested to finding that mutations in spitz, argos, anterior open/yan and pointed, all members of the EGFR signaling pathway, altered the transcriptional pattern of eya. Previous work has demonstrated that the EGFR pathway post-translationally regulates Eya activity in the developing eye through phosphorylation via Ras/MAPK at two sites within the transactivation domain. Experiments in both flies and in insect cell culture indicate that phosphorylation augments, but is not absolutely essential, for either the transcriptional activation potential of Eya or for the induction of ectopic eyes in forced expression assays (Salzer, 2010).

These findings suggest that the EGFR pathway is also required to regulate eya transcription. This is consistent with findings that eya expression is lost in mago^- clones, which reduce Ras signaling (Firth, 2009). Indeed, loss of aop/yan behind the morphogenetic furrow results in the higher levels of Eya and its facultative partner So. Both proteins are required for photoreceptor cell fate specification and maintenance. Elevated levels of Eya and So proteins in yan mutant clones are consistent with roles for Yan in suppressing photoreceptor cell fate during normal development. In yan clones, Eya protein levels are activated to significantly higher levels than that of So. One possible explanation for these results is that EGFR signaling may in fact regulate eya expression but not that of so. As EGFR signaling also regulates Eya activity, in a yan clone there may be a feedback loop that ultimately results in lowered levels of Eya phosphorylation. Reduced levels of the Eya phospho-protein, while still able to stimulate so transcription, may do so at a less efficient rate thereby leading to lower levels of ectopic So protein (Salzer, 2010).

Unexpectedly, it was found that dac, a putative downstream target of the So-Eya complex, is not up regulated in yan clones. Rather, dac expression is down-regulated when yan is removed. As So-Eya is thought to positively regulate dac expression this result is somewhat puzzling. The result does suggest that dac is regulated not only by the Eya-So complex but also by other mechanisms, possible through EGFR signaling and an intermediate repressor. The So-Eya-Dac subcircuit is under complex regulatory control. This study suggests that still greater complexity exists in the form of differential regulation by signal transduction cascades both at transcriptional and post-translational levels (Salzer, 2010).

Targets of Activity

Yan acts at the promoter level to repress transcription activation by Pointed, a positive regulator of the sevenless/Ras/MAPK pathway (O'Neill, 1994).

phyllopod (phyl) encodes a novel protein required for fate determination of photoreceptors R1, R6, and R7, the last three photoreceptors to be recruited into the ommatidia of the developing Drosophila eye. Genetic data suggests that phyl acts downstream of Ras1, raf, and yan to promote neuronal differentiation in this subset of photoreceptors. Ectopic expression of phyl in the cone cell precursors mimics the effect of ectopic activation of Ras1, suggesting that phyl expression is regulated by Ras1. phyl is also required for embryonic nervous system and sensory bristle development (Chang, 1995).

R7 photoreceptor fate in the Drosophila eye is induced by the activation of the Sevenless receptor tyrosine kinase and the RAS/MAP kinase signal transduction pathway. Expression of a constitutively activated JUN isoform in ommatidial precursor cells is sufficient to induce R7 fate independent of upstream signals normally required for photoreceptor determination. JUN interacts with the ETS domain protein Pointed to promote R7 formation. This interaction is cooperative when both proteins are targeted to the same promoter and is antagonized by Yan, a negative regulator of R7 development. Furthermore, phyllopod, a putative transcriptional target of RAS pathway activation during R7 induction, behaves as a suppressor of activated JUN (Treier, 1995).

prospero gene becomes transcriptionally activated at a low level in all Sevenless-competent cells prior to Sevenless signaling, and this requires the activities of Ras1 and two Ras1/MAP kinase-response ETS transcription factors, Yan and Pointed. Activation of pros transcription in all cells within the R7 equivalence group requires the down-regulation of Yan activity through phosphorylation by MAPK in R7 and cone cell precursors. Loss of pointed results in a reduction in the number of pros expressing cells. Two other nuclear factors, Seven in absentia (SINA) and Phyllopod are required for R7 determination, but are not absolutely for pros expression. However, the presence of phyl in cells is sufficient to induce them to express elevated levels of pros. phyl requires sina activity to stimulate pros expression. SINA protein can be shown to form a complex with PHYL (Kauffmann, 1996)

How multifunctional signals combine to specify unique cell fates during pattern formation is not well understood. Together with the transcription factor Lozenge, the nuclear effectors of the Egfr and Notch signaling pathways directly regulate D-Pax2 (shaven) transcription in cone cells of the Drosophila eye disc. Moreover, the specificity of shaven expression can be altered upon genetic manipulation of these inputs. Thus, a relatively small number of temporally and spatially controlled signals received by a set of pluripotent cells can create the unique combinations of activated transcription factors required to regulate target genes and ultimately specify distinct cell fates within this group. It is expected that similar mechanisms may specify pattern formation in vertebrate developmental systems that involve intercellular communication (Flores, 2000).

shaven is the Drosophila homolog of the vertebrate Pax2 gene. This locus is represented by at least two classes of mutant alleles: shaven (sv) and sparkling (spa). spa mutants show cone cell defects resulting from mutations in the fourth intron of the gene, which have led to the identification of a 926 bp SpeI fragment within this intron that includes the eye-specific enhancer (Flores, 2000).

In EGFR^ts third-instar larvae raised at 29°C for 36 hr prior to dissection, Shaven expression is lost in cone cell precursors. To restrict the loss of Egfr function to the undifferentiated cells posterior to the furrow and cells that acquire their fates during the second phase of morphogenesis, a lz-Gal4 driver was used to express a dominant-negative form of Egfr. In these discs, Shaven expression is lost from cone cell precursors, while neuronal patterning in the precluster is maintained. Shaven expression was further examined in mutants of genes encoding the nuclear components of the Egfr signaling pathway, the repressor Yan and the activator PntP2. Shaven expression is also lost in discs in which lz-Gal4 drives the expression of a nonphosphorylatable form of Yan refractory to the Egfr signal. Similarly, in the hypomorphic pnt¹²³⁰ mutant, a modest reduction of Shaven expression occurs in cone cell precursors, while a stronger reduction is observed upon expression of a dominant-negative form of PntP2. These experiments together suggest that the Egfr signaling pathway activates shaven expression in cone cell precursors by relieving Yan-mediated repression and stimulating PntP2 activation (Flores, 2000).

The above genetic analysis does not address whether the effects of Egfr signaling on shaven transcription are direct or indirect. Therefore, in vitro mutagenesis was used to examine potential direct effects. Six ETS domain consensus binding sites were found in the SME. EMSAs show that two of these sites (1 and 6) are bound by both Yan and PntP2. Yan also binds to two additional sites (2 and 4). All six ETS sites were mutated to 5'-TTAA/T-3' in the context of SME-lacZ, and the resulting SME^mETSx6-lacZ construct was transformed into flies. In these transgenic flies, ß-galactosidase expression is lost from cone cell precursors. Since PntP2 was found to bind only to Ets sites 1 and 6, a SME-lacZ construct in which only these sites were mutated (SME^mETS(1,6)-lacZ) was transformed into flies. ß-galactosidase expression in cone cells is completely eliminated. These in vitro and in vivo results together demonstrate that PntP2 directly controls shaven expression in cone cell precursors by binding to ETS domain sites in the SME (Flores, 2000).

Mutating Su(H) and ETS binding sites eliminates expression of the target gene in the cone cells, which demonstrates a direct role for these pathways in transcriptional activation of shaven. Clonal analysis was undertaken to establish the requirement of the Notch and Egfr pathways in shaven expression. Unfortunately, these pathways are necessary for proliferation and have many layers of function. Therefore a flip-out strategy was used to inhibit N and Egfr function in GFP-labeled single-cell clones. This was best achieved in clones induced by GMR-flp. The GMR enhancer is only active behind the furrow and only a single cell division takes place in this population of cells. As a result, the clone size is very small. In a wild-type background, single cells marked with GFP express Shaven. However, when these single cells also express EGFR^DN or N^ECN, they do not express Shaven. Thus, cone cells need functional Notch and Egfr receptors in order to express Shaven (Flores, 2000).

The results described so far suggest that shaven expression is limited to cells which (1) express Lz; (2) receive a sufficiently strong Egfr signal to both alleviate Yan-imposed repression and stimulate PntP2 activation, and (3) receive a N signal able to stimulate Su(H) activation. The tripartite control of shaven expression in the cone cell precursors requires that they receive all three inputs at the proper time in their development. Lz expression in cone cell precursors has been documented. Consistent with their reception of the Egfr signal, activated MAPK is detected in cone cell precursors at the time when they initiate Shaven expression. Dl is expressed in developing photoreceptor clusters at the time when the cone cell precursors express Shaven. Thus, the neuronal clusters signal through an inductive Dl/N pathway to activate shaven expression in the neighboring cone cell precursors. These results suggest that, in addition to expressing Lz, the cone cell precursors receive the Egfr and N signals at the time of fate acquisition and Shaven expression. Presumably, at least one of these three activation mechanisms is lacking in cells that do not express shaven. This hypothesis was tested through genetic manipulation of the system (Flores, 2000).

Undifferentiated cells immediately posterior to the furrow receive the N signal and express Lz, but they do not express Shaven. It is hypothesized that the absence of Shaven expression in these cells is caused by a lack of the Egfr signal. This hypothesis is consistent with the observation that Egfr signaling causes these cells to differentiate. Indeed, Shaven is ectopically expressed in undifferentiated cells that express an activated form of Egfr. Loss-of-function yan^e2D/yan^pokX8 discs also show ectopic expression of Shaven in undifferentiated cells. Similarly, in discs expressing SME^mETSx6-lacZ, in which the six ETS sites in the SME are mutated, ß-galactosidase is also expressed in undifferentiated cells. Presumably, relief of Yan repression is sufficient to activate some shaven in undifferentiated cells. In SME^mETS(1,6)-lacZ,where the Pnt binding sites are eliminated but two of the Yan binding sites are still intact, there is no expression of ß-galactosidase in the undifferentiated cells. These results suggest that while the undifferentiated cells posterior to the furrow express Lz and receive the N signal, they fail to express Shaven because they do not receive the Egfr signal and are therefore unable to relieve the Yan-imposed repression of shaven (Flores, 2000).

Regulated transcription of the prospero gene in the Drosophila eye provides a model for how gene expression is specifically controlled by signals from receptor tyrosine kinases. prospero is controlled by signals from the Egfr receptor and the Sevenless receptor. A direct link is established between Egfr activation of a transcription enhancer in prospero and binding of two transcription factors that are targets of Egfr signaling. Binding of the cell-specific Lozenge protein is also required for activation, and overlapping Lozenge protein distribution and Egfr signaling establishes expression in a subset of equivalent cells competent to respond to Sevenless. Sevenless activates prospero independent of the enhancer and involves targeted degradation of Tramtrack, a transcription repressor (Xu, 2000).

Thus, Egfr signaling is required to activate pros expression in the R7 equivalence group but is restricted from activating pros expression in other cells by the distribution of the transcription factor Lz. The transcriptional effectors of the Egfr pathway combinatorially interact with Lz at an eye-specific pros enhancer to restrict enhancer activity to the R7 equivalence group. It is suggested that this mechanism is a primary means by which pros transcription is restricted to the R7 equivalence group. This combinatorial mechanism supposes that Egfr signaling inactivates Yan and activates Pnt, but modification of these transcription factors is not sufficient to activate the enhancer. Lz is also required to activate the enhancer. The only cells that contain Lz, activated Pnt, and inactivated Yan are R1, R6, R7, and cone cells. Thus, the enhancer is activated in a subset of Egfr-responsive cells. A similar combinatorial mechanism regulates shaven expression in cone cells. shaven expression requires both Lz and Egfr-induced regulation of Yan and Pnt. However, Notch signaling through Su(H) is also required for shaven expression in cone cells. This third input may explain why shaven has a more restricted expression pattern than pros, given that cone cells receive a robust Notch signal (Flores, 2000). In muscle and cardiac cells, RTK signaling is similarly integrated with other signal inputs and tissue-restricted transcription factors to regulate enhancer activity of the even skipped gene (Halfon, 2000). Thus, differential expression of genes in response to an RTK/Ras signal appears to be controlled by each gene's capacity to bind and be regulated by different combinations of transcription factors (Xu, 2000).

A model is presented for the regulatory inputs into prospero. (1) In eye progenitor cells, the presence of Yan represses pros transcription through its binding to the enhancer and competitively excluding Pnt from binding to the same sites. (2) Lz begins to be produced in progenitor cells after the first wave of photoreceptor differentiation. However, Lz alone cannot activate the enhancer in progenitor cells that have not received a Spitz signal. (3) When a progenitor cell receives a Spitz signal, Egfr is activated. This inactivates Yan, allowing activated Pnt to bind to the enhancer. At the morphogenetic furrow, the enhancer is inactive despite Egfr-stimulated cells containing inactive Yan and active Pnt since progenitor cells in this region do not contain Lz, which is also required for enhancer activity. Hence, photoreceptors R2, R3, R4, R5, and R8 do not express pros. It is only in cells that receive a Spitz signal and contain Lz that the combination of Lz and Pnt bound to the enhancer activate the enhancer. (4) Ttk88 reduces the level of pros transcription through a mechanism independent of the eye enhancer. This repression may not be strong enough to block the eye enhancer in the R7 equivalence group but acts to limit its level of transcription. (5) When a progenitor cell receives both a Spitz and Boss signal, stronger or longer signal transduction induces Ttk88 inactivation. This Egf represses pros transcription and leads to a specific increase of Pros in R7 cells (Xu, 2000).

The ETS factors Yan and Pnt have been implicated as substrates for activated MAPK, whose activities are modified upon phosphorylation. Both Yan and Pnt bind to the same sites in the pros eye enhancer except for one site that is Yan-specific. Their effects on enhancer activity are antagonistic; Yan represses while Pnt activates. One model is that Yan represses transcription by outcompeting Pnt for their binding sites, thereby preventing Pnt from activating transcription. This model is attractive since it has been found that Yan has a 100-fold greater affinity than Pnt for ETS factor binding sites in vitro. If this difference between purified fusion proteins in vitro is extrapolated to the fly eye, it would explain how Yan can outcompete Pnt and repress transcription. Results from mutagenesis of the binding sites is also consistent with this model. Mutated binding sites cause the enhancer to be inactive, which is the result predicted if Yan merely prevents Pnt from interacting with those sites. If Yan were actively repressing transcription in a manner dependent upon binding, then mutated binding sites would cause derepression and ectopic expression. Although a model where the binding sites are obligatory for both active repression by Yan and activation by Pnt cannot be excluded, the competitive binding model is the simplest one consistent with these data (Xu, 2000).

From these data it is proposed that two RTKs, Egfr and Sev, regulate pros by activating the Ras1 intracellular pathway in R7 cells, but these RTKs regulate pros differentially. Egfr regulates pros by modifying Yan and Pnt, which act directly through the eye-specific enhancer. The Egfr signal in R7 cells appears to occur before Sev, and it sufficiently inactivates Yan and activates Pnt to switch on the enhancer before the Sev signal. This sufficiency is demonstrated in sev mutants where enhancer activity in R7 cells is no different from wild-type. In contrast, the Sev signal in R7 cells is not sufficient to switch on the enhancer in the absence of the Egfr signal since the enhancer is inactive in Egfr mutant R7 cells (Xu, 2000).

How do these RTKs selectively regulate particular transcription factors and thereby regulate different aspects of pros transcription? The most attractive model is that RTK selection reflects the timing or intensity of each signal. If it is timing, then there must be a time period of competence during which a factor is sensitive to any RTK signal, and the time period is different for each factor. Alternatively, the intensity of a signal may dictate which transcription factor activities are sensitive. For example, Yan and Pnt activities may be insensitive to signal strength that is less than or equal to the level achieved by Sev but not Egfr within R7 cells. Ttk88 activity may be insensitive to signal strength that is less than or equal to the level achieved by Sev or Egfr alone but not the combination of the two within R7 cells. Signal 'strength' may be determined by the level of Ras pathway activity or the length of time that the Ras pathway is active. Sensitivity of transcription factors might be set either by the affinities of these factors for binding sites in a gene such as pros, or by the ability of factors to be substrates for RTK-stimulated modification. Given that Yan and Pnt are modified by a very different mechanism from Ttk88, substrate sensitivity is a possible determinant. In summary, RTK signals may provide specificity to gene regulation based on quantitative variation in which threshold transcription responses are set by transcription factors that have different sensitivities to RTK signal strength (Xu, 2000).

In addition to a post-translational regulation of Head involution defective (Hid), the Ras/MAPK pathway promotes cell survival in Drosophila by downregulating the expression of hid. Conversely, downregulation of the Ras/MAPK pathway induces cell death by upregulating hid expression. hid transcript levels are downregulated in dominantly active Dras1- (Dras1^Q13) expressing embryos when assayed 3 hr after heat shock. In wild-type embryos, total HID mRNA levels do not change dramatically between stage 11, when Ras expression was ectopically induced, and stage 14, when HID mRNA levels were assayed. This eliminates the concern that developmental arrest might account for the observed difference in HID mRNA levels. It was observed that hid levels return to normal in Dras1^Q13 embryos by 5 hr after heat shock. Cell death also resumes in these embryos several hours later. This indicates that a transient increase in Ras activity leads to a transient suppression of hid expression, accompanied by a transient protection from naturally occurring cell death. HID mRNA levels were also assayed through an alternative procedure: whole mount in situ analysis. These results confirm that hid transcript levels decline in dominantly active Dras1- (Dras1^Q13) expressing embryos. This is particularly apparent in the midline glia, which strongly express hid. The survival of midline glia is known to depend on the activity of the Epidermal growth factor receptor pathway. To confirm that Ras regulation of hid utilizes the Raf/MAPK pathway, the effect of a constitutively active form of Draf (phlF22) on hid expression has been investigated. In situ analyses were performed on embryos expressing activated Draf under the control of the heat shock promoter. Heat-induced expression of phlF22 results in downregulation of hid transcript levels, suggesting that Ras functions through the Raf/MAPK pathway to downregulate hid expression (Kurada, 1998).

