Promoter Structure

The expression of Djun is controlled by multiple cis-acting elements in its promoter region and the 5' noncoding region of the transcription unit. A 43-bp 5' upstream promoter region is necessary for the transcription activity of Djun. Deletion of this fragment decreases transcriptional activity by 67-fold. This region includes a TATA box and a sequence similar to the Drosophila transcription factor 1 (DTF-1) consensus sequence (GCAACAT/GC/C). A large DNase I footprint covering both the DTF-1 binding site and the TATA box is detected in this region when incubated with nuclear extract from Drosophila embryos, suggesting interactions with related transcription factors. This 43-bp sequence alone, containing the DTF-1 binding site and TATA box, however, is not sufficient for transcription activity. An 80-bp sequence including the start of transcription has considerable basal activity. An intragenic region containing an AP-1 site and a CRE site modulates or fine tunes activity of the promoter. Its activity as an enhancer is reduced when moved upstream in either orientation. An extragenic region containing two AP-1 sites similarly affects promoter activity (Wang, 1994).

Targets of Activity

R7 photoreceptor fate in the Drosophila eye is induced by two events: the activation of the Sevenless receptor tyrosine kinase and the RAS/MAP kinase signal transduction pathway, induced by the transmembrane ligand Boss. 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. It is antagonized by YAN, another ETS domain protein and 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. These data suggest that JUN and Pointed act on common target genes to promote neuronal differentiation in the Drosophila eye, and that phyllopod might be such a common target (Treier, 1995).

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. Overexpression of a truncated type IV collagen has been shown to interfere with dorsal closure (Borchiellini, 1996). Interestingly, in vertebrates, transforming growth factor-beta and c-Jun regulate collagenase gene expression during wound healing, a process that also involves the closing of an epithelial sheath. By analogy, DJun activity may control a Drosophila collagenase that dissolves the extracellular matrix associated with the dorsal most cells, thereby permitting the cells to stretch laterally (Riesgo-Escovar, 1997a).

The expression of most members of the VH-1 family of PTPs is subject to tight transcriptional regulation. The same is likely to be true for puckered because it displays dynamic patterns of expression in the embryo and the adult. During and after germ band shortening, puc is expressed in the dorsal-most epidermal cells that play a leading role in the process of dorsal closure. In embryos mutant for the JNKK encoded by hemipterous or for the JNK encoded by basket, there is no puc expression in these cells, and dorsal closure fails in a manner similar to that produced by the overexpression of puc (Glise, 1995 and Riesgo-Escovar, 1996). These results suggest a model in which signaling through Hep and Bsk leads to the expression of effectors of dorsal closure and a regulator encoded by puc. The function of the latter is to exert a negative feedback on the signaling cascade of hep and bsk. Interestingly, in mutants for Djun (a likely target of JNK activity), puc expression is absent at the leading edge of the epidermis (N. Perrimon, pers. comm. to Martin-Blanco, 1998), suggesting a transcriptional link between the activity of the JNK encoded by bsk and the expression of puc. Thus, the activation of MAPKs is controlled by the balance between MAPK kinase and MAPK phosphatase activities during dorsal closure. In this system, Puckered seems to act in a feedback loop. Puckered expression is upregulated by DJun and in turn, Puckered inactivates MAPK, whose function is the activation of DJun downstream of Rac signaling (Martin-Blanco, 1998).

IkappaB kinase (IKK) and Jun N-terminal kinase (Jnk) signaling modules are important in the synthesis of immune effector molecules during innate immune responses against lipopolysaccharide and peptidoglycan. However, the regulatory mechanisms required for specificity and termination of these immune responses are unclear. Crosstalk occurs between the Drosophila Jnk and IKK pathways; this leads to downregulation of each other's activity. The inhibitory action of Jnk is mediated by binding of Drosophila activator protein 1 (AP1) to promoters activated by the transcription factor NF-kappaB. This binding leads to recruitment of the histone deacetylase dHDAC1 to the promoter of the gene encoding the antibacterial protein Attacin-A and to local modification of histone acetylation content. Thus, AP1 acts as a repressor by recruiting the deacetylase complex to terminate activation of a group of NF-kappaB target genes (Kim, 2005).

Raw mediates antagonism of AP-1 activity in Drosophila

High baselines of transcription factor activities represent fundamental obstacles to regulated signaling. This study shows that in Drosophila, quenching of basal activator protein 1 (AP-1) transcription factor activity serves as a prerequisite to its tight spatial and temporal control by the JNK (Jun N-terminal kinase) signaling cascade. These studies indicate that the novel raw gene product is required to limit AP-1 activity to leading edge epidermal cells during embryonic dorsal closure. In addition, evidence is provided that the epidermis has a Basket JNK-independent capacity to activate AP-1 targets and that raw function is required broadly throughout the epidermis to antagonize this activity. Finally, mechanistic studies of the three dorsal-open group genes [raw, ribbon (rib), and puckered (puc)] indicate that these gene products provide at least two tiers of JNK/AP-1 regulation. In addition to Puckered phosphatase function in leading edge epidermal cells as a negative-feedback regulator of JNK signaling, the three dorsal-open group gene products (Raw, Ribbon, and Puckered) are required more broadly in the dorsolateral epidermis to quench a basal, signaling-independent activity of the AP-1 transcription factor (Bates, 2008).

The initial molecular and genetic studies of the dorsal-open mutant raw revealed it to encode a widely expressed and novel gene product, required for the restriction of JNK/AP-1 activity to LE epidermal cells (Byars, 1999). The Raw protein sequence yielded no insights into its mechanism of function as the Raw sequence harbors none of the canonical motifs that are associated with nuclear localization, phosphorylation, membrane insertion, or protein secretion. Mechanistic studies of a novel protein can be challenging, but this study reports use of a variety of genetic strategies to probe Raw function and test models of AP-1 silencing. In particular (1) the epistatic relationship of raw to genes encoding well-characterized JNK-signaling components was assessed, (2) genes, which have designated the raw group, have been assessed that share an array of loss-of-function phenotypes, (3) the interaction phenotypes among the raw-group loci were determined, and (4) raw transgenics were generated, that were utilized to probe sites of Raw function. These analyses reveal that raw belongs to a small set of dorsal-open group genes that encode JNK/AP-1 pathway antagonists. The characterization of raw, and the raw group more generally, has led to a new appreciation of wide-ranging competence for AP-1 activity in early Drosophila embryos. As signal activation is critical for proper development, so also is its silencing (Bates, 2008).

The current study shows that although raw functions upstream of Jra as an AP-1 antagonist, its action is independent of the bsk-encoded kinase that is required to activate AP-1 activity in LE cells during closure. In addition, raw is required broadly in the epidermis to effect normal dorsal closure. Overall, these studies expose the importance of epidermal AP-1 silencing during embryogenesis and lead to an extension of existing models for dorsal closure, which have largely confined their focus to mechanisms of JNK/AP-1 activation in LE cells. In particular, the data indicate that Raw and the other raw-group gene products (Puckered and Ribbon) function to silence Basket JNK-independent AP-1 activity in the embryonic dorsolateral epidermis. AP-1 silencing, via the combined actions of the raw-group gene products, essentially wipes the epidermal slate clean and primes the system for activation via a still unidentified deterministic signal that acts only in LE cells (Bates, 2008).

The AP-1 abnormality in raw-group mutant embryos has not yet been molecularly defined. Previous studies provide compelling evidence that AP-1 overexpression in Drosophila embryos is not sufficient to disrupt either dorsal closure or development more generally. It seems unlikely, therefore, that elevated levels of the AP-1 transcription factor in raw-group mutants simply override a requirement for kinase activation in initiating an AP-1-dependent program of gene expression. Instead, it is speculated that AP-1 is aberrantly modified in raw-group mutant embryos. It might be that AP-1 escapes inactivation in mutants; either alternatively or additionally, AP-1 in mutants may be inappropriately activated via phosphorylation. In addition to Basket JNK, there are four other Drosophila MAP Kinases (p38a, p38b, Mpk2, and Rolled) that might provide dysregulated kinase activity in mutants. Consistent with this idea is the observation that the oogenesis phenotypes associated with raw (and puc) ectopic expression and mutation have considerable similarity with gain- and loss-of-function phenotypes associated with mutations in the p38 pathway that is required in the germ line for proper oogenesis. Finally, a kinase-dependent activation model for epidermal Jun provides the most parsimonious explanation for ectopic epidermal signaling observed in puc MPK-deficient embryos. From the perspective of regulated signaling more generally, however, lowering an AP-1 activity baseline in wild-type embryos will (1) provide a means for the clean on/off regulation of JNK/AP-1 that has been predicted in computer simulations and (2) make a less strenuous demand on the input activating signal (Bates, 2008).

The discovery that null alleles of raw and puc interact, with double mutants exhibiting an embryonic lethal phenotype distinct from their shared loss-of-function null phenotypes, revealed the independent contributions of raw and puc to embryogenesis, presumably through their effects on AP-1 antagonism. Drosophila overexpression studies have previously implicated several pathways in the parallel control of AP-1 activity, but this analysis represents the first direct demonstration of physiologically relevant, parallel regulatory pathways (Bates, 2008).

The genetic interaction that was documented between null alleles of raw and puc contrasts with the lack of a detectable interaction between null alleles of raw and rib. Moreover, the observation that raw and rib hypomorphs interact genetically during dorsal closure is consistent with previously published data, as well as with findings documenting (1) raw/rib interactions in several other epithelial tissues, including the nervous system, salivary gland, trachea, and gut (Blake, 1998; Blake, 1999) and (2) overlapping raw and rib expression patterns in Drosophila embryos (Byars, 1999). Together, results from these genetic and molecular studies point to roles for raw and rib in a single, previously unrecognized puc-independent AP-1 inactivation system (Bates, 2008).

In addition to providing evidence for raw-mediated global silencing of AP-1, this study underscores a simultaneous requirement for a biologically appropriate activator of JNK/AP-1 signaling. In this regard, expression of raw in LE cells failed to rescue raw-dependent defects in dorsal closure. Even more notable, however, was the observation that overexpression of raw+ in wild-type embryos, and in wild-type LE cells in particular, had no detrimental effects on embryonic development and dorsal closure. From a signaling perspective this result indicates that JNK-dependent AP-1 can be activated despite expression of the wild-type raw gene product, and thus Raw does not function as a binary switch for signaling. Although it is formally possible that LE expression of raw was initiated too late to disrupt JNK/AP-1 signaling and dorsal closure in the LE-gal4/UAS-raw+ transgenics, this interpretation is not favored since the LE-GAL4 driver used in this study has been shown previously to (1) be an effective driver of at least one gene that is required in LE epidermal cells for closure and (2) drive expression of a lacZ reporter in LE cells during dorsal closure (Bates, 2008).