Reduction in pointed (pnt) activity has been observed to enhance ectopic Hid induced cell death in the eye. The pointed transcription factor is a target of MAPK function and acts as a positive regulator in the R7 pathway. The pnt gene encodes two related proteins, pnt1 and pnt2. pnt2 operates downstream of the MAPK rolled in the Ras pathway. Therefore, the consequences of ectopic expression of pnt2 were examined. Embryos were generated that carry UAS-Pnt2 and a midline glia-specific Gal4 driver (52A-Gal4), resulting in the expression of pnt2 in the midline glia cells. Such embryos were tested for hid levels by whole-mount in situ analysis. Like embryos expressing activated Dras1 and activated Draf, pnt2-expressing embryos show decreased hid transcript levels, indicating that the Ras/MAPK pathway, acting through pnt, downregulates hid transcription (Kurada, 1998).

Since upregulation of the Ras/MAPK pathway promotes cell survival and downregulates hid expression, it was predicted that increased hid expression is the cause of the increased apoptosis observed when Ras activity is decreased. Ubiquitous expression of the negative regulator yan is able to induce massive embryonic apoptosis. In these same embryos HID mRNA levels are increased within 2 hr of yan^Act induction and continue to rise for many more hours. Thus, downregulation of Ras activity in the embryo results in increased hid transcription and apoptosis, and this transcription is regulated either directly or indirectly by yan. These results imply that Ras activation of MAPK and inactivation of yan is an important cell survival pathway in embryos (Kurada, 1998).

Blocking Epidermal growth factor receptor activity in the developing eye also enhances apoptosis. If hid is a target of Egfr/Ras/MAPK activity in this tissue, then hid levels should increase when Egfr activity is blocked. Expression of a dominant negative Egfr in the developing eye results in a band of increased hid transcription in the eye disc. This band lies several rows posterior to the furrow and corresponds well with the first developmental defects seen in these eye discs. In sum, these data implicate the downregulation of hid transcription as an important component of Egfr antiapoptotic activity. The post-transcriptional modification of Hid appears to be equally important (Kurada, 1998).

In undifferentiated cells of the larval eye imaginal disc, the transcriptional repressor Yan outcompetes the transcriptional activator Pointed for ETS binding sites on the prospero enhancer. During differentiation, the Ras signaling cascade alters the Yan/Pointed dynamic through protein phosphorylation, effecting a developmental switch. In this way, Yan and Pointed are essential for prospero regulation. Hyperstable Yan^ACT cannot be phosphorylated and blocks prospero expression. Lozenge is expressed in undifferentiated cells, and is required for prospero regulation. The eye-specific enhancer of lozenge has been sequenced in three Drosophila species spanning 17 million years of evolution and complete conservation of three ETS consensus binding sites was found. lozenge expression increases as cells differentiate, and Yan^ACT blocks this upregulation at the level of transcription. Expression of Lozenge via an alternate enhancer alters the temporal expression of Prospero, and is sufficient to rescue Prospero expression in the presence of Yan^ACT. These results suggest that Lozenge is involved in the Yan/Pointed dynamic in a Ras-dependent manner. It is proposed that upregulated Lozenge acts as a cofactor to alter Pointed affinity, by a mechanism that is recapitulated in mammalian development (Behan, 2002).

A genetic approach was used to examine Yan/Lz interactions; Yan is shown to temper lz expression. The degree of regulation is dependent upon the presence of the eye-specific enhancer. The complete conservation of three Ets binding sites across 17 million years of evolution is strong supporting evidence that this regulation is direct. At this time, however, the possibility that this regulation is indirect cannot be ruled out (Behan, 2002).

Separate inputs by Ets factors and Lz have been shown to be required for regulation of prospero and D-Pax2. The current data must be interpreted in this context. The lz^gal4 reporter system was used to show that hyperstable Yan^ACT is able to block lz expression at the level of transcription. The GMR and Sev ectopic expression systems have been used to tease out Yan control of lz apart from control of other genes (Behan, 2002).

A model is here proposed for prospero regulation by Prospero and Yan, with the added information that lz is also a target of Yan. Yan tempers Lz expression. In the undifferentiated cell, Yan represses prospero by directly binding to Ets sites. The transcriptional activator Pointed competes for the same DNA but with much less affinity. Lozenge transcription is tempered by Yan, but not entirely repressed. Upon activation of Egfr and Sevenless by their respective ligands Spitz and BOSS, Ras1 is stimulated. Ultimately, Yan and Pointed are phosphorylated, downstream of Ras1 but with opposite effects. Phosphorylated Yan is targeted for degradation. Phosphorylated Pointed binds DNA with a higher affinity. Yan repression of Lz is alleviated, and becomes upregulated by some other mechanism. Upregulated Lz binds with Pointed to mediate prospero transcription (Behan, 2002).

This begs the question, why the double level of control, i.e., with both Lozenge and Pointed required for prospero regulation? It is hypothesized that Lz, functioning as a transcription factor, is involved directly in the Ets developmental switch, acting as a cofactor to enhance the ability of Pointed to compete with Yan for Ets sites. What follows supports this argument. The developmental potential of R7 in the presence of one dose of sev-yan^ACT has been analyzed. In this mutant background, Pointed is phosphorylated normally by MAP kinase, but is unable to compete with the mutant hyperstable Yan. The result is that Prospero expression is never upregulated: Runt expression is not turned on, and R7 differentiation fails. Coexpression of GMR-[lz-c3.5] rescues Prospero expression. Furthermore, the ectopic R7 cells that develop in the GMR-[lz-c3.5] background follow their normal developmental pathway. Unlike the native R7 precursors, these ectopic R7 precursors express both upregulated Prospero and Runt. This may indicate a difference in the level of expression of the sev-yan^ACT transgene between the two cell types, or some other cell-specific factors involved in regulation. The results in both the endogenous and ectopic R7 cells indicate that the presence of Lz effects a change in the dynamic between Yan and Pointed (Behan, 2002).

Although this could be accomplished by a number of mechanisms, the hypothesis is favored that Lz induces a change in the ability of Pointed to bind to DNA. This is based on a mammalian paradigm. The mammalian homologs of Lz and Pointed are RUNX1 and Ets-1. Lz is 71% identical to RUNX1 in its homologous domains. Pointed is 95% identical to Ets-1 in the Ets DNA binding domain; Pointed and Ets-1 proteins are functionally homologous and can replace each other in vitro and in vivo. It has been shown that RUNX1 and Ets-1 bind cooperatively to separate, but nearby, DNA sites on the T cell receptor, and that this cooperativity can exist even when these sites are as far apart as 33 base pairs. Notably, RUNX1 and Ets-1 can not substitute for each other. Both inputs are required for stable ternary complex development. The presence of RUNX1 enhances Ets-1 DNA binding affinity by as much as 20 times in vitro. Regions of RUNX1 and Ets-1 outside of the DNA binding domains that are necessary for cooperative DNA binding, and similar regions exist in both Lz and Pointed proteins. Furthermore, it has been speculated that this type of cooperativity may exist in Drosophila eye development (Behan, 2002 and references therein).

Strikingly, in the prospero enhancer one Ets binding site is only 7 base pairs away from a Lz binding site. The genetic results reported in this study are consistent with Lz and Pointed acting cooperatively on the prospero enhancer. The double input of Lz and Pointed effectively competes with Yan^ACT. Clearly a dynamic exists between the three factors Lz, Yan and Pointed. Work in flies and mammals has shown that this dynamic is influenced by phosphorylation, competition, transcriptional regulation and cofactor availability (Behan, 2002).

Spatially and temporally regulated activity of Branchless/Breathless signaling is essential for trachea development in Drosophila. Early ubiquitous breathless (btl) expression is controlled by binding of Trachealess/Tango heterodimers to the btl minimum enhancer. Branchless/Breathless signaling includes a Sprouty-dependent negative feedback loop. Late btl expression is a target of Branchless/Breathless signaling and hence, Branchless/Breathless signaling contains a positive feedback loop, which may guarantee a continuous supply of fresh receptors to membranes of growing tracheal branch cells. Branchless/Breathless signaling activates MAP-kinase, which in turn, activates late btl expression and destabilizes Anterior-open (Yan), a repressor for late btl expression. Biochemical and genetic analysis has indicated that the minimum btl enhancer includes binding sites of Anterior-open (Ohshiro, 2002).

The minimum btl enhancer consists of B2 and B3 regions, the latter, a late enhancer. lacZ expression driven by B3 enhancer mimics btl late expression. The B3 enhancer may thus contain binding sites for Aop. Ets-domain containing proteins binds to DNA via conserved Ets domains and the canonical sequence of their targets is 5'GGA. The B3 enhancer contains an inverted repeat of GGA quite near 3' of CME2, a Trh/Tgo binding element. A study was made to determine Aop-capability for binding to the GGA pair. The Ets domain of Aop (ETS^AOP) was expressed in Escherichia coli cells and partially purified from cell extracts followed by electrophoresis mobility shift assay (EMSA). Fragment C contains only one GGA, while W is a portion of B3, containing the GGA pair. M1_¯M4 are mutants for GGA sites. ETS^AOP forms complexes with C and W. The W/ETS^AOP complex shows mobility apparently less than that of the C/ETS^AOP complex. Each W/ETS^AOP complex may contain two molecules of ETS^AOP, since W contains two GGAs. Consistent with this, W mutants with a single copy of GGA (M1 or M3) forms complexes with ETS^AOP whose mobility was similar to that of the C/ETS^AOP complex. No stable ETS^AOP/DNA complex is formed subsequent to disruption of two Ets sites, strongly suggesting that targets for Aop in B3 are the GGA pair, ETS1 and ETS2. ETS1 and ETS2 are concluded to be Aop targets required for in vivo transcription of btl (Ohshiro, 2002).

Unlike ETS^AOP, the Ets domain of Pnt (ETS^PNT) can bind to both W (wild-type B3 enhancer) and M1-M4 (mutant B3 enhancers), suggesting that Pnt is capable of binding to B3 through sequences other than ETS1 and ETS2 or mutated EST sequences. Consistent with this, significant btl-lacZ signals are detected in B3[M2]-lacZ embryos, expressing Aop^ACT (Ohshiro, 2002).

aop is expressed in most developing tracheal cells and its protein product is localized mainly in the nucleus, except for tip cells that receive the highest levels of Bnl signals. Misexpression of the activated form of Aop in tracheal cells virtually completely abolishes Btl signals and the absence of aop activity leads to btl misexpression in TC, from which btl expression is normally absent. Conversely, the overexpression of bnl, which stimulates Bnl/Btl signal transduction, brings about almost the complete elimination of Aop nuclear signals from most tracheal cells. The destabilization of Aop phosphorylated by the activated form of MAPK would be the most likely reason for this. It thus quite logically follows that the area of normal late btl expression is determined partly by an activity balance between ubiquitous Aop and Bnl/Btl signaling, whose activity should diminish gradually with distance from the Bnl source. Note that in contrast to Spry, whose expression is under the control of Bnl/Btl signaling, aop is transcribed constitutively in a Bnl/Btl-signaling-independent manner. btl is expressed even in growing trachea branch cells associated with nuclear Aop signals. High concentration of Pnt may overcome Aop-dependent repression and activate btl in these cells. Pnt is thought to compete with Aop to activate target gene expression. In pnt mutant embryos, btl expression is lost, again indicating that Pnt activates btl expression. Pnt transcription has been shown to be dependent on Bnl/Btl signaling and, accordingly, Bnl/Btl signaling is expected to activate Pnt expression in Bnl receiving cells. Thus, that the threshold value of Bnl/Btl signaling for btl expression is much lower than that for elimination of nuclear Aop is most likely due to the presence of this Pnt-mediated positive feedback regulation (Ohshiro, 2002).

EMSA along with an enhancer assay indicates that btl B3 enhancer, about 120 bp long and responsible for late btl expression contains an inverted repeat consisting of two Aop binding sites (GGAs; ETS1 and ETS2), suggesting that dimerized Aop binds to the B3 enhancer. Pnt is considered to compete with Aop for a common target sequence to activate target gene expression. pnt is essential for late btl expression. As with Aop, the Ets domain of Pnt (ETS^PNT) is capable of forming a complex not only with the authentic wild-type B3 enhancer, but with mutant forms of B3 enhancer as well. Pnt may thus be capable of binding to mutated ETSs or B3 sequences other than ETS1/ETS2. The B3 enhancer also contains a sequence similar to the Salr target in chorion s15. This putative Sal/Salr binding site is situated in the B3 enhancer just in the 3' vicinity of ETS1; ETS2 and the putative Sal/Salr binding site may partly overlap. Preliminary experiments have shown that Sal is capable of binding to the B3 enhancer (Ohshiro, 2002).

MAE, a dual regulator of the EGFR signaling pathway, is a target of the Ets transcription factors Pnt and Yan

Ets transcription factors play crucial roles in regulating diverse cellular processes including cell proliferation, differentiation and survival. Coordinated regulation of the Drosophila Ets transcription factors Yan and Pointed is required for eliciting appropriate responses to Receptor Tyrosine Kinase (RTK) signaling. Yan, a transcriptional repressor, and Pointed, a transcriptional activator, compete for regulatory regions of common target genes, with the ultimate outcome likely influenced by context-specific interactions with binding partners such as Mae (FlyBase name: ETS-domain lacking). Previous work in cultured cells has led to a proposal that Mae attenuates the transcriptional activity of both Yan and Pointed, although its effects on Pointed remain controversial. A new layer of complexity to this regulatory hierarchy is provided whereby mae expression is itself directly regulated by the opposing action of Yan and Pointed. In addition, Mae can antagonize Pointed function during eye development; a finding that suggests Mae operates as a dual positive and negative regulator of RTK-mediated signaling in vivo. Together these results lead to a proposal that a combination of protein-protein and transcriptional interactions between Mae, Yan and Pointed establishes a complex regulatory circuit that ensures that both down-regulation and activation of the RTK pathway occur appropriately according to specific developmental context (Vivekanand, 2004).

Because mae expression in wild-type embryos is reminiscent of the expression patterns of genes such as argos (aos) and orthodenticle (otd) that have been shown to be regulated by Epidermal growth factor receptor (EGFR) signaling, whether mae expression might be similarly regulated by the downstream EGFR pathway effectors, Yan and Pnt, was investigated. Analysis of the genomic region around mae reveals two clusters of ETS DNA binding consensus sites (EBS; defined as GGAA/T), one upstream of the transcription start site (MaeEBS1) and the other in the intron of mae (MaeEBS2), further suggesting that Yan and Pnt might regulate mae expression. To explore this possibility, in situ hybridization experiments were performed to determine whether mae expression is affected by altering the dosage of Yan and Pnt. As predicted, based on the presence of EBS clusters in the mae genomic region, mae expression is significantly increased in yan mutant embryos, while it is lost in pnt mutant embryos. Conversely, ubiquitous overexpression of Yan^ACT or Pnt results in down-regulation and up-regulation of mae expression, respectively. In addition to regulating mae expression in the embryo, Yan and Pnt also regulate mae expression in eye imaginal discs. Over-expression of PntP1 results in almost three-fold increase in mae levels, while over-expression of Yan^ACT results in a decrease. Taken together, these results suggest that mae expression is regulated by the Ets transcription factors Pnt and Yan in multiple developmental contexts (Vivekanand, 2004).

To determine whether Pnt and Yan regulate mae levels directly, the EBS clusters were cloned upstream of a minimal promoter and luciferase cDNA to generate two different MaeEBS-luciferase reporters, MaeEBS1-luciferase (upstream cluster) and MaeEBS2-luciferase (intronic cluster). This enabled an assessment of the effects of Pnt and Yan on these putative regulatory elements by performing transcription assays in Drosophila S2 cells. If Pnt and Yan directly regulate mae transcription, then the prediction would be that Pnt and Yan would bind to the EBSs and activate and repress transcription of the reporter, respectively (Vivekanand, 2004).

Both the upstream and the intronic EBS clusters behaved similarly in these luciferase reporter assays. Addition of the constitutively activated form of Pnt, PntP1, resulted in activation of the reporter, while co-transfection of Yan with PntP1 resulted in two to three fold repression in transcription. Similarly, co-transfection of PntP2 and RAS^V12 resulted in transcriptional activation of the reporter. The transcriptional modulation of the MaeEBS-luciferase reporters by Pnt and Yan supports the hypothesis that mae expression is directly regulated by Pnt and Yan in vivo (Vivekanand, 2004).

MAE has been shown to antagonize Pnt function, putting it in the unique position of being a dual positive and negative regulator of EGFR-mediated signals. Intriguingly, mae expression is itself regulated by Pnt and Yan, suggesting a whole new layer of feedback loops that fine-tune and down-regulate signaling (Vivekanand, 2004).

While overexpression of MAE blocks Yan's repression capability (Tootle, 2003), this occurs without altering Yan nuclear localization. Thus increased MAE expression appears to interfere directly with Yan-mediated transcriptional repression. An intriguing model to explain this finding originates from the observation that homotypic interactions mediated by the Pointed Domain (PD) of TEL, the mammalian ortholog of Yan, result in the formation of TEL polymer that may facilitate transcriptional repression by wrapping around the target DNA (Kim, 2001). Yan is similarly capable of self-association and the residues that are required for TEL polymerization have been conserved, suggesting Yan-Yan polymerization might similarly be critical for repression (Jousset, 1997 and Qiao, 2004). In this context, perhaps clusters of EBSs, similar to those described in mae, by recruiting multiple Yan molecules to a common target site may provide a scaffold for nucleating and promoting Yan polymerization (Vivekanand, 2004).

Such a model requires a mechanism to limit the extent of polymer formation, such that the cell can achieve efficient but reversible repression of target genes. Considering its multifaceted role in down-regulating Yan activity and its ability to bind the PD of Yan, MAE is a prime candidate to fill such a role. Consistent with this prediction, recent studies have found that PD-mediated polymerization of Yan is required for transcriptional repression and that MAE effectively 'caps' Yan oligomerization by occluding the residues required for polymerization (Qiao, 2004). Thus it is tempting to speculate that MAE's ability to abrogate Yan-mediated repression may reflect a role in 'depolymerizing' Yan at the DNA, an intriguing model that remains to be validated in vivo (Vivekanand, 2004).

In addition to antagonizing Yan activity, this work suggests that MAE also negatively regulates PntP2 function, thus positioning it uniquely within the RTK pathway as both a positive and negative regulator. For example, the phenotypes associated with misexpression of MAE in the Drosophila visual system are completely suppressed by co-expression of PntP2, arguing strongly that MAE can antagonize EGFR signaling in the eye by interfering with the activity of PntP2. While the photoreceptor loss and increased apoptosis phenotypes associated with MAE overexpression resemble the consequences of blocking Yan nuclear export and down-regulation, the reduced Yan expression observed in MAE-expressing eye disc argues against such an explanation. Furthermore, if MAE were inducing premature down-regulation of Yan in these cells, one would expect to observe ectopic photoreceptors, rather than the neuronal loss that actually occurs. Thus, although a direct effect on Yan cannot be excluded, the interpretation is favored that the primary consequence of MAE overexpression is reduction in activity of PntP2, and that the loss of Yan expression is a secondary outcome. It is important to note that both cell culture and in vivo experiments employ overexpression strategies that are subject to the caveats inherent to such analyses. Thus these experiments are viewed as an opportunity to reveal new mechanistic hypotheses that will provide an important foundation for future studies designed to unravel the complex regulatory circuitries that exist between MAE, Yan and Pnt in vivo (Vivekanand, 2004).

Induction of both positive and negative feedback loops by signal transduction pathways plays an important role in regulating the response to pathway activation. Activation of PntP2 by EGFR/RAS/MAPK results in the transcription of target genes including Argos and Kekkon1, which have been shown to negatively regulate the pathway. This study identifies another target of the Ets transcription factors Pnt and Yan, mae, which performs the dual role of promoting and inhibiting signaling by the EGFR/RAS/MAPK pathway. Based on the effects on mae expression pattern observed in pnt and yan mutants and in embryos and eye imaginal discs overexpressing Pnt and Yan, it is proposed that Pnt activates while Yan represses mae transcription (Vivekanand, 2004).

Based on MAE's ability to antagonize EGFR signaling output, activation of mae transcription by PntP2 provides a negative feedback loop that would prevent runaway pathway activation. While Kekkon-1 and the secreted antagonist Argos act at the level of the receptor to down-regulate signaling, the induction of mae transcription would ensure the down-regulation of the pathway by inhibiting the function of the effector PntP2. This would result in cell autonomous inhibition of the EGFR/RAS/MAPK pathway at the level of the transcription factor. Moreover, while the previously identified inhibitors Argos, Sprouty and Kekkon1 function solely as antagonists of RTK signaling, MAE is unusual in that it acts both as a positive and negative regulator of the pathway by inhibiting both Yan and PntP2 function (Vivekanand, 2004).

Because MAE negatively regulates both Yan and PntP2 function, imposing constraints on MAE protein levels becomes critical. This appears to be achieved by regulating mae expression levels directly by Yan and Pnt. For example, because excess MAE could potentially break up Yan-Yan polymer to such an extent that Yan would no longer able to repress appropriate target genes (Qiao, 2004), the negative regulation of mae expression by Yan sets up a situation whereby excessive levels of MAE do not accumulate. Thus in the absence of RTK signaling, repression of mae by Yan would ensure that only low MAE levels are present in the nucleus, allowing Yan to repress transcription. Emphasizing the importance of fine-tuning the expression levels of these three nuclear RTK pathway regulators and further complicating the circuitry, it has been suggested that Yan and Pnt may also directly regulate each other's transcription, setting up additional positive and/or negative regulatory loops. For example, the finding that overexpression of PntP2 leads to up-regulation of Yan in the eye disc is consistent with a feedback loop whereby the activity of PntP2, a positive pathway effector, attenuates its own activity by increasing expression of the Yan repressor. A great deal of future work will be needed to unravel the precise in vivo contexts in which these complex transcriptional regulatory networks operate (Vivekanand, 2004).

In conclusion, MAE joins the panoply of regulators of EGFR signaling that have been shown to play an important role in modulating and restricting the strength, range and duration of signaling events. By establishing negative feedback loops that act at multiple levels within a signal transduction cascade, a robust checkpoint is established to attenuate as well as prevent constitutive signaling by the RTK pathway (Vivekanand, 2004).

A microRNA mediates EGF receptor signaling and promotes photoreceptor differentiation in the Drosophila eye: Reciprocal interactions between Yan and mir-7

A critical question about signal transduction is how weak or transient activation of signaling pathways achieves a robust and long-term switch in gene expression. A microRNA is part of a mechanism that makes cells sensitive to signals in the Drosophila eye. Expression of miR-7 is activated in cells as they begin differentiating into photoreceptors. This is dependent on EGF receptor (EGFR) signaling that triggers ERK-mediated degradation of the transcription factor Yan. In nonstimulated cells, Yan represses miR-7 transcription, whereas miR-7 RNA represses Yan protein expression in photoreceptors, by binding to sequences within its mRNA 3'UTR. It is proposed that reciprocal negative feedback between Yan and miR-7 ensures mutually exclusive expression, with Yan in progenitor cells and miR-7 in photoreceptor cells. Expression is switched when EGFR signaling transiently triggers Yan degradation. This two-tiered mechanism explains how signal transduction activity can robustly generate a stable change in gene-expression patterns (Li, 2005).

To determine the role of miRNAs in Drosophila eye development, the expression patterns of individual miRNAs were determined by in situ hybridization. A distinctive staining pattern in eye imaginal discs was detected with a probe for miR-7 RNA. Analysis of miR-7 expression in the eye imaginal disc revealed miR-7 RNA in photoreceptor cells at the time when neuronal differentiation is first detected. In photoreceptors, staining was concentrated at interfaces with other photoreceptors and was restricted to the cytoplasm. This indicates that the mature miRNA was being detected since miRNA precursors are predominantly localized to nuclei. Although staining was detected in progenitor cells within the morphogenetic furrow, miR-7 RNA was weakly detected in progenitor cells located posterior to the furrow, in regions surrounding nascent photoreceptors. Staining of discs that overexpressed miR-7 or were mutant for the gene (miR-7^Δ1) confirmed that the expression pattern was specific for miR-7 RNA (Li, 2005).

Binding of most animal miRNAs to the 3'UTR of substrate mRNAs results in repression of protein synthesis. To determine if the miR-7 RNA expressed in photoreceptors is active for gene silencing, a reporter gene that is repressed by miR-7 activity was assayed. This reporter has GFP coding sequence fused to a 3'UTR containing two perfect miR-7 binding sites and is based on the observation that a miRNA will direct mRNA cleavage if the target transcript is perfectly complementary in sequence. Reporter gene expression was strongly reduced in photoreceptors, whereas a control reporter, which lacked miR-7 binding sites, showed no reduction in photoreceptor expression. Altogether, these results indicate that miR-7 is present and active in developing photoreceptors (Li, 2005).

To examine the function of miR-7 in photoreceptor development, mutations were generated in the miR-7 locus. The miR-7 sequence is embedded within the bancal (bl) gene, oriented in the same direction as the bl transcription unit. Based on extensive EST cDNA analysis (FlyBase), three longer mRNA transcripts A-C are generated by alternative splicing, and a shorter transcript (D) is generated from a second promoter. Using RT-PCR of mRNA derived from adults, it was confirmed that these transcripts are present. Three lines of evidence indicate that miR-7 is cotranscribed with bl. (1) No noncoding RNAs were detected that spanned miR-7 sequence within the locus. (2) An inverted repeat in the Drosophila pseudoobscura genome has a 44/47 sequence match with D. melanogaster miR-7. The orientation and position of this D. pseudoobscura repeat is identical with respect to the bl ortholog. Thus, the relationship between bl and miR-7 appears to be functionally significant, based on evolutionary conservation. (3) In situ hybridization of bl mRNA using probes for the long and short transcripts revealed patterns that are highly similar to the miR-7 expression pattern in eye imaginal discs (Li, 2005).

It is unclear if one or more alternative transcripts gives rise to miR-7 RNA. However, based on data from an insertional mutation, the short transcript is at least competent to produce functional miR-7 RNA. This mutation is the EP element insertion EP954, which is located 52 base pairs upstream of the second transcription start site. EP954 belongs to a class of P element vectors that allow transcription of sequences flanking the insertion site. EP954 is oriented such that ectopic transcripts driven by Gal4-inducible transcription would correspond to the short transcript of bl. Indeed, this results in elevated levels of mature miR-7 in eye discs (Li, 2005).

When Gal4 was specifically expressed in an eye that carried the EP954 mutation, it caused a roughening of the eye surface. To determine if this effect was specifically due to miR-7, 432 bp of genomic DNA containing the miR-7 sequence were cloned into a Gal4-inducible expression vector and this was transformed into flies. The fragment contained no bl coding sequence. Indeed, the eye phenotype observed in UAS-miR-7 animals was indistinguishable from that observed with EP954 (Li, 2005).

Using the UAS-miR-7 transgene, miR-7 was misexpressed in progenitor cells posterior to the morphogenetic furrow and its effect on early cell differentiation was examined with cell-specific markers. In the larval eye disc, ectopic R7 photoreceptor neurons were observed in ommatidia. There were no ectopic cone cells produced, hence miR-7 specifically stimulated photoreceptor differentiation. A similar effect was seen with EP954. When miR-7 was misexpressed, ectopic photoreceptors were also readily seen in ommatidia at a later stage of eye development. On average, there were 8.9 photoreceptors per ommatidium in these eyes (Li, 2005).

The ectopic formation of photoreceptors by miR-7 misexpression resembled that observed in yan loss-of-function mutants. Since miRNAs act by downregulating protein expression, it seemed possible that miR-7 was repressing Yan protein expression. Yan expression was examined with anti-Yan antibody and a strong reduction of Yan protein was observed in progenitor cells that misexpressed miR-7 RNA. Computational methods were used to identify putative miR-7 binding sites within the 3'UTR of yan mRNA. Four putative miR-7 binding sites were identified based on sequence complementarity to miR-7 RNA. Two of these sequences are conserved in the yan ortholog of D. pseudoobscura, suggesting that they are functionally important. To confirm that the downregulation of Yan expression is mediated by miR-7 binding to yan mRNA, a reporter gene was generated with GFP coding sequence fused to the yan 3'UTR. Expression of the GFP reporter was downregulated in photoreceptor cells. The wild-type and mutant reporters were equivalently expressed in progenitor cells. These results indicate that the putative miR-7 binding sites in the yan 3'UTR specifically repress expression in photoreceptors, which correlates with the presence of miR-7 RNA in these cells. When miR-7 RNA was misexpressed in progenitor cells, there was a gradual decrease of wild-type reporter expression in progenitor cells, whereas the mutant reporter remained unaffected. Altogether, these data indicate that miR-7 can directly downregulate Yan expression (Li, 2005).

To confirm the function of miR-7 in repressing Yan expression, a null allele in mir-7 was generated. EP954 was excised by P-transposase activity, and an excision line with a 6.8 kb deletion of flanking genomic DNA was established. The mutant miR-7^Δ1 is missing miR-7 sequence in addition to the last two exons of bl and two exons of hillarin, a neighboring gene. As predicted, the mutant was determined to be a miR-7 RNA null allele (Li, 2005).

Tests were performed to see whether miR-7^Δ1 is defective for Yan repression in eye discs. If miR-7 RNA normally downregulates Yan protein expression, then the Yan reporter would fail to be repressed in miR-7^Δ1 photoreceptor cells. Indeed, the wild-type Yan reporter was derepressed in miR-7^Δ1 photoreceptor cells, whereas expression of the mutant Yan reporter was not significantly affected. To determine whether this specific effect on the Yan reporter was also observed with endogenous Yan protein, Yan protein expression was examined in miR-7^Δ1 mutant eye discs. An increase was observed in the number of apically positioned cells from miR-7^Δ1 discs that had Yan protein present in their nuclei. Altogether, these results indicate that miR-7 directly downregulates Yan expression (Li, 2005).

Despite the effect of miR-7^Δ1 on Yan expression, differentiation of photoreceptors and overall eye development of miR-7^Δ1 mutant animals appeared almost normal. The normal development of mutant photoreceptors suggested that basal Yan protein seen in miR-7^Δ1 mutants is not sufficient to effectively block photoreceptor differentiation. One possible reason is that miR-7 acts redundantly to inhibit Yan and promote photoreceptor differentiation (Li, 2005).

The existence of a protein-turnover mechanism to downregulate Yan suggested that it might act in parallel with miR-7 to repress Yan and thereby promote differentiation. Therefore, it was determined if miR-7^Δ1 affects photoreceptor differentiation when Yan protein turnover is blocked. The Yan^Act transgene has been mutated in all of its product's ERK phosphorylation sites, and its 3'UTR contains the most proximal miR-7 binding site (Rebay, 1995). Indeed, expression of Yan^Act protein is negatively regulated by miR-7, as evident by enhanced Yan protein staining in apically positioned Yan^Act miR-7^Δ1 mutant cells. Flies expressing Yan^Act have smaller eyes than normal, and ommatidia had on average 5.2 photoreceptors per ommatidium. This effect is due to the reduced protein turnover of the altered Yan protein. The inhibitory effect of Yan^Act on photoreceptor differentiation is dramatically enhanced in a miR-7^Δ1 mutant background. Early photoreceptor differentiation is particularly inhibited in the double mutant such that only an average of 3.7 photoreceptors per ommatidium were observed at later stages of eye development. The interaction between miR-7^Δ1 and Yan^Act was due to loss of miR-7 because coexpression of a miR-7 transgene completely rescued the enhancement caused by miR-7^Δ1. These results are consistent with a role for miR-7 in negatively regulating Yan expression and, consequently, stimulating photoreceptor differentiation (Li, 2005).

The Notch signaling pathway plays an important role in eye development, and several validated targets of miR-7 repression are genes that are direct transcriptional targets of the Notch pathway. These include E(spl)m3, E(spl)m4, E(spl)mγ, E(spl)m5, Bearded, hairy, Tom, and Bob. Yan is also transcriptionally regulated by Notch. None of the above immediate early genes (with the exception of yan) have been found to function in photoreceptor differentiation. However, it was nevertheless possible that miR-7 represses unidentified Notch target genes, which contribute to differentiation. To test this possibility, miR-7 expressed with constitutively active Notch (UAS-N^ΔE) in progenitor cells, and it was observed that the double mutant eye phenotype resembled the sum of each mutant phenotype alone. This result suggests that the eye phenotypes observed with miR-7 are due to effects in a pathway parallel with Notch (Li, 2005).

Activation of EGFR is critical for downregulation of Yan, suggesting that the effect of miR-7 RNA on Yan might be dependent on EGFR. Therefore, in situ hybridization of miR-7 RNA was performed in an EGFR mutant background. When a constitutively active mutant form of EGFR was misexpressed in progenitor cells, miR-7 RNA expression was strongly upregulated in these cells. This indicated that EGFR signaling stimulates miR-7 expression. To determine if EGFR signaling is required for miR-7 transcription, EGFR signaling was blocked with a dominant-negative form of EGFR. This results in differentiation of the first two or three photoreceptors but no further photoreceptor differentiation. Then the UAS-miR-7 transgene was expressed in eye disc cells under transcriptional control of Gal4. This resulted in enhanced photoreceptor differentiation even in the background of the dominant-negative EGFR. Since the miR-7 transgene is transcriptionally uncoupled from EGFR regulation and yet still induces photoreceptors, this result argues that the effect of EGFR on miR-7 expression is mediated at least in part through transcription control (Li, 2005).

Two transcriptional effectors of EGFR signaling in the eye disc are Yan and Pointed. Yan represses transcription of target genes, except when EGFR signaling leads to its turnover. Possibly, Yan mediates the effect of EGFR on miR-7 expression by repressing miR-7 in unstimulated progenitor cells. To test this hypothesis, miR-7 expression was examined in yan mutants. A yan¹ loss-of-function mutant has ectopic miR-7-positive cells present in ommatidia. miR-7 expression was examined in the Yan^Act mutant, which is resistant to ERK-dependent degradation. Levels of miR-7 RNA were dramatically reduced in photoreceptors expressing Yan^Act. Together, these data indicate that Yan normally represses miR-7 in progenitor cells and ectopically represses miR-7 in photoreceptors when ERK-dependent degradation is blocked (Li, 2005).

While it was clear that Yan represses miR-7 transcription, it was not clear what factor(s) activates miR-7 transcription once Yan was degraded by EGFR/ERK signaling. Yan acts as a repressor of other genes by binding to enhancers and preventing two related ETS-domain factors called Pointed-P1 and Pointed-P2 from binding and activating transcription. Possibly, one or both isoforms of the Pointed transcription factor competed with Yan for binding and activation of miR-7 expression. If so, then overexpression of Pointed should outcompete Yan even in progenitor cells and elevate miR-7 expression, as has been observed for other genes. When Pointed-P1 was overexpressed, there was a profound increase in miR-7 expression. In contrast, miR-7 expression was unchanged when Pointed-P2 was overexpressed. Thus, Pointed-P1 is an activator of miR-7 expression in the eye disc (Li, 2005).

To determine whether Yan downregulates miR-7 expression directly, conserved ETS binding sites were sought in genomic DNA upstream of miR-7. Using a hidden Markov algorithm that detects clusters of sequence motifs, a statistically significant cluster of putative ETS binding sites was detected less than 2 kb upstream of miR-7 in an intron of bl. This sequence cluster contains six sites that are also conserved 2 kb upstream of miR-7 in the related species D. pseudoobscura. The conserved sequences possibly represent a transcriptional regulatory region that interacts with ETS factors such as Yan. To determine if these sites interact with Yan, electrophoresis mobility shift assays were performed in vitro. Purified GST-Yan protein binds with high affinity to a labeled oligonucleotide that contains a Yan binding site from the prospero enhancer. Unlabeled oligonucleotides containing each of the six sites upstream of miR-7 were used as competitors for binding between GST-Yan and the labeled oligonucleotide. Competition analysis revealed that four of the six sites had a binding affinity for GST-Yan that was comparable to the high-affinity Yan binding site. Binding was specific since an oligonucleotide with a mutated ETS motif failed to compete for GST-Yan binding. Thus, Yan directly interacts with conserved sequence elements upstream of miR-7 (Li, 2005).

Therefore, there is reciprocal negative regulation between Yan protein and miR-7 RNA in retinal cells. miR-7 directly represses the expression of Yan, and Yan directly represses miR-7 expression. This feedback loop explains the apparent mutually exclusive expression patterns of Yan and miR-7. Undifferentiated progenitor cells express Yan, which inhibits miR-7 transcription, while cells undergoing photoreceptor differentiation express miR-7, which inhibits Yan protein synthesis. Through this reciprocal feedback, each gene then logically exerts positive feedback on itself, which reinforces its respective expression pattern (Li, 2005).