The finding that raw expression in LE cells is not sufficient to inactivate AP-1 activity in a cell-autonomous fashion is consistent with models for independent, developmentally regulated triggers of JNK signaling. Indeed, there is abundant experimental support for developmentally regulated activation of JNK signaling in LE cells. JNK/AP-1 activation likely follows an amalgamation of signals, both from the amnioserosa and the epidermis, both in the form of cytoskeletal components and signaling molecules. Among the best candidates with postulated roles in JNK/AP-1 activation are small GTPases, nonreceptor tyrosine kinases, and integrins. Thus, despite the broad epidermal competence for AP-1 signaling that has been shown in this work, the activation signal is itself limited to only LE cells and functions via an unknown mechanism. Importantly, AP-1 antagonism by raw cannot override its signal-dependent activation in the LE (Bates, 2008).

dpp, when expressed pan-epidermally, leads to a raw-like phenotype: embryonic lethality associated with ventral cuticular defects. In a direct assessment of equivalence of raw loss-of-function and dpp gain-of-function ventral cuticular phenotypes, whether pan-epidermal expression of brinker (brk) can rescue raw-dependent defects in the ventral cuticle was tested. The Dpp signaling modifier Brinker functions by negatively regulating dpp target genes (Bates, 2008).

This study found that although brk is normally expressed in nonoverlapping lateral and ventral domains of the embryonic epidermis, it is undetectable in the epidermis of embryos homozygous for a null allele of raw. It was also found that although brk+ fails to rescue raw-dependent defects in dorsal closure, it does rescue raw-dependent defects in the ventral cuticle. Together, these data point to an important role for dpp, brk, and/or their target genes in development of the ventral epidermis (Bates, 2008).

What cannot be discerned from these studies is (1) how the nonoverlapping epidermal domains of dpp and brk are established and maintained and (2) if and how epidermal dpp and brk interact during normal embryonic development. In this regard, a previous finding that LE dpp is not autoregulatory makes it unlikely that brk functions in direct fashion to set the LE dpp expression boundary. Even more significant is the finding that cuticles derived from Jra raw double mutants exhibit defects in dorsal closure, but not ventral cuticular patterning (BYARS, 1999). Indeed, these data highlight the requirement for functional Jun in generating ventral cuticular defects in raw mutant embryos. Taken together then, these data suggest that the effects of JNK/AP-1-activated dpp in the dorsal epidermis of raw mutant embryos are far reaching, extending even to the most ventral regions of the embryo (Bates, 2008).

Having established a dependence upon Jun for raw-dependent ventral cuticular defects, it is postulated that the absence of brk in raw mutant embryos is a direct consequence of ectopic JNK/AP-1 activity in the dorsal epidermis of these mutants. It is suspected that ectopic JNK/AP-1 activity leads secondarily to ectopic dpp activity, and that in its turn ectopic dpp activity leads finally to brk repression. An alternative view, that raw might have dual regulatory roles in the epidermis, seems less likely although it is not absolutely excluded by this strictly genetic analysis. In this regard, in addition to its function as a JNK/AP-1 antagonist in the embryonic dorsal epidermis, raw might function independently as a trigger of brk expression in the ventral epidermis. Clearly, the mechanism of raw function and the relationship of dpp to brk in eliciting properly formed ventral cuticle warrant further investigation (Bates, 2008).

In Drosophila, as in all animals, signaling pathways are finely regulated at several levels. Although there are multiple tiers of regulation operating on the JNK/AP-1 signaling cascade, surprisingly little of the regulation of this pathway is known. This study of the functions and interactions of a subset of dorsal-open group genes (raw, rib, and puc) has shed some additional light on both old (puc-mediated) and new (raw/rib-mediated) mechanisms of JNK/AP-1 antagonism. These data indicate that Raw functions to silence Basket JNK-independent AP-1-mediated transcription and to set the stage for JNK-dependent regulation of transcription. The suggestion that spatial restriction of the JNK/AP-1 signal requires antagonists, as well as activators, is not without precedent in other signaling systems. Many signaling pathways have already been shown to be multilayered and to depend heavily on negative regulation to terminate developmental events, and/or control both the distance and speed that a signal can move (e.g., Nodal). In addition, and as was suggested is the case for the Drosophila JNK/AP-1 pathway, reducing basal levels of a signaling pathway can augment the effects of its signaling responses (e.g., Hedgehog and Lef1) (Bates, 2008).

Finally, given the numerous associations of improper JNK/AP-1 activity with human disease, it seems apparent that many cell types have the capacity to signal via the JNK/AP-1 pathway. Presumably, this capacity is diminished (and then tightly regulated) during normal vertebrate development and aging. Viewed from this perspective, characterization of Raw as an essential AP-1 antagonist establishes a clear basis for future studies of AP-1 regulation (Bates, 2008).

Transcriptional regulation of Profilin during wound closure in Drosophila larvae

Injury is an inevitable part of life, making wound healing essential for survival. In postembryonic skin, wound closure requires that epidermal cells recognize the presence of a gap and change their behavior to migrate across it. In Drosophila larvae, wound closure requires two signaling pathways [the Jun N-terminal kinase (JNK) pathway and the Pvr receptor tyrosine kinase signaling pathway] and regulation of the actin cytoskeleton. In this and other systems, it remains unclear how the signaling pathways that initiate wound closure connect to the actin regulators that help execute wound-induced cell migrations. This study shows that chickadee, which encodes the Drosophila Profilin, a protein important for actin filament recycling and cell migration during development, is required for the physiological process of larval epidermal wound closure. After injury, chickadee is transcriptionally upregulated in cells proximal to the wound. JNK, but not Pvr, mediates the increase in chic transcription through the Jun and Fos transcription factors. Finally, it was shown that chic-deficient larvae fail to form a robust actin cable along the wound edge and also fail to form normal filopodial and lamellipodial extensions into the wound gap. These results thus connect a factor that regulates actin monomer recycling to the JNK signaling pathway during wound closure. They also reveal a physiological function for an important developmental regulator of actin and begin to tease out the logic of how the wound repair response is organized (Brock, 2012).

The traditional model of the actin cytoskeleton in cell migration, based on in vitro cell culture and biochemical assays, provides a useful framework for the mechanics of how cell migration is regulated. However, there is need for in vivo studies in order to answer important questions that are not addressed by the current model: 1. Is there a role for Profilin-mediated recycling during wound-induced migration of differentiated cells in vivo? 2. Is there a role for transcriptional regulation of actin regulators during such migrations? This latter question emerges because the basic model generally assumes that migratory cells have an intact actin-regulatory apparatus that needs only to be activated to initiate and sustain migration. While this assumption may be correct for migrating cells in developmental contexts one could imagine that initially non-migratory differentiated cells may need more than their resting complement of actin regulators in order to effect long-distance migration (Brock, 2012).

Unwounded larval epidermal cells have an even distribution of actin and Profilin throughout the cytoplasm and are thought to be non-migratory. These fully differentiated epithelial cells secrete an apical cuticle and a basal lamina. They respond to the physiological signal of tissue damage by partially dedifferentiating and becoming migratory. This study shows that the leading edge cells form multiple actin-based structures including a discontinuous cable, filopodia, and lamellipodia. A working model is proposed where the basal levels of Profilin are sufficient to make actin-based structures, but wound-induced transcription of chic is required for the cells to efficiently migrate. The lack of actin-based structures at the wound edge in cells lacking Profilin would indicate that if Formin-mediated actin nucleation is involved in wound healing, it likely requires Profilin. An epidermal sheet lacking detectable Profilin fails to close wounds or form any actin-based structures at the wound edge whereas a sheet containing only a basal level of Profilin (i.e., one that is lacking proteins that transcriptionally regulate Profilin after wounding, such as JNK, Fos, or Jun) forms actin structures at the wound edge, but is ultimately unable to efficiently migrate and close the wound. This model is complicated by the fact that cells lacking JNK, Fos, or Jun also have defects in dedifferentiation, as these cells do not stop secreting cuticle following wounding. Thus, the possibility cannot completely excluded that the lack of wound closure is due to defects in dedifferentiation. However, it is entirely possible that upregulation of actin-binding regulators is an important component of the dedifferentiation process, as this involves returning to a state during which these cells were competent to migrate (Brock, 2012).

Current wound closure models have identified two signaling pathways that are important for healing. One is Pvr signaling, where the secreted VEGF-like ligand Pvf1 activates the Pvr receptor. Currently, only a few proteins are suspected of being downstream of Pvr signaling, but Profilin is not among them. Given that epidermal cells lacking Pvr are unable to mobilize actin to the wound edge, Pvr is likely upstream of actin regulatory proteins that initiate actin polymerization at the leading edge of migrating cells. The second pathway is JNK signaling, which is required for closure but not for actin polymerization at the wound edge. Naively, it was anticipated that wound-induced chic expression would be regulated by Pvr since epidermal expression of UAS-chicRNAi also blocks actin accumulation at the wound edge. Surprisingly, this is not the case. chiclacZ expression is instead regulated by JNK signaling, as it is in the developing embryo during DC . This data reveals that although the JNK signaling pathway is not required for actin nucleation at the wound edge it contributes to actin dynamics through regulating expression of chic and perhaps other genes important for migration (Brock, 2012).

How does JNK signaling activate chic transcription after wounding? Although the upstream signal for the JNK signaling pathway is still unknown, the kinase cascade is well-defined and is thought to culminate with the phosphorylation of the transcription factors, DJun and DFos. These two proteins are commonly thought to act as a dimer (AP-1) to mediate transcriptional activation of target genes. In the early DC studies chickadee expression was shown to depend on the JNK signaling pathway. This study did not address the roles of DJun and DFos in particular, although these transcription factors are required for DC. In wound healing contexts, however, it appears that DFos can act without DJun to activate a ddc-wound reporter and a msn-lacZ wound reporter. This study found that both DJun and DFos are required to activate chic. Additionally, two consensus binding sequences for the AP-1 transcription factor (TGANTCA) are located upstream of the chic start codon (depending on the message isoform the sites are located in the 5’UTR, the first intron, or the promoter region), indicating that it is at least possible that the upregulation of chic transcription is directly accomplished by Jun and Fos. The consensus sequence is also located upstream of the human Pfn1, indicating that there is potential for this regulation to be conserved. This suggests that in the migrating cells at the wound edge, DFos can act either as a homodimer, with unidentified binding partners, or with DJun to regulate the necessary transcriptional targets (Brock, 2012).