The switch in expression patterns appears to be triggered by the EGFR signaling pathway. Both Yan and miR-7 expression are regulated by EGFR but in opposing directions. EGFR signaling activates the rapid turnover of Yan protein via ERK-mediated phosphorylation. In the case of miR-7, EGFR signaling positively regulates its transcription. It is proposed that miR-7 transcription is induced by EGFR through degradation of Yan protein, resulting in derepression of miR-7 transcription. Yan is an ETS-domain transcription factor that represses transcription by a passive mechanism involving competition with Pointed-P1 and Pointed-P2 for binding to specific DNA sites. Pointed-P1 and Pointed-P2 activate target genes, but only when Yan has been downregulated by ERK. It is proposed that miR-7 transcription is induced by Yan and Pointed using a similar mechanism. This model is supported by the presence upstream of the miR-7 coding sequence of a highly conserved cluster of DNA sites that bind Yan protein with high affinity, and by finding that miR-7 RNA expression is strongly activated by Pointed-P1 (Li, 2005).

The miR-7/Yan feedback loop allows switching between one state (Yan+) and a second state (miR-7+), which correlates with the cell changing from a progenitor state to a photoreceptor identity. This may represent an example of a bistable system. Such systems exist almost exclusively in one of two possible states that are stabilized by feedback loops. Feedback loops can either be positive or double negative in nature, and their strength determines the reversibility of the system. In the case of the transition from the Yan+ state to the miR-7+ state, this switch is triggered by the EGFR signal. The bistable nature of the feedback mechanism ensures that the response is enhanced relative to the signal. This could be for one or more reasons. (1) The bistable switch could convert a weak or variable signal into a strong uniform response. In this sense, it ensures that variation in signaling activity between different cells has less impact on their ability to uniformly respond. (2) The bistable switch might respond to a transient signal and translate it into a stable response. Indeed, Yan degradation is dependent on ERK kinase that is transiently activated by EGFR signaling (after about 8 hr) clearing cells of detectable Yan protein. However, cells continue to be depleted of Yan protein even though Yan mRNA transcription remains active. Possibly, miR-7 downregulates Yan protein synthesis during and after ERK signaling to ensure the continuous absence of Yan protein until its mRNA pool is eventually reduced by transcription shutoff. Duration of the miR-7 effect might be dependent upon whether the feedback loop is short lived, only relying upon the pool of miR-7 RNA transiently synthesized or longer lived due to persistent synthesis of new miR-7 even after the signal has dissipated. Although the participation of Pointed-P1 suggests the latter mechanism at work, future study will provide a fuller understanding of the switch (Li, 2005).

The miR-7/Yan feedback loop reinforces a developmental decision, and as such, it regulates downstream effectors of the differentiation process. Which component of the loop provides regulatory output? Yan provides some output since it has been established that Yan directly controls the transcription of at least two genes involved in later steps of retinal cell differentiation. It is conceivable that miR-7 also provides regulatory output. Could some of the effects of miR-7 be executed upon the Notch pathway, which controls aspects of photoreceptor differentiation? Genetic interactions between Notch and miR-7 suggest that they act in parallel pathways affecting differentiation. Several genes that are transcriptional targets of the Notch pathway are also repressed by miR-7. However, none of these except yan have been shown to function in photoreceptor differentiation. Moreover, computational analysis of candidate miR-7 targets has not identified Notch or other components of its pathway (Li, 2005).

Despite the feedback loop, Yan and miR-7 alone are not sufficient to account for their mutually exclusive expression. Yan protein is still greatly reduced in miR-7 mutant photoreceptors. Moreover, miR-7 RNA is not reduced in Yan^Act photoreceptors to the degree observed normally in progenitor cells. This latter observation could explain why the miR-7 mutation enhances the effect of Yan^Act on photoreceptor differentiation since low residual levels of miR-7 might still be effective at inhibiting Yan^Act expression. If this is the case, what is the point of normally upregulating miR-7? An important consideration is that miR-7 RNA is still detectable in Yan^Act photoreceptors. This could be due to several reasons: (1) the repression of miR-7 in progenitors could be due to Yan plus other inhibitory factors; (2) photoreceptors might have positive-acting factors that are not active in progenitors; (3) Yan^Act ectopically expressed in photoreceptors might not itself be a perfect reproduction of endogenous Yan within progenitors (Li, 2005).

Although miR-7 regulates Yan in photoreceptors, this relationship is not used in all cell-fate decisions controlled by Yan. Expression of Yan^Act in non-neuronal cone cells inhibits cone-specific gene transcription. In contrast, miR-7 appears to play no direct role in cone cell development. miR-7 RNA was not detected in differentiating cone cells. Nor did the miR-7 gene promote cone fates. Rather, miR-7 appears to promote a photoreceptor fate at the expense of a cone cell fate. Thus, downregulation of Yan in cone cells does not involve miR-7. It is possible that Yan is repressed in these cells through a different miRNA. It is also possible that cone cells do not use miRNA-dependent repression of Yan. Two lines of evidence support this idea; (1) cone cells are much more sensitive to Yan^Act than photoreceptors, suggesting that ERK-dependent repression of Yan plays a more dominant role in cone cells; (2) low levels of Yan protein are detected in normal cone cells whereas Yan is undetectable in photoreceptors. Together, it suggests that robust downregulation of Yan may not be as critical in cone cells as in photoreceptors or that downregulation occurs at a later stage (Li, 2005).

In conclusion, miR-7 acts in a reciprocal negative feedback loop with a transcription factor to control cell-fate decisions that are triggered by signal transduction activity. It remains to be seen how generally miRNAs will be involved in this type of mechanism. But the potential of rapidly evolving miRNA regulation could be important for evolving new regulatory circuits and, ultimately, new patterns within body plans (Li, 2005).

Germ line differentiation factor Bag of Marbles is a regulator of hematopoietic progenitor maintenance during Drosophila hematopoiesis: Expression of Bam or mir-7 in hematopoietic progenitors leads to a reduction of Yan protein

Bag of Marbles (Bam) is a stem cell differentiation factor in the Drosophila germ line. This study demonstrates that Bam has a crucial function in the lymph gland, the tissue that orchestrates the second phase of Drosophila hematopoiesis. In bam mutant larvae, depletion of hematopoietic progenitors is observed, coupled with prodigious production of differentiated hemocytes. Conversely, forced expression of Bam in the lymph gland results in expansion of prohemocytes and substantial reduction of differentiated blood cells. These findings identify Bam as a regulatory protein that promotes blood cell precursor maintenance and prevents hemocyte differentiation during larval hematopoiesis. Cell-specific knockdown of bam function via RNAi expression revealed that Bam activity is required cell-autonomously in hematopoietic progenitors for their maintenance. microRNA-7 (mir-7) mutant lymph glands present with phenotypes identical to those seen in bam-null animals and mutants double-heterozygous for bam and mir-7 reveal that the two cooperate to maintain the hematopoietic progenitor population. By contrast, analysis of yan mutant lymph glands revealed that this transcriptional regulator promotes blood cell differentiation and the loss of prohemocyte maintenance. Expression of Bam or mir-7 in hematopoietic progenitors leads to a reduction of Yan protein. Together, these results demonstrate that Bam and mir-7 antagonize the differentiation-promoting function of Yan to maintain the stem-like hematopoietic progenitor state during hematopoiesis (Tokusumi, 2011).

The findings on Bam, mir-7 and Yan suggest a mechanism for the interaction of these regulators in the control of blood cell homeostasis. The role of Yan is to direct a quiescent hematopoietic progenitor, through a primed intermediate progenitor state, towards a blood cell differentiation fate as crystal cell, plasmatocyte or lamellocyte. The final differentiation status of these cells would be subject to the function of distinct lineage-determining transcription factors. By contrast, Bam and mir-7 cooperate to negatively modulate yan mRNA translation in the quiescent hematopoietic progenitor, thus maintaining the initial prohemocyte state. Although details of this possible translational repression remain to be parsed out, it is likely to include an as yet unidentified protein partner of Bam that would facilitate mir-7 and yan mRNA packaging within an inhibitory RNA-induced silencing complex in prohemocytes (Tokusumi, 2011).

Changes in Notch signaling coordinates maintenance and differentiation of the Drosophila larval optic lobe neuroepithelia

A dynamic balance between stem cell maintenance and differentiation paces generation of post-mitotic progeny during normal development and maintenance of homeostasis. Recent studies show that Notch plays a key role in regulating the identity of neuroepithelial stem cells, which generate terminally differentiated neurons that populate the adult optic lobe via the intermediate progenitor cell type called neuroblast. Thus, understanding how Notch controls neuroepithelial cell maintenance and neuroblast formation will provide critical insight into the intricate regulation of stem cell function during tissue morphogenesis. This study shows that a low level of Notch signaling functions to maintain the neuroepithelial cell identity by suppressing the expression of pointedP1 gene through the transcriptional repressor Anterior open. Increased Notch signaling, which coincides with transient cell cycle arrest but precedes the expression of PointedP1 in cells near the medial edge of neuroepithelia, defines transitioning neuroepithelial cells that are in the process of acquiring the neuroblast identity. Transient up-regulation of Notch signaling in transitioning neuroepithelial cells decreases their sensitivity to PointedP1 and prevents them from becoming converted into neuroblasts prematurely. Down-regulation of Notch signaling combined with a high level of PointedP1 trigger a synchronous conversion from transitioning neuroepithelial cells to immature neuroblasts at the medial edge of neuroepithelia. Thus, changes in Notch signaling orchestrate a dynamic balance between maintenance and conversion of neuroepithelial cells during optic lobe neurogenesis (Weng, 2012).

A deregulated conversion of neuroepithelial cells into neuroblasts perturbs formation of the neuronal network and will almost certainly lead to visual impairment of the adult fly. Thus, a dynamic balance between neuroepithelial cell maintenance and differentiation plays a pivotal role during morphogenesis of the optic lobe. This study provides evidence that changes in Notch signaling regulate the dynamic balance between maintenance of neuroepithelial cells and formation of neuroblasts. A low level of Notch signaling maintains the neuroepithelial cell identity by triggering Aop-dependent repression of the pntP1 gene. Transient up-regulation of Notch signaling in transitioning neuroepithelial cells raises their threshold of response to PntP1 preventing them from precociously converting into immature neuroblasts. Finally, abrupt down-regulation of Notch signaling together with a high level of PntP1 trigger the conversion from transitioning neuroepithelial cells into immature neuroblasts at the medial edge of neuroepithelia. Thus, interplay between changes in Notch signaling and transient up-regulation of pntP1 orchestrates synchronous and progressive formation of neuroblasts in a medial-to-lateral orientation across the entire neuroepithelial swath (Weng, 2012).

Lack of Notch reporter transgene expression throughout neuroepithelia located laterally from transitioning neuroepithelial cells has been perplexing in light of recent studies reporting that Notch signaling is necessary for maintenance of their identity. One possibility might be that these Notch reporter transgenes including E(spl)mγ-GFP might not contain all necessary regulatory response elements to respond to Notch signaling in most neuroepithelial cells. Alternatively, the level of Notch signaling might simply be too low to activate the expression of the Notch reporter transgene. The second hypothesis is favored for the following reasons. Since over-expression of Notch_intra is sufficient to trigger robust cell autonomous expression of E(spl)mγ-GFP in neuroepithelia located laterally from transitioning neuroepithelial cells, this transgene does contain all necessary regulatory elements to respond to a high level of Notch signaling. Furthermore, the Notch ligand Delta is expressed in a low level throughout neuroepithelia located laterally from transitioning neuroepithelial cells and Delta likely functions to trans-activate Notch signaling in these cells. In the context of trans-activation of Notch signaling by Delta, the level of the ligand correlates with the level of signaling output. Taken together, it is concluded that maintenance of the neuroepithelial cell identity requires a low level of Notch signaling (Weng, 2012).

It is proposed that Notch maintains the identity of neuroepithelial cells by activating Aop-dependent repression of the pntP1 gene. The Suppressor of Hairless protein, which is necessary for activating transcription of Notch targets genes, directly binds to the promoter of the aop gene. Furthermore, removing the Notch or aop function triggered premature conversion of neuroepithelia into neuroblasts whereas over-expressing Notch or aop prevented conversion of neuroepithelial cells into neuroblasts. Most importantly, over-expression of aop suppressed premature differentiation of Notch mutant neuroepithelial cells. Finally, heterozygosity of the pntP1 gene completely suppressed premature conversion of neuroepithelial cells into neuroblasts in a hypomorphic aop mutant genetic background. These data lead to the conclusion that Notch signaling maintains the identity of neuroepithelial cells by activating an Aop-dependent repression of pntP1. In the future, analyses of Notch and pntP1 double mutants will be necessary to confirm this regulatory mechanism (Weng, 2012).

Down-regulation of Notch signaling is necessary for formation of neuroblasts, so transient up-regulation of Notch signaling in transitioning neuroepithelial cells appears rather counterproductive. One possibility might be that up-regulation of Notch signaling paces the conversion from transitioning neuroepithelial cells into neuroblasts by increasing their threshold of response to PntP1. Consistent with this hypothesis, constitutively activated Notch signaling prevented transitioning neuroepithelial cells from becoming converted into neuroblasts despite expressing PntP1. This hypothesis was further supported by co-expression of pntP1 overcoming the blockade by constitutively activated Notch signaling and restoring conversion of transitioning neuroepithelial cells into neuroblasts. Thus, it is proposed that up-regulation of Notch signaling in transitioning neuroepithelial cells raises their threshold of response to PntP1 and functions to prevent them from becoming converted into immature neuroblasts precociously. Such an elaborated mechanism only permits transitioning neuroepithelial cells expressing the highest level of PntP1 to convert into immature neuroblasts. This mechanism is consistent with a recent study reporting that the EGF ligand is processed and secreted by cells near the medial edge of the optic lobe neuroepithelia. As a result of simple diffusion, transitioning neuroepithelial cells at the medial edge of neuroepithelia will be exposed to the highest level of the EGF ligand and will express the highest level of PntP1. As such, EGF signaling likely creates a vector field establishing the directionality of conversion from neuroepithelial cells into neuroblasts whereas Notch signaling refines the functional output of EGF signaling by raising the threshold response to PntP1 (Weng, 2012).

Many important questions arise from this highly plausible mechanism by which the interplay between Notch and EGF signaling paces synchronous conversion of neuroepithelial cells into neuroblasts one row at a time. This model will predict that immature neuroblasts immediately adjacent to transitioning neuroepithelial cells should secrete the processed EGF ligand. However, the antibody specific for the Rhomboid (Rho) protease required for proteolytic activation of the EGF protein is currently unavailable and a genomic fragment encompassing the rho-1 locus tagged with YFP did not show detectable expression in the larval optic lobe. Alternatively, a recent study shows that pntP1 is a direct target of Notch in vivo. Thus, up-regulation of Notch signaling might directly activate transcription of the pntP1 gene in transitioning neuroepithelial cells. Since Notch signaling becomes abruptly down-regulated at the medial edge of neuroepithelia, it is highly possible that the threshold of response to PntP1 also becomes lowered in the same cells. Thus, the pre-existing level of PntP1 protein will likely be sufficient to trigger the conversion from transitioning neuroepithelial cells into immature neuroblasts. More experiments including identification of the cell type from which the processed EGF ligand is released and a direct test to confirm the role of EGF signaling during conversion of neuroepithelia into neuroblasts will be key to distinguish these two possible mechanisms (Weng, 2012).

The fat-hippo signaling mechanism controls tissue growth by regulating proliferation and cell death and promotes timely differentiation of optic lobe neuroepithelial cells). While inactivation of fat-hippo signaling delays conversion of neuroepithelia into neuroblasts, removal of the downstream effecter yorkie only accelerates the conversion near the medial edge of the optic lobe neuroepithelia. Thus, fat-hippo signaling likely functions as a gatekeeper to prevent over-growth of optic lobe neuroepithelia by triggering transient cell cycle arrest. Intriguingly, transient cell cycle arrest precedes increased Notch signaling in transitioning neuroepithelial cells. Detailed studies in the future will be necessary to determine whether activation of the fat-hippo signaling might contribute to increased Notch signaling in transitioning neuroepithelial cells (Weng, 2012).

The ETS domain transcriptional repressor Anterior open inhibits MAP kinase and Wingless signaling to couple tracheal cell fate with branch identity

Cells at the tips of budding branches in the Drosophila tracheal system generate two morphologically different types of seamless tubes. Terminal cells (TCs) form branched lumenized extensions that mediate gas exchange at target tissues, whereas fusion cells (FCs) form ring-like connections between adjacent tracheal metameres. Each tracheal branch contains a specific set of TCs, FCs, or both, but the mechanisms that select between the two tip cell types in a branch-specific fashion are not clear. This study shows that the ETS domain transcriptional repressor anterior open (aop) is dispensable for directed tracheal cell migration, but plays a key role in tracheal tip cell fate specification. Whereas aop globally inhibits TC and FC specification, MAPK signaling overcomes this inhibition by triggering degradation of Aop in tip cells. Loss of aop function causes excessive FC and TC specification, indicating that without Aop-mediated inhibition, all tracheal cells are competent to adopt a specialized fate. Aop plays a dual role by inhibiting both MAPK and Wingless signaling, which induce TC and FC fate, respectively. In addition, the branch-specific choice between the two seamless tube types depends on the tracheal branch identity gene spalt major, which is sufficient to inhibit TC specification. Thus, a single repressor, Aop, integrates two different signals to couple tip cell fate selection with branch identity. The switch from a branching towards an anastomosing tip cell type may have evolved with the acquisition of a main tube that connects separate tracheal primordia to generate a tubular network (Caviglia, 2013).