In Drosophila embryonic models of wound closure both the contractile actin cable and filopodial processes are important for wound closure, but their relative contributions are still unclear. There has been debate over whether the cable mediates closure through contraction, through serving as a platform for extension of processes into the wound gap, or through a combinaton of these functions. From the data shown in this study it seems that actin-based contraction is not a major contributor to larval wound closure. First, the actin concentrations that appear at larval wound edges are discontinuous. Second they do not appear to be locally contractile given that the cells behind prominent concentrations do not obviously taper toward the wound. This is similar to what has been observed in the embryonic Xenopus epithelium where actin cables form but differently shaped wounds do not round up as would be expected from cable contraction. Thus it would appear that in larvae the actin concentrated at the wound edge primarily facilitates process extension into the wound gap (Brock, 2012).

This study has establish a connection between a known wound-induced signaling pathway, JNK signaling, and Profilin-mediated regulation of the actin cytoskeleton. It is speculated that transcriptional induction of actin-regulators may be a general feature of cell migration in differentiated cells as suggested by a recent study of cells undergoing EMT. By connecting upstream signaling pathways to downstream actin dynamics, this work begins to unravel the logic of how the cellular movements required for wound closure are orchestrated (Brock, 2012).

Protein Interactions

Phosphorylation of DJUN by the MAP kinase Rolled regulates photoreceptor differentiation. DJUN can, in fact, sequester Rolled protein from a crude extract, indicating a specific interaction. D-JUN is phosphorylated on three conserved MAPK sites. A DJun mutant that carries alanines in place of the Rolled phosphorylation sites acts as a dominant suppressor of photoreceptor cell fate if expressed in the eye imaginal disc. In contrast, a mutant in which phosphorylation sites are replaced by phosphate-mimetic Asp residues can promote photoreceptor differentiation (Peverali, 1996).

The protein kinase activities of p38a (Mpk2) and p38b were examined by using an in vitro assay. The two Drosophila p38 MAPKs were expressed as GST fusion proteins in bacteria and purified. In vitro kinase assays demonstrate that both p38a and p38b can phosphorylate Drosophila Jun and mammalian ATF2. The Drosophila p38 isoforms can also phosphorylate myelin basic protein, which is phosphorylated more effectively by PKA. These results reveal that while Drosophila p38s can phosphorylate ATF2, Drosophila Jun also serves as a substrate but to a lesser extent. In contrast, Drosophila Basket (JNK) recognizes both substrates with the same efficiency (Z. Han, 1998).

Drosophila AP-1 consists of two proteins (DFOS and DJUN) that have functional and structural properties in common with mammalian Fos and Jun proto-oncogene products. The predicted amino acid sequences of DFOS and DJUN proteins reveal that both proteins contain a bipartite domain consisting of a leucine repeat and an adjacent DNA-binding basic region, both of which are characteristic of members of the AP-1 family. DFOS, in contrast to the mammalian cFOS proteins, recognizes the AP-1 site on its own and activates transcription in vitro in the absence of DJUN or JUN. Heteromeric complexes formed between DFOS and DJUN bind the AP-1 site better than either protein alone. The two proteins activate transcription synergistically in vitro (Perkins, 1990).

DJUN, in cooperation with mouse c-FOS, can trans-activate an activator protein 1(AP-1) DNA binding site when introduced into mammalian cells. These data suggest that DJUN, much like its mammalian homolog, may activate transcription of genes involved in regulation of cell growth, differentiation, and development. Furthermore, the identification of DJUN allows one to exploit the genetics of Drosophila to identify genes in signal transduction pathways involving DJUN and thus c-jun (Zhang, 1990).

The Drosophila fat facets (faf) gene encodes a deubiquitination enzyme with a putative function in proteasomal protein degradation. Mutants lacking zygotic faf function develop to adulthood, but have rough eyes caused by the presence of one to two ectopic outer photoreceptors per ommatidium. faf interacts genetically with the receptor tyrosine kinase (RTK)/Ras pathway, which induces photoreceptor differentiation in the developing eye. faf also interacts with pointed: the extra-photoreceptor phenotype observed in faf mutants is clearly suppressed by pointed mutation; many more ommatidia have six outer photoreceptors in a trapezoidal arrangement characteristic of wildtype ommatidia. yan mutation in combination with faf strongly enhances the faf phenotype. Reducing the D-Jun activity suppresses the faf mutant phenotype. In sevenless;faf double mutants, R7 cells, normally absent in sevenless mutants, form in 60% of the ommatidia. Thus, faf can alleviate the requirement for sev in the R7 precursor. These results indicate that RTK/Ras signaling is increased in faf mutants, causing normally non-neuronal cells to adopt photoreceptor fate. Consistently, the protein level of at least one component of the Ras signal transduction pathway, the transcription factor D-Jun, is elevated in faf mutant eye discs when the ectopic photoreceptors are induced. It is proposed that defective ubiquitin-dependent proteolysis leads to increased and prolonged D-Jun expression, which together with other factors contributes to the induction of ectopic photoreceptors in faf mutants. These studies demonstrate the relevance of ubiquitin-dependent protein degradation in the regulation of RTK/Ras signal transduction in an intact organism (Isaksson, 1997).

The transcriptional activation potential of proteins can be assayed in chimeras containing a heterologous DNA-binding domain that mediates their recruitment to reporter genes. This approach has been widely used in yeast and in transient mammalian cell assays. This approach was applied to assay the transactivation potential of proteins in transgenic Drosophila embryos. A chimera between the DNA-binding bacterial LexA protein and the transactivation domain from yeast GAL4 behaves as a potent synthetic activator in all embryonic tissues. In contrast, a LexA chimera containing Drosophila Fos (Dfos) requires an unexpected degree of context to function as a transcriptional activator. Evidence to suggest that this context is provided by Djun and Mad (a Drosophila Smad), and that these partner factors need to be activated by signaling from Jun N-terminal kinase and decapentaplegic, respectively. Because Dfos behaves as an autonomous transcriptional activator in more artificial assays systems, these data suggest that context-dependence of transcription factors may be more prevalent than previously thought (Szuts, 2000).

Which factors provide the context for Dfos function? Several lines of evidence implicate JNK and Dpp signaling and their transcriptional target factors Djun and Mad as the essential context. (1) The only embryonic cells in which LexFos functions reliably and robustly to stimulate transcription are the dorsal leading edge cells which experience both of these signals. (2) Neither of the LexFos derivatives (LexFosN, LexFosC) function in these cells, strongly implicating the basic leucine zipper domain of LexFos (the only domain absent from both derivatives) in its function. As this domain mediates dimerization with Djun, the only known dimerization partner of Dfos in Drosophila, this indicates that the activity of LexFos depends on Djun. Recall that Djun is present and activated by JNK signaling in the leading edge cells. (3) JNK signaling as mimicked by overexpression of constitutively acitive Drac* or Dcdc42*, potently synergizes with LexFos to mediate widespread transactivation in the embryo. A very similar widespread synergy has also been seen between LexFos and Jun*, a mutant form of c-Jun that mimics signal-activation of this protein. The embryonic territories in which these synergies are observed appear to correspond to sites of Dpp stimulation. Consistent with this, a limited synergy between Dpp and LexFos has also been observed in some embryonic cells. These synergies strongly implicate JNK and Dpp as necessary context signals for LexFos function. (4) LexFos activity strictly depends on the context sequence in the MadL target reporter; under no conditions does it transactivate a reporter that contains four tandem LexA binding sites (albeit LexGAD very efficiently does so). The context sequence in MadL essentially consists of a binding site for the Dpp response factor Mad, which is thus a likely partner for the putative LexFos/Djun* dimer (Szuts, 2000).

These results indicate that JNK-activated Djun and Dpp-activated Mad may be critical and widespread context partners of Dfos. Consistent with this, Dfos function is required for dorsal closure of the embryo and, by implication, functions normally in cells that experience JNK and Dpp signaling. In the embryonic midgut, Dfos functions in cells that experience Dpp and Egfr signaling. Because the LexFos/JNK synergy in the mesoderm implies that JNK signaling is normally absent from this tissue, this suggests that the normal partner of Dfos in the midgut visceral mesoderm may be a factor, as yet unidentified, that is activated by Egfr signaling. Interestingly, synergy between the c-Jun/c-Fos dimer and TGF-beta activated Smad has also been observed in mammalian cells. Furthermore, Jun proteins have recently been shown to bind directly to Smad3/4. Thus, the partnership between signal-activated Jun/Fos dimers and Smads may be fairly widespread and fundamental (Szuts, 2000).

Transrepression of AP-1 by nuclear receptors

Mammalian cell culture studies have shown that several members of the nuclear receptor super family such as glucocorticoid receptor, retinoic acid receptor and thyroid hormone receptor can repress the activity of AP-1 proteins (referring to Drosophila Kayak and Jun) by a mechanism that does not require the nuclear receptor to bind to DNA directly, but that is otherwise poorly understood. Several aspects of nuclear receptor function are believed to rely on this inhibitory mechanism, which is referred to as transrepression. This study presents evidence that nuclear receptor-mediated transrepression of AP-1 occurs in Drosophila melanogaster. In two different developmental situations, embryonic dorsal closure and wing development, several nuclear receptors, including Seven up, Tailless, and Eagle antagonize AP-1. The inhibitory interactions with nuclear receptors are integrated with other modes of AP-1 regulation, such as mitogen-activated protein kinase signaling. A potential role of nuclear receptors in setting a threshold of AP-1 activity required for the manifestation of a cellular response is discussed (Gritzan, 2002).

The best understood AP-1-dependent process in Drosophila development is a coordinated cell sheet movement known as dorsal closure. During DC, lateral epidermal cells migrate dorsally and close the epidermis on the dorsal side of the embryo. Failure to undergo DC results in a characteristic dorsal open phenotype, the cuticle of affected embryos displays a dorsal hole. Mutations in genes encoding the Drosophila homologs of JNKK, (JNK, Jun and Fos) all give rise to similar dorsal open phenotypes. Thus, it is thought that DC requires activation of Jun/Fos heterodimers by a JNK-type MAPK cascade. Embryos homozygous for kay1, a fos null allele are devoid of zygotic Fos activity and DC fails. A large dorsal hole forms and the cuticle collapses. In an embryo homozygous kay2, a hypomorphic fos-allele, AP-1 activity is reduced but not eliminated. Correspondingly, the DC phenotype is weaker. The embryo displays a small dorso-anterior hole (Gritzan, 2002).