This work has investigated how the choice between the two types of specialized tip cells in the tracheal system is controlled. The transcriptional repressor Aop plays a key role in linking tracheal tip cell fate selection with branch identity. First, a novel tube morphogenesis phenotype is described in aop mutants, which is due to the massive mis-specification of regular epithelial cells into specialized tracheal tip cells. aop is specifically required for controlling tracheal cell fate, whereas aop, like pnt, is dispensable for primary tracheal branching, thus uncoupling roles of RTK signaling in cell fate specification and cell motility. The finding that tracheal branching morphogenesis proceeds normally in the presence of excess tip cell-like cells suggests that collective cell migration is surprisingly robust and that mis-specified cells apparently do not impede the guided migration of the tracheal primordium. Second, it was demonstrated that in the absence of inhibitors of MAPK signaling (aop and sty), all tracheal cells are competent to assume either TC or FC fate. The transcriptional repressor Aop globally blocks both TC and FC differentiation, but high-levels of MAPK signaling in tip cells relieve Aop-mediated inhibition, thus permitting differentiation. Third, the results suggest that in the DT region Aop limits FC induction through a distinct mechanism by antagonizing Wg signaling in addition to MAPK signaling. Conversely, in the other branches, Aop limits TC differentiation by blocking MAPK-dependent activation of Pnt. Fourth, it was shown that the region-specific choice between the two cell fates in the DT is determined by Wg signaling and by the selector gene salm. Based on these results, a model is proposed in which a single repressor, Aop, integrates MAPK and Wg signals to couple tip cell fate selection with branch identity. High levels of Bnl signaling trigger Pnt activation and Aop degradation in tracheal tip cells. It is proposed that in the DT, unlike in other tracheal cells, MAPK-induced degradation of Aop releases inhibition of Wg signaling. This is consistent with recent work showing an inhibitory effect of Aop on Wg signaling, possibly through direct interaction of Aop and Arm, or through Aop-mediated transcriptional repression of Wg pathway component. The current work extends the evidence for this unexpected intersection between two major conserved signaling pathways, suggesting that this function of Aop is likely to be more widespread than previously appreciated. The findings also provide an explanation for the puzzling observation that, in pnt mutants, TCs are lost, while FCs become ectopically specified. As pnt is required for expression of the feedback inhibitor sty, loss of pnt is expected to lead to MAPK pathway activation and consequently to increased Aop degradation. This would release Aop-mediated repression of Wg signaling, resulting in extra FCs, whereas TCs are absent because of the lack of pnt-dependent induction. This suggests that excessive FC specification in the DT of aop and sty mutants is mainly due to deregulated Wg signaling, rather than to de-repression of pnt-dependent MAPK target genes. Consistent with this notion, it was shown that pnt is not required for Delta and Dys expression in tracheal cells, although constitutively active Aop^ACT represses their expression (Caviglia, 2013).

The results further show that salm function constrains the fate that is chosen by cells when released from the Aop inhibitory block. MAPK signaling triggers Aop degradation in all tip cells, but only in the absence of salm does this signal lead to TC induction. In salm-expressing cells, degradation of Aop releases Wg signaling, resulting in FC specification. Thus, salm biases the choice between two morphologically different types of seamless tubes. This is reminiscent of the role of salm in switching between different cell types in the peripheral nervous system and in muscles. salm expression is sufficient to repress TC formation. The genetic results, consistent with biochemical data showing that Salm acts as a transcriptional repressor, suggest that salm promotes FC fate by repressing genes involved in TC development. However, salm is not sufficient to overcome the requirement for Wg signaling in FC induction, indicating that Wg does not act solely via salm to induce FC fate. Indeed, FC induction requires genes whose expression is independent of salm (esg, dys). In addition, it is proposed that a feedback loop between Wg signaling and salm expression maintains levels of Wg signaling in the DT sufficiently high to induce FC fate. Taken together, these results suggest that the default specialized tip cell fate, and possibly an ancestral tracheal cell state, is TC fate. Although FCs and TCs differ in their morphology, they share a unique topology as seamless unicellular tubes. FCs and TCs might therefore represent variations of a prototypical seamless tube cell type. Salm might modify cellular morphology by repressing TC genes, including DSRF, which mediates cell elongation and shape change. Intriguingly, Wg-dependent salm expression in the DT of dipterans correlates with a shift towards FC as the specialized fate adopted by the tip cells of this branch. This study has shown that salm expression inhibits TC fate, while promoting the formation of a multicellular main tracheal tube by inhibiting cell intercalation. It is therefore tempting to speculate that the salm-dependent switch from a branching towards an anastomosing tip cell type in the DT may have evolved with the acquisition in higher insects of a main tube that connects separate tracheal primordia to generate a tubular network. It will be of great interest to identify the relevant target genes that mediate the effect of Salm on tube morphology and tip cell fate (Caviglia, 2013).

The mechanisms of tip cell selection during angiogenesis in vertebrates are beginning to be understood at the molecular level. However, the signals that control the formation of vascular anastomoses by a particular set of tip cells are not known. Intriguingly, the development of secondary lumina in aop mutants is reminiscent of transluminal pillar formation during intussusceptive angiogenesis, which is thought to subdivide an existing vessel without sprouting. Although the cellular basis for this process is not understood, it is conceivable that specialized endothelial cells are involved in transluminal pillar formation. This work provides a paradigm for deciphering how two major signaling pathways crosstalk and are integrated to control cell fate in a developing tubular organ. It will be interesting to see whether similar principles govern tip cell fate choice during tube morphogenesis in vertebrates and invertebrates (Caviglia, 2013).

3D chromatin interactions organize Yan chromatin occupancy and repression at the even-skipped locus

Long-range integration of transcriptional inputs is critical for gene expression, yet the mechanisms remain poorly understood. This study investigated the molecular determinants that confer fidelity to expression of the heart identity gene even-skipped (eve). Targeted deletion of regions bound by the repressor Yan defined two novel enhancers that contribute repressive inputs to stabilize tissue-specific output from a third enhancer. Deletion of any individual enhancer reduced Yan occupancy at the other elements, impacting eve expression, cell fate specification, and cardiac function. These long-range interactions may be stabilized by three-dimensional chromatin contacts that were detected between the elements. This work provides a new paradigm for chromatin-level integration of general repressive inputs with specific patterning information to achieve robust gene expression (Webber, 2013).

In addition to the muscle/heart enhancer targeted by the Yan–Pnt switch (MHE), a genome-wide occupancy study of Yan mapped three other regions to the eve locus. The four Yan-bound domains are referred as D1, D2, D3/MHE, and D4. To explore the contribution of individual Yan-bound regions to mesodermal eve expression specific deletions were recombineered into a functional genomic bacterial artificial chromosome (BAC) construct encompassing the entire 16.4-kb eve locus carrying an in-frame YFP tag. As enhancer-blocking activity has previously been identified between the MHE and Ter94 gene, it was reasoned that the D4 Yan-bound element was more likely to contribute to Ter94 than to eve regulation, and it was excluded from the analysis. Transgenes were generated, and altered function was assessed by examining expression in the Eve-positive cardiogenic precursors. The simple prediction for deletion of a functional Yan-bound region is that loss of repression should increase expression of the target gene; however, if the deletion also removes binding sites for critical activators, then reduced expression is expected. Either scenario predicts reduced ability to complement an eve-null. Alternatively, if elements are functional yet fully redundant under optimal conditions, no change in expression or viability is predicted (Webber, 2013).

The control eve-YFP transgene fully complemented an eve-null background, with 86% rescue to adulthood, and was expressed in a pattern identical to that of endogenous eve. Thus, in stage 11 embryos, Eve-YFP was prominent in the muscle and cardiogenic precursors as well as in clusters of neurons in the CNS. Deletion of the D1, D2, or D3 Yan-bound regions altered expression and reduced genetic rescue efficiency, indicating important contributions to regulation of eve. As predicted based on loss of key activating inputs, deletion of the D3/MHE region significantly reduced mesodermal expression, with Eve^ΔD3-YFP expression detectable, on average, in only one cell per cluster. For the Eve^ΔD1-YFP and Eve^ΔD2-YFP transgenes, although expression in the mesoderm appeared qualitatively normal, quantification of the YFP signal revealed significant increases in both the mean intensity and embryo-to-embryo variation relative to the wild-type Eve-YFP control. The reduced ability of the deletion transgenes to rescue an eve-null suggests that these Yan-bound elements are not redundant. The deleted regions do not include known stripe enhancers, and cuticle preparations of dead embryos from the rescue experiments did not show segmentation defects. Thus, the reduced viability is unlikely attributed to axial patterning defects (Webber, 2013).

Precise regulation of eve expression in the cardiogenic mesoderm is essential for acquisition of heart cell identity and ultimately for cardiac function. Consistent with this, the elevated expression of Eve^ΔD1-YFP reflected in part an increase in the number of Eve-positive mesodermal cells specified, raising the possibility that compromised heart development might contribute to reduced fitness. To test this, pupal heart rates were measured. In agreement with a previous study, the heart rate of eve^−/−; eve^ΔD3-YFP animals was significantly reduced relative to the eve^−/−; eve-YFP control. eve^−/−; eve^ΔD1-YFP larvae also had a reduced heart rate, suggesting that the ectopic cell fate specification detected compromises the ensuing developmental program. Deletion of the D2 region did not significantly change the number of Eve-positive mesodermal cells specified, and the heart rate measured in eve^−/−; eve^ΔD2 pupae was comparable with that of the control (Webber, 2013).

Adult heart function is commonly assessed with assays that measure the general vigor and activity of the fly. In both a flight assay and an assay measuring the negative geotaxis response before and after heat stress, eve^−/−; eve^ΔD3-YFP and eve^−/−; eve^ΔD1-YFP flies again performed poorly relative to eve^−/−; eve-YFP control animals. eve^−/−; eve^ΔD2-YFP animals exhibited a response intermediate to that of the eve^−/−; eve^ΔD1-YFP and control eve^−/−; eve-YFP animals in the geotaxis assay prior to heat stress. However, after stress, the activity of eve^−/−; eve^ΔD2-YFP flies was significantly below that of the control. Together, these results confirm the physiological importance of the Yan-bound D1 region and support the hypothesis that it mediates repressive inputs important for stabilizing mesodermal eve expression within levels required to support normal cell fate acquisition during heart development. As the D2 deletion partially overlaps a neuronal enhancer, further analysis will be required to determine whether the reduced fitness and activity result from either compromised neural function, subtle cardiac defects, or a combination of the two (Webber, 2013).

To obtain more direct evidence that compromised Yan-mediated repression contributes to increased expression and reduced fitness of the deletion mutants, the effectiveness was tested of twist-Gal4-driven overexpression of a constitutively active form of Yan, Yan^ACT, in repressing expression of the deletion transgenes. If Yan-mediated repressive inputs act exclusively on the MHE, then Yan^ACT should repress mesodermal expression of Eve^ΔD1-YFP and Eve^ΔD2-YFP but not Eve^ΔD3-YFP. Alternatively, if Yan-mediated regulation across multiple genomic elements contributes to stable eve expression, then all deletion transgenes should be sensitive to Yan^ACT. The results support the latter scenario, as Yan^ACT fully repressed all three eve-YFP deletion transgenes (Webber, 2013).

It was next asked whether heterozygosity for yan or pnt might, respectively, enhance or suppress the loss of robustness measured for the eve deletion constructs. Using either a null allele or a small deficiency, it was found that heterozygosity for yan increased the mean intensity and variation of wild-type Eve-YFP expression and further increased the intensity of Eve^ΔD2-YFP expression. While yan heterozygosity did not significantly increase Eve^ΔD1-YFP expression, the number of Eve-positive cells specified was increased. These changes also impact fitness, as yan heterozygosity further reduced the genetic rescue efficiency of the eve transgenes. Conversely, heterozygosity for pnt suppressed the increased intensity and variability of Eve^ΔD1-YFP and Eve^ΔD2-YFP expression. A reduced pnt dose also improved the negative geotaxis response following heat shock. Together, these results suggest that the D1 and D2 elements are required for robust regulation of eve expression in the muscle and cardiogenic precursors such that loss of Yan-mediated repression upon deletion of either element permits inappropriate Pnt-mediated activation of eve, presumably through the MHE, leading to increased and more variable expression. Such destabilization of the Yan–Pnt switch makes the system prone to aberrant cell fate specification and ultimately reduces fitness (Webber, 2013).

To explore further the properties of the D1 and D2 Yan-bound regions, it was asked whether they define autonomous cis-regulatory elements or whether their activity requires the endogenous genomic context. Reporter constructs carrying sequences spanning the chromatin immunoprecipitation (ChIP)-defined Yan-bound regions were generated, and expression was examined in stage 11 embryos. In contrast to the control MHE reporter that expressed specifically in the expected mesodermal clusters, the D1 and D2 reporters showed weak ubiquitous expression. A slightly stronger signal was detected in the mesoderm for both reporters but in domains broader than the three cell clusters seen with the MHE reporter. Removing yan increased the intensity but not the specificity of the expression patterns of the D1 and D2 reporters, suggesting that the individual elements can be recognized and repressed by Yan outside of the context of the intact eve locus. The autonomous ability of each element to recruit Yan was confirmed by ChIP-PCR, although quantitative PCR (qPCR) analysis revealed that occupancy was reduced relative to that measured at the endogenous locus (Webber, 2013).

Because the D1 and D2 Yan-bound elements do not appear to provide independent additive inputs capable of directing mesodermal eve expression, it was asked whether their primary role might be to integrate Yan-repressive inputs across the intact locus. It was hypothesized that long-range interactions between these regions might stabilize Yan binding, thereby providing a buffering mechanism that sets the precise MHE-driven eve expression pattern needed for accurate cell fate specification. If correct, then deletion of any individual Yan-bound region not only should abolish occupancy at the site of deletion, but might also perturb binding at the remaining elements. To test this, Yan occupancy was assessed by ChIP-qPCR in embryos homozygous for the eve deficiency and carrying the deletion transgenes. Supporting the model of coordinated stabilization of Yan occupancy, deletion of any individual Yan-bound domain reduced Yan occupancy by about twofold at the remaining two eve elements but did not alter enrichment at a distant locus. Thus, precise regulation of mesodermal eve expression relies on long-range coordination between cis-regulatory elements rather than additive inputs from autonomous enhancers (Webber, 2013).

One possible mechanism is that the 3D chromatin environment facilitates interactions between the D1, D2, and D3 elements. Using proximity ligation and PCR analysis (referred to as chromatin conformation capture [3C]), a long-range interaction was detected between a D1–D2 fragment and the D3/MHE fragment. Sequencing confirmed the expected identity of the amplified ligated fragment. Control primers located just outside of the eve locus did not yield a 3C product with the D1–D2 fragment. Furthermore, the 3C interaction was not detected in adult heads, suggesting context-specific chromatin contacts. Finer-scale mapping in both wild-type and yan mutant embryos should provide further insight into how the 3D chromatin environment facilitates cooperative recruitment of Yan across the locus and how this in turn both stabilizes the Yan–Pnt switch in optimal conditions and buffers against genetic or environmental variation that might limit Yan (Webber, 2013).

In conclusion, the results suggest a novel regulatory mechanism in which, rather than contributing patterning information, the D1 and D2 enhancers act to dampen the expression control mediated by the D3/MHE. More broadly, this raises the possibility that even when transcription factor-bound regions identified through ChIP sequencing (ChIP-seq) fail to give clear expression patterns as autonomous reporters, they may still contribute to gene regulation in vivo. In the case of Yan, it is speculated that its intrinsic polymerization ability allows it to exploit the 3D chromatin environment to organize long-range repressive complexes that coordinate information across multiple enhancers (Webber, 2013).

Many parallels can be drawn between Yan/Pnt regulation of the transition from uncommitted progenitor to specified cell fate within a developing tissue and the control of the pluripotent state in mammalian cells. Given the potential for natural, genetic, or environmental variation to perturb the timing, accuracy, or stability of such transitions, mechanisms to impart robustness to gene expression are almost certainly required and very likely conserved across species. Thus, the regulatory paradigm established in this study may be broadly relevant (Webber, 2013).

Protein Interactions

Phosphorylation by Rolled/MAPK affects the stability and subcellular localization of Yan, resulting in a rapid regulation of yan activity. Overexpression of MAPK phosphorylation site-deficient Yan in the developing eye blocks photoreceptor neuron differentiation (Rebay, 1995). In cells with activated RAS protein the subcellular distribution of YAN protein is altered; the protein appears almost entirely cytoplasmic (Rebay, 1995).

Epidermal growth factor receptor induces pointed P1 and inactivates Yan protein in the embryonic ventral ectoderm. Two other candidate genes for EGF-R regulation are ventral nervous system defective and Fasciclin III. Ectopic expression of secreted Spitz results in expression of orthodenticle within the entire ventral ectoderm, suggesting that ventral expression of otd is normally induced by higher levels of EGF-R activity. Of the two pointed transcripts, only pntP1 is expressed in the ventral ectoderm. It first appears prior to gastrulation in the entire neuroectoderm region. In pointed null mutants, the expression of orthodenticle, argos and tartan in these cells is abolished or significantly reduced. Since Pointed P1 is thought to be a constitutively active transcription factor, with no requirement for modulation of its activity by the EGF-R/MAP kinase signaling pathway, a direct induction of pntP1 transcription by EGF-R appears possible. Early pntP1 expression is EGF-R independent, but at stage 9/10, expression of pnt is not observed in the ventral ectoderm of Egf-R mutants. yan, which encodes a negative regulator of ETS transcriptional activators, is first detected at stage 5/6, where it is found in the dorsal ectoderm. yan expression declines in a dorsal-ventral gradient and is not found in mesoderm. Expression of yan does not depend on EGF-R, as it is unaltered in Egf-R mutants. In yan mutants, the ventralmost markers orthodenticle, argos and tartan show a clear expansion. Absence of the Yan protein may thus allow the Pointed P1 protein, which is expressed earlier in a broader domain, to efficiently induce ventralization. In the absence of Egf-R and yan, the early EGF-R-independent expression of pntP1 is capable of triggering otd expression. An activated form of Yan, which is unable to undergo phosphorylation by MAP kinase, was expressed in wild-type embryos. Indeed, the expression of orthodenticle and argos is significantly reduced or abolished, in the region where activated Yan is expressed (Gabay, 1996).

During normal tracheal development, secondary and terminal branching genes are induced at the ends of growing primary branches by localized expression of Branchless. Because the ectopic branches in sprouty mutants are formed by the prestalk cells located near the cells that are normally induced to branch, the extra branches could arise from overactivity of the Bnl pathway. To test whether sty functions by limiting the Bnl pathway or by preventing branching in some other way, an examination was made of downstream effectors in the Bnl pathway that regulate the later branching events (Hacohen, 1998).

One such effector is pointed (pnt), a downstream target of several receptor tyrosine kinase pathways. pnt expression is induced by Bnl at the ends of primary branches and promotes secondary and terminal branching. Similarly, the DSRF gene and three other marker genes (Terminal -2,-3, and -4) are induced at the ends of growing primary branches; all promote terminal branching. In sty mutants, all five downstream effectors are expressed in expanded domains that include the prestalk cells, which later form ectopic branches. The DSRF marker is activated at the same time as in the normal branching cells (Hacohen, 1998).