To test whether Drosophila NRs can antagonize AP-1, a variety of AP-1 constructs were in the embryonic epidermis. Interestingly, expression of some, but not all, NRs tested result in DC phenotypes of different strengths. Expression of Svp in the dorsal epidermis under the control of pnrGal4 results in a DC phenotype reminiscent of that of kay2 homozygotes. This finding is consistent with a suppression of AP-1 activity by Svp. Similarly, expression of Tll under the control of a heat shock promoter causes a weak dorsal open phenotype. The differentiation of ventral cells does not seem to be disturbed by Tll expression since the pattern of denticles in this part of the epidermis appears grossly normal. Thus, Tll expression specifically affects the dorsal epidermis where AP-1 activity is required. The expression of Knrl under the control of pnrGal4 elicits stronger DC phenotypes with the dorsal hole frequently extending over several segments (Gritzan, 2002).

If the DC defects caused by NR expression reflect a negative effect on AP-1, the defects should be sensitive to changes in Fos or Jun activity. In genetic interaction experiments, the dorsal open phenotypes caused by NR expression were compared in a wild-type background and in embryos with altered levels of AP-1 activity. Embryos heterozygous for kay1 carry only one copy of the fos gene. While these embryos are phenotypically normal, their levels of AP-1 are reduced and they might therefore be more susceptible to a further decrease of this activity. If expression of NRs causes DC defects by antagonizing AP-1, it should have stronger phenotypic consequences in embryos heterozygous for kay1 than in wild type embryos. Indeed, while expression of Eagle (Eg) in a wild type background mostly results in DC phenotypes of intermediate strength, NR expression in embryos heterozygous for kay1 typically elicits complete failure of DC, indicative of a severe reduction of AP-1 activity. Since embryos of both genotypes display somewhat variable phenotypes, the effect of kay1 heterozygosity is best appreciated by quantitative analysis. A clear reduction in size, a collapsed folded cuticle and a dorsal hole extending over at least half of the body length are described as characteristics of a strong DC phenotype. Embryos with a smaller dorso-anterior hole and normal body size were scored as showing weak DC phenotypes. This analysis confirms that Eg expression has more severe phenotypic consequences in kay1 heterozygotes than in wild type embryos and supports the suggestion that NRs cause defective DC by suppressing AP-1 activity (Gritzan, 2002).

In a complementary experiment, the effect of Eg expression was examined in embryos with increased AP-1 activity. In embryos heterozygous for pucE69, the levels of the CL100 phosphatase Puckered (Puc) which specifically inactivates JNK are reduced. Thus, in contrast to kay1 heterozygotes, embryos of this genotype have elevated levels of JNK, and consequently AP-1, activity. If the phenotypic outcome of NR expression in the embryo is mediated by transrepression of AP-1, the DC defects should be weaker in puc heterozygotes than in a wild type background. Quantitative analysis reveals that the frequency of strong DC phenotypes is indeed greatly reduced in pucE69 heterozygotes expressing Eg compared to Eg expression in a wild type background. Expression of the NR Knrl in the various backgrounds yields essentially identical results. These data support the hypothesis that several Drosophila NRs can antagonize AP-1 as has been shown for mammalian NRs (Gritzan, 2002).

Based on the results of the DC assays, it cannot be determined whether the antagonism between AP-1 and NRs is caused by the downregulation of direct AP-1 target genes by NRs or whether the effect is more indirect. To address this issue, the effect of Drosophila NRs on bona fide AP-1 target genes was monitored. However, direct AP-1 target genes have not yet been clearly defined in Drosophila. While it is known that dpp and puc expression in DC requires AP-1, it cannot be excluded that this effect is indirect. To circumvent this problem, a mammalian cell culture system was used. Transrepression of AP-1 by mammalian NRs was first described in the context of collagenase transcription. Extensive studies of the collagenase promoter have identified AP-1 as one of its primary regulators. Activation of the GR down-regulates AP-1-mediated transcription of collagenase. It was asked whether Drosophila NRs behave similarly in this well-defined assay. Transcriptional activation by AP-1 was measured using a reporter construct in which transcription of the firefly luciferase gene is controlled by the upstream region of the human collagenase gene. Comparing luciferase activity in the presence and absence of Drosophila NRs, it was found that both Eg and Tll efficiently antagonized AP-1 activity in a dose-dependent manner. The observed effects are quantitatively comparable to those reported for the GR. Taken together, these data strongly suggest that Drosophila NRs are competent for AP-1 transrepression. Furthermore, the cell culture assay demonstrates that Drosophila NRs can antagonize mammalian AP-1 and implies that the mechanism of transrepression is conserved between Drosophila and mammals (Gritzan, 2002).

Does modulation of AP-1 activity by NRs occur only in situations where AP-1 is regulated by JNK or does this type of regulation also operate in different contexts? A function for Fos downstream of ERK has been demonstrated in the differentiation of wing veins. Extra vein material can result from elevated levels of ERK, as in flies carrying a gain-of-function allele of the rolled gene, which encodes Drosophila ERK. This allele, called rolledSevenmaker (rlSem), encodes a form of ERK with increased resistance to inactivation by dephosphorylation. Expression of a dominant-negative form of Fos in the wings of rlSem flies results in loss of ectopic vein material. Conversely, overexpression of Fos enhances the extra-vein phenotype caused by rlSem (Gritzan, 2002).