The transcriptional repressor Yan is another critical target of Bnl signaling. As in other RTK pathways, activation of the Btl receptor leads to MAPK-dependent phosphorylation and degradation of Yan, which is necessary to activate the later programs of tracheal branching. Normally, Yan is degraded only in the tip cells of the outgrowing primary branches. In sty mutants, Yan is degraded in an expanded domain that coincides with the expanded domains of pnt and DSRF expression. A yan-lacZ transcriptional reporter continues to be expressed normally, implying that down-regulation of Yan occurs posttranscriptionally as in other RTK pathways. The results show that sty loss of function mutations enhance all known downstream effects in this Bnl pathway. An engineered gain of function condition, in which the sty gene product is overexpressed during embryonic stages 10-12, severely blocks induction of downstream effectors and branching by Bnl. The reciprocal is also true: overexpression of Bnl can overcome the opposition of sty and induce secondary and terminal branching throughout the tracheal system. Thus, sty behaves genetically as a competitive inhibitor of the Bnl pathway (Hacohen, 1998).

Dorsal closure requires two signaling pathways: the Drosophila Jun-amino-terminal kinase (DJNK) pathway and the Decapentaplegic pathway. The changes in cell shape in the lateral epidermis occur in two phases. In the first phase, the cells of the leading edge begin to stretch dorsally. In a second phase, the remaining cells ventral to the first row change shape. DJNK, known as Basket, controls dorsal closure by activating DJun and inactivating the ETS repressor Aop/Yan by phosphorylation. The role of Aop/Yan is to hold decapentaplegic transcriptionally silent until Aop/Yan is inactivated by phosphorylation. These phosphorylation events regulate dpp expression in the most dorsal row of cells. Interestingly, mutants in components of the DJNK and Dpp pathways affect the two phases of dorsal closure differently. Whereas loss-of-function mutations in either DJNK or DJun block the cell shape changes of all cells, mutations in thick veins and punt block only the second phase. Thus it is concluded that Dpp functions to instruct more ventrally located cells to stretch. These results provide a causal link between the DJNK and Dpp pathways during dorsal closure (Riesgo-Escovar, 1997).

The fate of the R7 photoreceptor cell in the developing eye of Drosophila is controlled by the Sevenless (SEV) receptor tyrosine kinase. SEV activates a highly conserved signal transduction cascade involving the proteins Ras1 and Raf and the Rolled/mitogen-activated protein (RL/MAP) kinase. The ETS domain protein encoded by the P2 transcript of the pointed (PNT) gene is a nuclear target of this signaling cascade which acts downstream of RL/MAP kinase. The PNTP2 protein is phosphorylated by RL/MAP kinase in vitro at a single site and this site is required for its function in vivo. MAP kinase controls neural development through phosphorylation of two antagonizing transcription factors of the ETS family: Yan and PNT (Brunner, 1994).

50 different proteins have been reported to be ERK substrates. These include signaling proteins likely to function upstream of ERK [such as Son-of-sevenless (Sos), guanine nucleotide exchange factor and MEK]; signaling proteins likely to function downstream of ERK (such as the protein kinase pp90rsk); transcription factors (such as c-Fos, GATA-2, c-Myc); ETS proteins (including Elk-1, LIN-1, and Aop/Yan), and proteins involved in a wide variety of other processes. These findings suggest that ERK plays a central role in signal propagation and feedback regulation. Furthermore, ERK is a transition point between signaling proteins and regulators of differentiation, suggesting it makes an important contribution to the specificity of RTK-Ras-ERK signaling pathways. Although a large number of ERK substrates have been identified, the understanding of ERK function remains fragmentary, since ERK probably phosphorylates different substrates in different cell types and the cellular context of most substrates has yet to be defined. In addition, many ERK substrates probably have not been identified. Little is known about how ERK recognizes such a diverse group of substrates. Although the structure of ERK was determined using X-ray crystallography, this approach has not revealed how ERK interacts with substrate proteins, because the structure of ERK bound to a substrate has yet to be determined. Studies of residues in substrate proteins that are phosphorylated by ERK and assays of peptide substrates have identified a serine or threonine followed by a proline (S/TP) as the minimal consensus sequence for phosphorylation by ERK. In addition, a proline at position 2 is favorable, whereas a proline at position 1 is unfavorable (the phosphoacceptor S/T is position 0). However, this information is not sufficient to explain how ERK recognizes specific proteins as substrates, because many proteins that contain S/TP sequences are not phosphorylated by ERK. Studies of the interaction of JNK with its substrate c-Jun have identified a sequence (that is, the delta domain) positioned amino-terminal to the S/TP sites that is required for efficient phosphorylation. This has led to the hypothesis that a docking site on the substrate protein that is separate from the phosphorylation sites mediates the interaction with JNK. Other protein kinases, such as cyclin-cdk2, also appear to interact with a docking site on substrate proteins . Although >50 proteins have been reported to be ERK substrates, only recently has one such docking site for ERK been identified. This docking site, a domain of Elk-1 called the D box, is similar in sequence to the delta domain of c-Jun, and these two domains are functionally interchangeable, suggesting that the delta domain/D box is a docking site for both ERK and JNK (Jacobs, 1999 and references).

The identification and characterization of a different docking site, the amino acid sequence FXFP, mediates interactions with ERK but not JNK. These two docking sites define three classes of substrates: proteins that contain only FXFP, only the delta domain/D box, or both. These findings suggest that a modular system of docking sites regulates interactions of the different MAP kinases with various substrates. In substrates that contain both docking sites, the sites function additively to create a high-affinity interaction with ERK. Thus, this system also modulates the affinity of substrates for ERK and may determine which residues are phosphorylated. This information is used to develop peptide inhibitors of ERK and identify new ERK substrates, including the kinase suppressor of the ras (KSR) family of protein kinases (Jacobs, 1999).

The Caenorhabidtis elegans LIN-1 protein contains an ETS DNA-binding domain and presumably regulates transcription. LIN-1 appears to be regulated directly by ERK, since LIN-1 is efficiently phosphorylated by Erk2 in vitro and lin-1 is regulated negatively by RTK-Ras-ERK pathways in vivo. Six gain-of-function mutations have been identified and characterized that impair the ability of lin-1 to be regulated negatively by RTK-Ras-ERK pathways and disrupt vulval development. Each mutation alters or eliminates FQFP, a sequence located in the carboxy-terminal region of LIN-1, suggesting that this motif is important for LIN-1 regulation. The sequences of other ETS proteins were analyzed and FQFP was found in vertebrate Elk-1, SAP-1a, and Net/ERP/SAP-2, all highly related proteins that comprise the Elk subfamily of ETS proteins. FQFP is positioned near the carboxyl terminus of a conserved region (named the C box), which contains multiple S/TP motifs that are phosphorylated by ERK. In addition, FQFHP was found in a comparable position of Drosophila Aop/Yan. Aop/Yan also appears to be regulated directly by ERK. This combination of sequence and functional similarities has led to a proposal that LIN-1 and Aop/Yan are members of the Elk subfamily of ETS proteins. Based on these observations, it is hypothesized that FQFP is an evolutionarily conserved docking site that mediates ERK binding to these ETS proteins. According to this model, the lin-1(gf) mutations diminish phosphorylation of LIN-1 by ERK because they alter or eliminate FQFP, resulting in constitutively active LIN-1 (Jacobs, 1999 and references).

Mae is a signalling intermediate that directly links the MAPK signalling pathway to Yan

The evolutionarily conserved Ras/mitogen-activated protein kinase (MAPK) cascade is an integral part of the processes of cell division, differentiation, movement and death. Signals received at the cell surface are relayed into the nucleus, where MAPK phosphorylates and thereby modulates the activities of a subset of transcription factors. A new component of this signal transduction pathway called Mae (for modulator of the activity of Ets) has been cloned and characterized. Mae is a signalling intermediate that directly links the MAPK signalling pathway to its downstream transcription factor targets. Phosphorylation by MAPK of the critical serine residue (Ser 127) of the Drosophila transcription factor Yan depends on Mae, and is mediated by the binding of Yan to Mae through their Pointed domains. This phosphorylation is both necessary and sufficient to abrogate transcriptional repression by Yan. Mae also regulates the activity of the transcriptional activator Pointed-P2 by a similar mechanism. Mae is essential for the normal development and viability of Drosophila, and is required in vivo for normal signalling of the epidermal growth factor receptor. This study indicates that MAPK signalling specificity may depend on proteins that couple specific substrates to the kinase (Baker, 2001).

Phosphorylation of transcription factors by MAPK is a key link between cell signalling and the control of gene expression. The Ets family of transcription factors regulates cell growth and differentiation, and the activities of many of its 35 members are modulated through phosphorylation by MAPK4. To identify signalling intermediates, proteins were isolated that interact with the Drosophila Ets transcription factor Yan. Yan functions downstream of several receptor tyrosine kinase (RTK) pathways, where it regulates the differentiation of many precursor cells. Experiments in tissue-culture cells suggest that Yan functions as a transcriptional repressor whose activity is attenuated rapidly by MAPK phosphorylation (Baker, 2001).

Using full-length yan complementary DNA as a bait, a yeast two-hybrid screen was conducted of an 18-h Drosophila embryo library, from which a full-length cDNA clone was recovered. The only region of homology between Yan and the predicted Mae protein is a Pointed (Pnt) domain located at the carboxy terminus. The Pnt domain (also called the SAM domain) defines a subfamily of Ets proteins, including Ets-1, Ets-2, GABP and TEL from vertebrates, and Drosophila Yan and Pnt-P2. It is also found in Polycomb-group proteins, where it mediates protein-protein interactions, and in MAPK kinase kinases that are components of the MAPK cascade (Baker, 2001).

Since Yan represses transcription by binding to consensus Ets DNA-binding sites, whether Mae interferes with Yan binding to DNA in a gel mobility shift assay was tested. Mae does not bind to the consensus Ets DNA-binding site, but it prevents Yan from doing so. This inhibition is mediated through the Pnt domain-dependent binding of Mae to Yan. DNA binding by Yan requires a functional Ets DNA-binding domain, but not intact phosphorylation sites or a functional Pnt domain, because Yan^ACT (in which all nine MAPK consensus sites are mutated to alanine) binds the DNA site normally, as does Yan(G84P) in which an amino acid conserved in all Pnt domains is mutated (Baker, 2001).

An assay was established of transcriptional repression by Yan. Because Drosophila S2 cells express mae endogenously, the yan and mae constructs were expressed in Cos-7 cells. Yan represses the activity of a TK-luciferase reporter tenfold. Repression depends on the Ets DNA consensus site, and on the Ets DNA-binding domain in Yan. As expected, Mae alone does not affect reporter activity because it lacks an Ets DNA-binding domain (Baker, 2001).

RTK signalling downregulates Yan activity through phosphorylation mediated by Ras and the Erk/Rolled MAPK. Expression of a constitutively active form of Ras (12H-Ras) completely abrogates transcriptional repression by Yan, but only in the presence of Mae. Consequently, Mae is required for Ras to inactivate Yan in Cos-7 cells. An excess of Mae alone can reduce the repressive effect of Yan (3-fold), presumably through formation of Mae-Yan heterodimers. However, low quantities of Mae, which alone inhibit the repressive effect of Yan by 30%, are sufficient to mediate the complete, Ras-dependent inactivation of Yan. Furthermore, the non-phosphorylatable Yan^ACT mutation is insensitive to Ras/Mae-induced inhibition, indicating that Mae inhibits Yan by promoting its phosphorylation by MAPK. Indeed, Yan mutants that cannot bind Mae -- Yan(G84P) and Yan(46-107) -- are also resistant to inactivation by Ras/Mae (Baker, 2001).

Although Yan has nine sites of MAPK phosphorylation, Ser 127 is the critical regulatory site of Yan because mutation to alanine creates a constitutive repressor that is refractory to downregulation by Ras signalling6. To test whether Mae mediates phosphorylation of Ser 127 of Yan, in vitro phosphorylation of Yan by activated Erk was examined. Activated Erk phosphorylates wild-type Yan independently of Mae, and, as expected, is unable to phosphorylate the constitutive repressor Yan^ACT, which has all nine MAPK phosphorylation sites mutated. Notably, Yan(S127), in which all consensus phosphorylation sites except Ser 127 are mutated to alanine, is resistant to phosphorylation by Erk alone, even with very high amounts (up to 1 microg) of the kinase. Yan(S127) is efficiently phosphorylated by Erk and Mae together, however, indicating that Mae is needed for phosphorylation of this Ser 127 residue (Baker, 2001).

This phosphorylation depends on the Pnt domain of Yan, indicating that the ability of Mae to promote Erk-directed phosphorylation of Ser 127 is dependent on binding of Mae to Yan. Yan(S127) represses transcription of the luciferase reporter as efficiently as the wild-type protein, and is also completely susceptible to inactivation by ¹²H-Ras/Mae. Therefore, Mae is required for MAPK to abolish transcriptional repression by Yan, by mediating phosphorylation of the critical regulatory residue Ser 127 (Baker, 2001).

Inhibition of Yan repression by RTK signalling in Drosophila is accompanied by activation of a transcription factor encoded by the pointed (pnt) gene. pnt encodes two alternative Ets transcription factors: P1, a constitutively active transcriptional activator whose expression is induced by Ras/MAPK signalling; and P2, a transcriptional activator that, like Yan, contains an amino-terminal Pnt domain and whose activity is stimulated through phosphorylation by the Ras/MAPK pathway. Activation of Pnt-P2 by Erk also seems to depend on Mae: the two proteins physically associate and this interaction is dependent on the Pnt domain. Pnt-P2 is a weak activator in this transcription assay and is not stimulated by Ras alone. However, Ras and Mae together augment its transcriptional activity fourfold. Thus, Mae also regulates Pnt-P2 activity, presumably by promoting its phosphorylation by MAPK (Baker, 2001).

These results show that Mae is needed in cultured cells to permit regulation of Ets transcription factor activity by Erk. This requirement was not evident in previous experiments on Yan because Drosophila S2 cells were used in which endogenous mae is expressed at high levels (Baker, 2001).

To study Mae function in vivo, in situ hybridization was used to examine Maeexpression in early Drosophila embryos. In stage 6-7 embryos, mae is expressed in bilaterally symmetric anteroposterior stripes, 3-4 cells wide, that flank the ventral midline. These stripes of expression narrow to a width of two cells and one cell adjacent to the midline by stage 9 and stage 11, respectively. mae expression is very similar to the expression pattern of epidermal growth factor receptor (EGFR) signalling regulators such as rhomboid (rho), vein, argos, yan and pnt, and it marks the ventral neurectodermal zone that is patterned by EGFR signalling. mae is subsequently expressed in tracheal pits, in ventral denticle domains, and in areas such as the optic lobe and the medial domains of the brain, all of which are sites of EGFR activity (Baker, 2001).

To investigate further the in vivo role of mae, the enhancer-trap lines l(2)k06602 and l(2)k12907, which contain single P-element insertions that are responsible for the lethal phenotype, were analyzed. These P-elements insert into the 5' untranslated region (UTR) of the mae gene [0.8-kilobases (kb) and 50-base-pairs (bp) upstream of the mae initiation codons in l(2)k0660 and l(2)k12907, respectively]. ß-Galactosidase in early l(2)k06602 and l(2)k12907 embryos is, like mae, expressed in the ventral neurectoderm, tracheal pits and ventral denticle belts. Furthermore, homozygous mae mutant embryos (25% of embryos derived from either heterozygous l(2)k06602 or l(2)k12907 parents) lack detectable mae expression. These results show that the P-element insertions affect development because they disrupt mae expression (Baker, 2001).

A role for Mae in EGFR signalling is further supported by certain features of the mae mutant phenotype. Embryonic patterning is affected in mae mutant embryos, especially towards the midline of the anterior abdominal segments. Ventral denticle belts are thinner, and rows of denticles, particularly the first, are missing or misorientated. maek12907 and maek06602 embryos each show a similar phenotype when hemizygous for Df(2R)PC4, a deficiency for the region, indicating that the patterning defects result from lack of mae expression. Preferential disruption of midline patterning in anterior abdominal segments is reminiscent of the phenotype of rho and pnt mutant larvae (Baker, 2001).

mae seems to be a downstream component of the EGFR signalling pathway. Its pattern of expression depends on Rho, the spatially restricted limiting component of EGFR signalling, but is not required for rho expression. Furthermore, in the l(2)k12907 enhancer-trap line in which mae expression is abolished, lacZ marks the mae transcriptional domains (and thereby domains of EGFR signalling), and this domain is broadened along the ventral midline. Similarly, the expression domain of argos is broadened in early stage mae mutant embryos, although levels of expression, notably in later stage embryos, seem to be reduced. argos is a downstream target of EGFR signalling whose expression is also disrupted in Drosophila embryos that are mutant for regulators of EGFR signalling such as Ras, Yan, Pnt and Rho (Baker, 2001).

The above experiments show that Mae mediates inactivation of Yan by MAPK phosphorylation of the critical regulatory Ser 127 residue. Presumably, binding of Mae causes a local change in the conformation of Yan, which exposes Ser 127 to MAPK. Further evidence for this idea is that Mae binding to the Pnt domain in Yan also affects the ability of Yan to bind DNA through its Ets domain. Erk can associate with Mae and Yan in GST pull-down assays but, unlike Mae binding to Yan, this interaction is weak and is not evident in co-precipitation assays. It is proposed that Mae binding to Yan or Pnt-P2 allows MAPK to phosphorylate their critical residues in a transitory ternary enzyme-substrate complex (Baker, 2001).

This is the first report of a requirement for an intermediary protein linking MAPK to its substrate, and it suggests a mechanism for achieving tissue-specific responses to the generic RTK/Ras/MAPK signalling pathways; that is, determination of the MAPK substrate through tissue-specific adapter/coupling proteins. Cells will respond differently to MAPK according to the adapter/coupling proteins that they express. Future work will help test whether phosphorylation of other MAPK substrates also depends on adapter/coupling proteins (Baker, 2001).

The transcriptional repressor Yan prevents inappropriate responses to receptor tyrosine kinase signaling by outcompeting Pointed for access to target gene promoters. The molecular mechanism underlying downregulation of Yan involves CRM1-mediated nuclear export. A novel role in this context is defined for MAE, a co-factor previously implicated in facilitating MAPK phosphorylation of Yan. In addition to promoting Yan downregulation, MAE also participates in an inhibitory feedback loop that attenuates Pointed-P2 activation. Thus, it is proposed that MAE plays multiple independent roles in fine-tuning the levels of Pointed and Yan activity in accordance with changing RTK signaling conditions (Tootle, 2002).

MAPK-mediated recognition and phosphorylation of Yan at Serine 127 is thought to be facilitated by a protein called Modulator of the Activity of ETS (MAE) (Baker, 2001). Mechanistically, MAE binds to Yan via a protein-protein interaction motif found at the N terminus of Yan and the C terminus of MAE (Baker, 2001), referred to as the Pointed Domain (PD). Interestingly, it has been suggested (Baker, 2001) that MAE binds to the PD of PNT-P2, and enhances the transcriptional activation of PNT-P2; this has led to the proposal that MAE promotes PNT-P2 phosphorylation by MAPK. Thus, it has been speculated that by promoting phosphorylation events that simultaneously downregulate Yan and upregulate PNT-P2, MAE facilitates downstream responses to RTK signaling (Tootle, 2002).

Although it is clear that MAPK phosphorylation initiates Yan downregulation, the ensuing events, with respect to both Yan and PNT-P2, remain poorly understood. This study shows that nuclear export, via CRM1, is an essential step in downregulating Yan both in cell culture and in vivo. In this context, the PD of Yan plays a dual role in maintenance of nuclear localization in the absence of signaling and regulation of nuclear export upon RAS/MAPK activation. By manipulating the levels of mae expression in cells co-expressing specifically designed structural variants of Yan, it has been demonstrated that MAE plays a crucial role in mediating the nuclear export of Yan, independent of its role in promoting MAPK phosphorylation. Consistent with previous reports (Baker, 2001), it has been found that overexpression of MAE decreases transcriptional repressor activity of Yan. However, whereas the transcriptional activity of PNT-P2 was proposed to be stimulated by MAE co-expression, it has now been found that overexpression of MAE inhibits the ability of PNT-P2 to activate transcription. Thus, it is proposed that MAE mediates downregulation of both Yan and PNT-P2. In the case of Yan, MAE facilitates MAPK-mediated phosphorylation and subsequent nuclear export, while in the case of PNT-P2, MAE could participate in a negative feedback loop that attenuates transcriptional activity (Tootle, 2002).

Although redistribution of Yan from the nucleus to the cytoplasm upon RAS/MAPK activation in S2 cultured cells is suggestive of nuclear export, it is formally possible that this shift results from degradation of Yan in the nucleus, coupled with a failure of newly synthesized and phosphorylated Yan to enter the nucleus. To determine if the cytoplasmic accumulation of Yan in RAS^V12 stimulated S2 cultured cells is a consequence of nuclear export, it was asked whether blocking the nuclear export machinery would result in nuclear retention of Yan. Yan, which is predicted to be 78 kDa, is too large to diffuse through the nuclear pore, and thus its export must occur by facilitated transport. CRM1, a common exportin, mediates translocation of nuclear export sequence (NES) containing proteins from the nucleus. It was found that in RASV12-stimulated S2 cultured cells, Yan is retained in the nucleus in the presence of Leptomycin B (LMB), a drug that specifically binds and inhibits CRM1, or dsRNA interference (RNAi) to knock down crml expression. These data indicate that the cytoplasmic accumulation of Yan induced by RAS/MAPK activation is the result of CRM1-dependent nuclear export (Tootle, 2002).

In unstimulated or undifferentiated cells, Yan localizes to the nucleus. For both Yan and its mammalian ortholog TEL, the DNA-binding domain serves as a nuclear localization sequence (NLS). Upon RTK stimulation, Yan is actively exported from the nucleus via CRM1 recognition of its N-terminal NES motif. The presence of both NLS and NES motifs within Yan raises the question of how each domain is either recognized or masked under different signaling conditions (Tootle, 2002).

These results led to the proposal that proper Yan subcellular localization involves dynamic regulation of its DNA-binding affinity via modulation of protein-protein interactions in response to changing RTK signaling levels. Consistent with this model, it has been found that nuclear localization requires that Yan be bound to the DNA, since a mutation that abolishes DNA binding, yan^{Mut ETS}, results in CRM1-dependent cytoplasmic accumulation of Yan. The PD, an N-terminal protein-protein interaction motif, also plays a pivotal role in determining the subcellular localization of Yan, since loss of the PD (yan^DeltaNES3+PD) results in partial CRM1-mediated export in the absence of signaling. In addition, Yan^DeltaNES3+PD exhibits a 30% decrease in repression activity relative to wild-type Yan, suggesting a weaker or less productive interaction with DNA. Together these data suggest that PD-mediated protein-protein interactions may be crucial in facilitating productive DNA binding and/or masking inappropriate CRM1 recognition of the NESs (Tootle, 2002).

The finding that PD-mediated interactions are crucial for the transcriptional repression ability of Yan agrees with similar experiments with TEL, but disagrees with the results of (Baker, 2001) who find that compromised PD function has no significant effect on the transcriptional repression of Yan. Presumably, this discrepancy reflects the use of the mammalian Cos7 cell line to study Yan (Baker, 2001), as opposed to the more physiologically relevant Drosophila S2 cell line used in the current experiments (Tootle, 2002).

One explanation for how the PD of Yan might be involved in DNA-binding affinity, transcriptional repression and maintenance of nuclear localization comes from structural studies of the PD of TEL. This work suggests that DNA binding and transcriptional repression may be mediated by a PD-PD homo-oligomeric complex of TEL that wraps the target DNA around itself, and because yan has been shown to self-associate via its PD, it is possible that oligomerization of Yan could be critical for DNA binding/nuclear localization (Tootle, 2002).

In addition to promoting homotypic Yan-Yan interactions, PD-mediated binding to heterologous proteins may also influence Yan localization and activity. MAE, the only protein known to interact with the PD of Yan (Baker, 2001), appears to serve such a function. Co-immunoprecipitation experiments have confirmed that MAE can bind to Yan in the absence of signaling, and show that the complex is destabilized in the presence of RAS/MAPK activation. However, because MAE inhibits Yan-mediated transcriptional repression, it is expected that, in the absence of signaling, not all Yan will be bound to MAE. The finding that MAE can also be co-immunoprecipitated with PNT-P2, suggests a mechanism for sequestering MAE away from Yan to allow efficient repression and prevent inappropriate differentiation in the absence of signaling (Tootle, 2002).

Upon activation of the RAS/MAPK cascade, dual phosphorylated MAPK enters the nucleus and phosphorylates Yan, triggering a cascade of events that ultimately leads to the removal of transcriptional repression. MAE is needed for MAPK-mediated phosphorylation of Yan at Serine 127 in vitro, the same site previously shown to be critical for initiating Yan downregulation both in cell culture and in vivo. This study sheds new light on the sequence of steps in this process. CRM1-mediated nuclear export is a necessary step in downregulation of Yan. How is this achieved? A model is supported whereby in response to pathway stimulation, the PNT-P2-MAE complex is phosphorylated, releasing PNT-P2 to activate transcription and MAE to interact with Yan. Binding to MAE inhibits the transcriptional repression of Yan, and may facilitate phosphorylation of serine 127 by activated MAPK, although the order in which these two events happen remains to be determined. These data suggest MAE then plays a third role in presenting Yan to CRM1, thereby promoting nuclear export (Tootle, 2002).

In support of this model, loss of mae function, both in vivo and in cell culture, restricts Yan to the nucleus. However, since MAPK phosphorylation of Yan is a prerequisite for export and requires MAE, these result could simply reflect a failure of Yan to be phosphorylated. Arguing against this, RNAi of mae also results in nuclear retention of Yan^{Mut ETS}, which normally localizes to the cytoplasm in a CRM1-dependent manner, even in the absence of RAS stimulation. Thus, in a situation where MAPK phosphorylation is not involved, MAE plays an active role in presenting Yan to CRM1. Therefore the favored interpretation is that MAE has an essential function in regulating nuclear export, independent of its earlier postulated role in facilitating MAPK phosphorylation of Yan (Tootle, 2002).

These same two events mediated by MAE, MAPK phosphorylation and CRM1 recognition of Yan, in turn lead to destabilization of the Yan-MAE complex. For example, inhibition of CRM1-mediated export results in MAE remaining nuclear when co-transfected with Yan, even upon RAS^V12 stimulation. Because MAE localization is not directly regulated by CRM1 or by RAS pathway activation, this result is interpreted to indicate that CRM1 is needed to disrupt the Yan-MAE complex. It has been shown that in certain cases, phosphorylation of the cargo protein is necessary for CRM1 recognition. In agreement with this, in the presence of RAS^V12, MAE remains nuclear when expressed with Yan^ACT, which has all the MAPK phosphoacceptor residues mutated to alanine. This leads to the model that phosphorylation of Yan, when in the Yan-MAE complex, leads to interaction with the exportin CRM1. This in turn disrupts the Yan-MAE complex, with Yan being actively exported by CRM1, and MAE being free to diffuse uniformly throughout the cell (Tootle, 2002).

The ultimate outcome of this complex series of events is abrogation of Yan-mediated repression of target genes and freeing the promoters for interaction with Pointed. In unstimulated cells, unphosphorylated PNT-P2 localizes to the nucleus in a complex with MAE, but is effectively out competed for binding to target gene promoters by Yan. Upon activation of the RAS/MAPK cascade, phosphorylation of PNT-P2 transforms it into a potent transcriptional activator. In vitro experiments show that MAE binding to PNT-P2 leads to activation of transcription, and this is assumed to occur via MAE promoting MAPK phosphorylation, and hence activation, of PNT-P2. It has been shown that PNT-P2 contains a MAPK binding site, suggesting PNT-P2 interacts directly with MAPK without requiring a facilitator protein. Consistent with this second scenario, it has been found that MAE inhibits PNT-P2 transcriptional activation. However, it is formally possible that MAE could have dual and antagonistic roles with respect to PNT-P2 regulation, first stimulating its activity by promoting MAPK phosphorylation and later limiting its ability to activate transcription. Definitive validation of either model will require in vivo analysis of the role of MAE with respect to PNT-P2 regulation (Tootle, 2002).

Superficially, this proposed role in antagonizing PNT-P2 function seems to disagree with the finding that loss of mae function suppresses the rough eye phenotype of Sev-RAS^V12. However, in the absence of MAE, Yan cannot be downregulated. Thus, the effect of loss of mae function on PNT-P2 regulation is irrelevant in this context, since the target sites will still be occupied by Yan. However, the dual function of MAE as both a positive and a negative regulator of RTK signaling may explain the relatively weak suppression of Sev-RAS^V12 and the fact that it has not been isolated in any of the numerous RTK pathway-based genetic modifier screens (Tootle, 2002).

In summary, these data lead to a model in which, in unstimulated cells, Yan binds with high affinity to the DNA and blocks PNT-P2 from contacting and activating the promoters of downstream target genes. Upon stimulation by RAS, MAPK phosphorylation of Yan and PNT-P2 allows CRM1 to interact with and export Yan, in a process that disrupts Yan and MAE binding and disrupts the PNT-P2-MAE complex, allowing PNT-P2 to bind to the DNA and activate transcription. Free MAE could then interact again with PNT-P2, resulting either in its removal from the DNA, inhibition of transcriptional activation or interaction with a phosphatase that returns it to an inactive state. Thus, a negative feedback loop would be created to prevent runaway signaling by PNT-P2. An alternative, and not necessarily mutually exclusive, mechanism with respect to PNT-P2, is that the interaction of MAE with PNT-P2 might prevent efficient phosphorylation by MAPK, thereby limiting the pool of activated PNT-P2 and keeping the signaling response in check. It is likely that additional co-factors that bind MAE, Yan and/or PNT-P2 will be required for fine-tuning activation and downregulation in response to changing RTK signaling conditions (Tootle, 2002).

Precise regulation of RTK pathway activity appears crucial for achieving a proper balance between cellular proliferation, differentiation and survival in all metazoan animals. Excessive or continuous activation of the pathway has been linked to carcinogenesis in mammals, underscoring the importance of tightly controlled signaling. For example, numerous deletions and translocations involving TEL, the mammalian ortholog of Yan, have been associated with leukemias, and in some cases with solid tumors. Striking similarities exist between the regulation of TEL and Yan. Like Yan, TEL localizes to the nucleus, where it functions as a transcriptional repressor. Yan and TEL both require the PD for maintaining nuclear localization and transcriptional repression. Both proteins become phosphorylated in response to activation of signaling cascades. Although the functional consequences of TEL phosphorylation remain to be investigated, the current results predict that phosphorylation may downregulate TEL repression activity (Tootle, 2002).

In the context of TEL downregulation, it is interesting to note that no mammalian orthologs of mae have been identified yet. However, a second mammalian TEL-like gene, referred to as TEL2 or TELB, has been isolated. TEL2 also functions as a transcriptional repressor, is capable of oligomerizing with itself and with TEL, and may thus serve as a regulator of TEL. Of particular interest with respect to this work defining the role of MAE, TEL2 encodes six splice variants, one of which, TEL2a, yields a protein with just the PD. TEL2a closely resembles the structure of MAE, and BLAST results show that the PD of MAE is most closely related to the PD of TEL2, with 39% identity and 51% similarity. Thus, it seems likely that TEL2a may regulate TEL activity by a mechanism similar to that used by MAE for regulating Yan. With respect to the interactions that have been demonstrated between PNT-P2 and MAE, it will be interesting to investigate whether TEL2a also interacts with and regulates other PD containing ETS family transcriptional activators, such as ETS1, the mammalian ortholog of PNT-P2 (Tootle, 2002).

Derepression by depolymerization: structural insights into the regulation of Yan by Mae

Yan, an ETS family transcriptional repressor, is regulated by receptor tyrosine kinase signaling via the Ras/MAPK pathway. Phosphorylation and downregulation of Yan is facilitated by a protein called Mae. Yan and Mae interact through their SAM domains. Repression by Yan requires the formation of a higher order structure mediated by Yan-SAM polymerization. Moreover, a crystal structure of the Yan-SAM/Mae-SAM complex shows that Mae-SAM specifically recognizes a surface on Yan-SAM that is also required for Yan-SAM polymerization. Mae-SAM binds to Yan-SAM with approximately 1000-fold higher affinity than Yan-SAM binds to itself and can effectively depolymerize Yan-SAM. Mutations on Mae that specifically disrupt its SAM domain-dependent interactions with Yan disable the derepression function of Mae in vivo. Depolymerization of Yan by Mae represents a novel mechanism of transcriptional control that sensitizes Yan for regulation by receptor tyrosine kinases (Qiao, 2004).

This study describes new mechanism for the regulation of the polymeric transcriptional repressor Yan, a repressor that must be exquisitely sensitive to RTK stimulation at the cell surface. In particular, Mae regulates Yan by blocking Yan polymerization. As shown in detail from crystal structure of the complex, Mae-SAM utilizes its ML surface to interact with the EH-surface of Yan-SAM. The binding mode is an almost perfect mimic of the binding mode in the Yan-SAM polymer. Mae-SAM does not possess a functional EH surface, however, so it cannot be incorporated into the polymer. The interaction seen in the crystal structure was validated biochemically and the biological relevance was demonstrated by the finding that Mae variants with mutations in the observed interface between Yan-SAM and Mae-SAM fail to downregulate Yan. Mae-SAM binds with much higher affinity than Yan-SAM, so it can compete effectively for polymer formation. Further experiments will be required to understand the origin of the affinity differences between Yan and Mae since it is not obvious from the structures alone (Qiao, 2004).

Although nuclear export appears to be an important part of Yan downregulation, abrogation of repression activity in response to Mae-mediated depolymerization of Yan does not require nuclear export. Mechanistically, loss of self-association could cause a reduction in Yan's ability to polymerize on DNA, leading to derepression of transcriptional targets. It is possible that Mae also prevents DNA binding by direct inhibition of the DNA binding domain of Yan. Although Mae can directly inactivate Yan, nuclear export probably works in consort with depolymerization. Earlier results showing that phosphorylation is required for nuclear export suggest that phosphorylation may act as a trigger for this relocalization (Qiao, 2004).

These results also provide new insight into the role of Mae in Yan phosphorylation. It has been proposed that Mae acts to recruit MAPK to Yan and may also induce a conformational change in Yan to expose the site of phosphorylation. The fact that Mae depolymerizes Yan suggests a mechanism by which Mae can activate Yan for phosphorylation. The key phosphorylation site on Yan, Ser127, is very close to the SAM domain and would therefore most likely be inaccessible to the kinase in the polymer. Depolymerization would therefore be necessary for the kinase to act. Thus, by depolymerizing Yan, Mae can play a multifaceted role in Yan downregulation. Mae depolymerization could prevent spreading, expose the site of phosphorylation, and facilitate the recruitment of MAPK to Yan (Qiao, 2004).

From the proposed regulatory model, one might expect that Mae and Yan would exhibit complementary patterns of expression (i.e., that Mae expression would be high where Yan expression is low and vice versa). This might help to ensure that Mae does not interfere with Yan-mediated repression prior to RTK stimulation. It is noted that this is not a requirement of the model since Mae could be subject to additional layers of regulation. Nevertheless, antibody staining and in situ hybridation data are consistent with this expectation. For example, in the germ band elongating embryo, Yan is largely absent from the midline of the CNS, while Mae is expressed most strongly at the midline. Furthermore, in the developing eye disc, Mae is primarily expressed in differentiated photoreceptors, while Yan is downregulated as photoreceptor differentiation occurs (Qiao, 2004 and references therein).

Polymerization of SAM domains is essential for the repressive functions of both Yan and TEL. Moreover, the SAM domains of the unrelated polycomb group transcriptional repressors, Ph and Scm, also form polymers. This polymerization could be used to generate a large transcriptionally silenced chromosomal domain. In the case of repression by TEL and Yan, the SAM domain-containing factor also contains a sequence-specific DNA binding domain, namely the ETS domain. Binding of Yan and TEL to specific sites via the ETS domain could serve to nucleate a polymer, which would then spread by the oligomerization of the SAM domain. In contrast, Ph and Scm do not contain obvious sequence-specific DNA binding motifs, so their initial binding to the template may require protein-protein interactions with other sequence-specific transcriptional repressors. For example, the job of the polycomb group proteins is to maintain the repressed transcriptional state established by segmentation gene-encoded transcriptional repressors such as Hunchback. Perhaps one role of the segmentation gene-encoded repressors is to recruit (either directly or indirectly) SAM domain-containing polycomb group proteins, which can then spread along the template via polymerization. In support of this idea, the polymeric SAM domain of Scm is essential for long range repression (Qiao, 2004 and references therein).

Spreading perhaps via corepressor polymerization has also been implicated in heterochromatic silencing. For example, telomeric silencing in budding yeast by the Sir3/Sir4 corepressors and centromeric silencing in fission yeast, Drosophila, vertebrates, and plants by HP1/Swi6 corepressors may involve corepressor spreading. In the absence of appropriate boundary elements, and/or in the presence of abnormally high concentrations of the corepressors, increased spreading results in the silencing of genes in the adjacent euchromatic regions. The precise mechanism of spreading in these systems remains unknown, although polymerization seems to be a likely mechanism for this type of long range repression (Qiao, 2004).

To the extent that polymerization is utilized in gene silencing, mechanisms for regulating the extent of polymerization must exist in the cell. The current results demonstrate that one mechanism for regulating the extent of polymerization is to utilize a protein that can only bind to one end of the polymer, which effectively blocks further extension. Thus, the results provide initial structural insight into what may prove to be a common mechanism in the regulation of polymeric transcriptional repressors (Qiao, 2004).

One important feature of Yan-mediated silencing distinguishes it from both polycomb silencing and heterochromatic silencing. Both polycomb and heterochromatic silencing represent epigenetically stable states that are normally maintained throughout the lifetime of an organism and even from one generation to the next. In contrast, Yan-mediated silencing is a comparatively unstable state that must be rapidly reversed during the normal developmental cycle in response to RTK activation. It is speculated that, by limiting the extent of Yan polymerization, Mae may help to generate a state in which targets of Yan repression are poised for rapid derepression (Qiao, 2004).

Antagonistic regulation of Yan nuclear export by Mae and Crm1 may increase the stringency of the Ras response

Phosphorylation of Yan, a major target of Ras signaling, leads to Crm1-dependent Yan nuclear export, a response that is regulated by Yan polymerization. Yan SAM (sterile {alpha} motif) domain mutations preventing polymerization result in Ras-independent, but Crm1-dependent Yan nuclear export, suggesting that polymerization prevents Yan export. Mae, which depolymerizes Yan, competes with Crm1 for binding to Yan. Phosphorylation of Yan favors Crm1 in this competition and counteracts inhibition of nuclear export by Mae. These findings suggest that, prior to Ras activation, the Mae/Yan interaction blocks premature nuclear export of Yan monomers. After activation, transcriptional up-regulation of Mae apparently leads to complete depolymerization and export of Yan (Song, 2005).

Yan polymerization is mediated by two hydrophobic SAM domain interfaces termed the mid-loop (ML) and end-helix (EH) surfaces, which bind one another to form a head-to-tail polymer. Mutagenesis of key residues in these surfaces (e.g., Ala86 on the ML surface or Val105 on the EH surface) converts Yan from a polymer into a monomer (Song, 2005).

In unstimulated Drosophila S2 cells, wild-type Yan remains in the nucleus, while introduction of constitutively active Ras (Ras^V12) results in Yan export. In contrast, the two monomeric mutants, Yan^A86D and Yan^V105R, are both exported from the nucleus even in the absence of Ras signaling. Nuclear localization was quantified by categorizing cells according to whether they displayed predominant nuclear localization, predominant cytoplasmic localization, or localization in both the nucleus and cytoplasm. Approximately 90% of cells expressing wild-type Yan displayed predominant nuclear localization. In contrast, only ~20% of cells expressing monomer Yan displayed predominant nuclear localization, with ~50% displaying localization in both the nucleus and cytoplasm, and ~30% displaying predominant cytoplasmic localization. Export of monomeric Yan mutants is fully Crm1-dependent, since cotransfection of dsRNA against Crm1 results in predominant nuclear localization of monomeric Yan in 90% of the cells. Conversely, overexpression of Crm1 enhances monomeric Yan export; >90% of Yan^A86D-expressing cells display predominant cytoplasmic localization of monomeric Yan (Song, 2005).

Since unstimulated S2 cells may nonetheless contain low levels of Ras signaling, it was determined whether or not monomer export requires the critical phosphoacceptor Ser127 residue in Yan. Monomeric Yan harboring the S127A mutation is still exported to the cytoplasm, although to a lesser extent than is monomeric Yan without the S127A mutation. Once again, the export was completely dependent upon Crm1 as demonstrated by Crm1 RNA interference (RNAi). In conclusion, the export of monomeric Yan is dependent on Crm1. Ras signaling stimulates but is not strictly required for export of the monomeric protein (Song, 2005).

The Mae SAM domain contains a functional ML surface that binds the Yan SAM EH surface, blocking Yan polymerization. Mae lacks a functional EH surface and so cannot bind to the Yan ML surface. Because the intrinsic affinity of Yan EH for Mae ML is three orders of magnitude greater than the intrinsic affinity of Yan EH for Yan ML, complete depolymerization of Yan occurs whenever Mae is present in stoichiometric excess over Yan. Under these conditions, all the Yan is driven into Yan:Mae heterodimers (Song, 2005).

If nuclear export of the Yan monomer is due to the exposure of Crm1-binding site(s) in the monomer that are buried in the Yan polymer, the Yan:Mae interaction might also mask the Crm1-binding site(s), resulting in the nuclear retention of the monomer. In accord with this idea, cotransfection of Mae prevents the nuclear export of Yan^A86D, a monomeric Yan mutant that retains the ability to bind Mae. The nuclear retention of the Yan monomer is completely dependent upon the interaction between Yan and Mae, since a Mae mutant with a defective ML surface, which is unable to bind to Yan, fails to interfere with the export of Yan^A86D. In addition, the localization of Yan^V105R, a monomeric Yan mutant that cannot bind Mae, is not altered by cotransfection of wild-type Mae (Song, 2005).

If Mae masks the Crm1-binding site(s) on Yan, then Crm1 might compete with Mae for binding to Yan. In this case, Crm1 overexpression would be expected to antagonize nuclear retention of Yan by Mae. In addition, if Yan phosphorylation facilitates Yan export by helping Crm1 to compete with Mae, cotransfection of Ras^V12 should also antagonize Mae-mediated Yan nuclear retention. To test these possibilities Yan^A86D and Mae were coexpressed in the presence of Ras^V12, Crm1, or both. Overexpression of Mae results in predominant nuclear localization of monomeric Yan in ~85%-95% of the cells. Coexpression of Crm1 reduces this proportion to ~20%-30%, dependent on the dose of transfected Mae. Coexpression of Ras^V12 alone has a much smaller effect on Mae-mediated Yan nuclear retention -- the proportion of cells with predominant nuclear localization was ~65%-85%. The level of Ras^V12 expression used in these experiments is sufficient to drive essentially all Yan from the nucleus in the absence of cotransfected Mae. Coexpression of Ras^V12 and Crm1 results in additive or slightly greater than additive effects on monomeric Yan nuclear retention, supporting the idea that Ras signaling facilitates Crm1-mediated Yan export (Song, 2005).

To test directly for competition between Crm1 and Mae, coprecipitation assays were employed. Because the interaction between Crm1 and its targets is affected by multiple factors (e.g., it is stabilized by RanGTP in the nucleus and destabilized by RanGDP in the cytoplasm, and may depend upon additional adaptors) S2 cell nuclear extracts were employed as a source of Crm1. Transiently expressed Flag-tagged Yan was purified on anti-Flag agarose beads, and then incubated with S2 nuclear extract. Crm1 precipitates with the Flag-Yan-bound beads. The bound Crm1 is displaced from the beads upon addition of the wild-type MBP-MaeSAM domain, but not upon addition of MBP-MaeSAM^A141D, in which the SAM domain contains a point mutation that disrupts the ML surface (Song, 2005).

Next it was determined whether phosphorylation of Yan enhances the coprecipitation of Crm1. Flag-tagged wild-type Yan was transiently expressed in S2 cells in the absence or presence of Ras^V12 and then tested in coprecipitation assays for binding to Crm1 in S2 cell nuclear extracts. Like untagged Yan, Flag-tagged Yan localizes to the nucleus in the absence of Ras signaling and is exported to the cytoplasm in the presence of Ras signaling. The amount of Ras^V12 used in these experiments was sufficient to drive nearly all the Yan protein into the cytoplasm, indicating that Yan phosphorylation is likely quantitative. This was further verified by anti-Flag immunoblots showing a quantitative mobility shift of Flag-Yan upon coexpression with Ras^V12. The Crm1-Yan interaction was more stable when the Yan was expressed with the activated form of Ras, as shown by the coprecipitation and Mae competition assays. This enhanced interaction was likely dependent on phosphorylation of Yan on Ser127; coexpression with activated Ras has no effect on the ability of Flag-tagged Yan^S127A to bind Crm1 (Song, 2005).

In contrast to the results in which a Crm1-containing nuclear extract was imployed, purified Crm1 does not show significant affinity for Yan, indicating that the nuclear extract provides additional components that stabilize the interaction. This is consistent with studies on exportins showing that they often require adaptor molecules for high-affinity binding to their targets. In addition, the GTP-bound form of Ran is required for the stable binding of Crm1 to its targets, and therefore GMP-PNP was included as a standard component in all the Crm1 binding assays (Song, 2005).

A model is proposed in which Mae plays multiple roles in regulating the Crm1:Yan interaction. In cells that have not received the RTK signal, polymeric Yan is in equilibrium with depolymerized Yan. The formation of the depolymerized Yan is likely favored by the presence of a small amount of Mae in the nucleus of unstimulated cells, which binds and blocks one of the Yan polymerization interfaces (the Yan EH surface). It is suspected that by promoting limited Yan depolymerization, Mae ensures that unstimulated cells will be poised to receive the RTK signal. However, at this stage Mae also appears to block access of Crm1 to depolymerized Yan. This is important since if depolymerized Yan was accessible to Crm1, the resulting export of Yan would pull the equilibrium away from the polymer, leading to further Yan export and ultimately to premature expression of Yan target genes (Song, 2005).

It is essential to maintain Mae at low levels in undifferentiated cells, because Mae-mediated Yan depolymerization is sufficient to result in derepression, even in the absence of Ras signaling. Maintenance of low levels of Mae prior to differentiation appears to be at least partially ensured by the Yan-dependent repression of Mae. Upon activation of Ras, Ser127 in the Yan-Mae dimer is phosphorylated by the activated Rolled MAPK, favoring Crm1 in the competition with Mae for binding to Yan. The resulting increased Yan export is expected to relieve the transcriptional repression of Mae, and the accumulation of additional Mae is then expected to result in further Yan depolymerization, phosphorylation, and nuclear export. This feedback loop ensures that while Yan targets will be stably repressed prior to differentiation, they will be completely derepressed upon reception of the RTK signal. Furthermore, this feedback loop could serve to amplify a small change in the level of the RTK ligand, thereby allowing for sharp threshold responses to graded extracellular ligands (Song, 2005).

Downregulation of vertebrate Tel (ETV6) and Drosophila Yan is facilitated by an evolutionarily conserved mechanism of F-box-mediated ubiquitination

The vertebrate Ets transcriptional repressor Tel (ETV6) and its invertebrate orthologue, Yan, are both indispensable for development, and they orchestrate cell growth and differentiation by binding to DNA, thus inhibiting gene expression. To trigger cell differentiation, these barriers to transcriptional activation must be relieved, and it is established that posttranslational modifications, such as phosphorylation and sumoylation, can specifically impair the repressive functions of Tel and Yan and are crucial for modulating their transcriptional activity. To date, however, relatively little is known about the control of Tel and Yan protein degradation. In recent years, there has been a concentrated effort to assign functions to the large number of F-box proteins encoded by both vertebrate and invertebrate genomes. This study reports the identification and characterization of a previously unreported, evolutionarily conserved F-box protein named Fbl6. Both human fb16 and Drosophila fbl6 cDNA were isolated, and it was shown that the encoded Fbl6 protein binds to both Tel and Yan via their SAM domains. Both Tel and Yan are ubiquitinated, a process which is stimulated by Fbl6 and leads to proteasomal degradation. It has been established that the sumoylation of Tel on lysine 11 negatively regulates its repressive function and that the sumoylation of Tel monomers, but not that of Tel oligomers, may sensitize Tel for proteasomal degradation. This study found that Fbl6 regulates Tel/Yan protein stability and allows appropriate spatiotemporal control of gene expression by these repressors (Roukens, 2008b).

The ancestral Ets repressor Yan and its vertebrate orthologue, Tel (ETV6), play pivotal roles in the control of cell differentiation. Because these factors directly and negatively regulate gene expression, deciphering the mechanisms that regulate their activity is crucial for understanding the spatiotemporal control of cell differentiation. Sumoylation of an N-terminal lysine residue encoded by Tel (TelK11) serves to inhibit repression of gene expression by Tel (Roukens, 2008a). This study yas further explored Tel/Yan posttranslational regulation and reports that both Tel and Yan protein downregulation is promoted by an evolutionarily conserved F-box-protein-dependent mechanism. Specifically, both Tel and Yan can be ubiquitinated and that ubiquitination is facilitated by Fbl6, which sensitizes these proteins for degradation. It is, of course, possible that other F-box proteins may also play a role in regulating Tel/Yan activity, perhaps in a tissue-specific fashion, and future studies should clarify this (Roukens, 2008b).

The Tel/Yan SAM domain is required for association with the F-box protein Fbl6. The SAM domain of both Tel and Yan is required for the binding of Fbl6. The SAM domain defines a subfamily of Ets transcription factors, including Tel, Tel2, Ets1, and Ets2 from vertebrates as well as Yan and Pointed P2 from Drosophila, raising the possibility that at least some of these factors are also targeted by Fbl6 or a related F-box protein. Indeed, it was found that human Fbl6 also associates with the Tel-related Tel2 protein and promotes its ubiquitination. Furthermore, biochemical interactions have been detected between Drosophila Fbl6 and the SAM domain-containing proteins Pointed P2 but not P1, which lacks the SAM domain) and Mae in tissue culture cells. However, no associations were detected between Fbl6 and either Ets1 or Ets2, each of which harbors a SAM domain, suggesting that there is specificity, perhaps structural, that determines these interactions. Many other families of proteins express SAM domains, such as the polycomb group proteins and components of the mitogen-activated protein kinase cascade. It should be interesting to investigate whether Fbl6 (or related F-box proteins) plays a role in regulating their activity (Roukens, 2008b).

Tel monomers are especially labile and prone to ubiquitination. The data support the idea that Tel/Yan monomers are more susceptible to ubiquitination and degradation than the oligomeric forms of these proteins are. Apparently, and paradoxically, the biochemical interactions between Tel/Yan monomers and Fbl6 were found to be far weaker than the interactions between Tel/Yan oligomers and Fbl6. However, these interactions were strongly stabilized in the presence of proteasome inhibitors, suggesting that the failure to find strong associations between these proteins resulted from the instability of the Tel/Yan monomer and of the Fbl6 complex. Consistent with this, it was found that Tel monomers are more readily ubiquitinated than wild-type Tel. It is worth noting that, in general, wild-type Tel is detected in cells as a phosphorylated or nonphosphorylated protein species and that the phosphorylation of Tel appears to further sensitize it to Fbl6-mediated degradation. In contrast, phosphorylated forms of monomeric Tel are evidently especially unstable and are detected only in the presence of proteasome inhibitors. It has previously been described that Tel is negatively regulated by extracellular signal-regulated kinase-induced phosphorylation of serine residues 213 and 257 (Maki, 2004). Phosphorylation of these sites could be a trigger for promoting Tel ubiquitination, although in ubiquitination assays, it was not possible to detect an obvious effect of mutating these residues or an additional putative mitogen-activated protein kinase target, serine residue 22 in the N terminus of Tel. Previous work also supports the contention that Tel/Yan monomers are relatively labile (Roukens, 2008a). Monomeric Tel was detectably and very efficiently sumoylated in cells only in the presence of proteasome inhibitors, suggesting that this species of Tel is normally very unstable. The current model of Tel function holds that Tel monomers directly associate via their conserved SAM domains and that the resulting DNA-bound oligomers oppose the transcription-activating apparatus. Since Fbl6 also interacts with the Tel SAM domain, perhaps self-associated Tel oligomers are relatively more resistant to F-box-mediated degradation than are Tel monomers because of reduced accessibility to exposed SAM domains. In the future, it should be of considerable interest to elucidate how Tel sumoylation and ubiquitination are precisely integrated to regulate Tel activity (Roukens, 2008b).

Fbl6 regulates Yan protein stability. This work suggests that Fbl6 regulates Yan protein levels in vivo. Drosophila embryos that lack fbl6 expression contain significantly elevated levels of Yan protein. Consistent with this, the levels of expression of a downstream target of Yan named argos was sharply reduced. Surprisingly, no obvious morphological phenotypes, including during photoreceptor development (such as clear alterations in normal ommatidium development) or wing development, were found either in embryos, in larvae, or in adult flies that lack fbl6 expression. Moreover, no detectable phenotypic consequences were found for embryos that were deficient in fbl6 expression and also in expressing loss-of-function mutations of mae or the recently identified miR7 microRNA, both of which are also negative regulators of Yan activity in vivo. This may reflect genetic redundancy with other F-box-containing proteins or perhaps a broader role for Fbl6 as a regulator of other proteins that might counteract the function of Yan and therefore prevent any obvious mutant phenotypes resulting from elevated Yan protein levels. Indeed, in tissue culture cells, biochemical interactions were found between Fbl6 and known antagonists of Yan function, including the SAM domain-containing proteins Pointed P2 ) (but not P1, which lacks the SAM domain) and Mae. Also, the nature of Tel/Yan degradation may be tissue specific and mediated by distinct or overlapping protein-degrading processes. For example, it has been suggested previously that the PEST sequences of Yan may be determinants of Yan stability; these sequences may attract, or act independently of, the F-box-dependent ubiquitination system. In addition, altering the concentration of ubiquitination effectors can have a substantial impact on the function of the targeted proteins, which may influence whether or not a morphological phenotype is discernible. For example, different levels of Mdm2 have profoundly different impacts on p53 function; low levels promote p53 monoubiquitination and subsequent nuclear export, whereas high Mdm2 levels lead to polyubiquitination and proteasomal degradation (Roukens, 2008b).

Normal cell development and differentiation rely upon an orchestrated program of actions that must be controlled precisely, both spatially and temporally. This study has describe an evolutionarily conserved pathway of Tel/Yan downregulation. This complements recent work on Tel sumoylation and previous studies that delineated the function of these unique repressors. Future work aims to uncover further regulatory features of these crucial cellular mechanisms and to provide a unified framework for how these different processes impinge on Tel/Yan and collaboratively elicit an appropriate cellular response (Roukens, 2008b).

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