32B Gal4, UAS Sem flies express the RlSem form of ERK in the wing from a UAS-driven transgene. As a consequence of elevated levels of ERK activity, these animals develop ectopic wing vein material. Reducing fos gene dosage in this system strongly suppresses the vein phenotype, consistent with the proposed role of Fos as an ERK effector. Thus, 32B Gal4 UAS Sem flies provide a suitable system to examine how genetic manipulations of AP-1 activity affect vein differentiation. To investigate a potential role of the Drosophila NRs in this process, one copy of kni, eg, tll or svp was removed in 32B Gal4, UAS Sem flies. Reducing kni function does not influence the vein phenotype. However, heterozygosity for any of the other three receptors tested reproducibly leads to a mild enhancement of the ectopic vein differentiation. As an unambiguously scoreable criterion to statistically evaluate phenotypic effects, the presence of ectopic vein material posterior to L5 was chosen. This area of the wing is relatively resistant to the formation of extra vein material. Quantitative analysis clearly shows that whereas the formation of extra vein material posterior to L5 in 32B Gal4 UAS Sem flies is suppressed by reducing fos activity, it is enhanced by a reduction of eg, svp or tll function. These data suggest that all three NRs antagonize AP-1 activity in wing vein differentiation, conceivably in a redundant manner (Gritzan, 2002).

It is speculated that the modulation of AP-1 activity by NRs contributes to what has recently been termed signal consolidation. Cells have to place a value on incoming signals (e.g. EGF-induced ERK activity) such that they are either answered by a biological response (e.g. the execution of a transcriptional program) or disregarded as noise. It is proposed that the modulation of AP-1 activity by NRs facilitates the interpretation of the EGF signal in wing vein differentiation by defining a threshold of ERK activity. Cells in which ERK activity does not reach this threshold do not mount an AP-1-dependent transcriptional response to the EGF signal. When transrepressional control is impaired (as in the svp, tll double mutant clones) the threshold is lowered and more cells than appropriate interpret EGF-induced ERK activity as a consolidated signal. This leads to the formation of ectopic vein material. This model is supported by the finding that the ectopic vein tissue observed in clones of tll and svp mutant tissue did arise close to the position of the endogenous veins and not randomly throughout the clonal area. Thus, the regulation of AP-1 by NRs appears to convey cell-intrinsic information (Gritzan, 2002).

Chameau HAT and DRpd3 HDAC function as antagonistic cofactors of JNK/AP-1-dependent transcription during Drosophila metamorphosis

Gene regulation by AP-1 transcription factors in response to Jun N-terminal kinase (JNK) signaling controls essential cellular processes during development and in pathological situations. The histone acetyltransferase (HAT) Chameau and the histone deacetylase DRpd3 act as antagonistic cofactors of DJun and DFos to modulate JNK-dependent transcription during pupal thorax metamorphosis and JNK-induced apoptosis in Drosophila. It has been demonstrated, in cultured cells, that DFos phosphorylation mediated by JNK signaling plays a central role in coordinating the dynamics of Chameau and DRpd3 recruitment and function at AP-1-responsive promoters. Activating the pathway stimulates the HAT function of Chameau, promoting histone H4 acetylation and target gene transcription. Conversely, in response to JNK signaling inactivation, DRpd3 is recruited and suppresses histone acetylation and transcription. This study establishes a direct link among JNK signaling, DFos phosphorylation, chromatin modification, and AP-1-dependent transcription and its importance in a developing organism (Miotto, 2006).

Whether Chm directly binds to DFos and/or DJun was investigated using GST pull-down assays. The C-terminal half of Chm (amino acids 494-812), which contains the MYST domain, displays strong in vitro affinity for an N-terminal fragment of DFos (including the N terminus and the basic DNA-binding domain), and binds DJun as well, although less efficiently. Conversely, the cytoplasmic kinase Basket (Bsk)/DJNK does not bind to the His-Chm fusion protein. The Chm N terminus (amino acids 20-400) does not associate with DFos or DJun. Similar experiments with GST-fused DFos deletion mutants identified the basic region of DFos as the predominant Chm-interacting domain, although significant association with the C-terminal part of DFos was also observed. Immunoprecipation assays followed by Western analyses confirmed that these interactions occur in vivo. Both DFos and DJun coprecipitate with Myc-Chm from nuclear extracts of larvae expressing a Myc-tagged version of the protein. DFos is eluted from the immunoprecipitate at higher salt concentrations than DJun; this indicates a more stable association with Chm and that Chm to DFos interaction can occur in the absence of DJun. Confirming the specificity of the assay, nuclear proteins unrelated to JNK signaling, the chromatin-associated protein Modulo (Mod), the homeodomain transcription factors Ultrabithorax (Ubx) and Engrailed (En), as well as the basic helix-loop-helix (bHLH) factor DMyc are not precipitated by Myc-Chm. In reciprocal experiments, an anti-TAP antibody coprecipitates Myc-Chm from nuclear extracts of larvae ubiquitously expressing Myc-Chm and TAP-DFos or TAP-DJun. The results of the in vitro and the in vivo experiments, taken together, show that Chm, DFos and DJun can directly interact and form multimeric protein complexes in larvae (Miotto, 2006).

Interaction between Drosophila bZIP proteins Atf3 and Jun prevents replacement of epithelial cells during metamorphosis

Epithelial sheet spreading and fusion underlie important developmental processes. Well-characterized examples of such epithelial morphogenetic events have been provided by studies in Drosophila, and include embryonic dorsal closure, formation of the adult thorax and wound healing. All of these processes require the basic region-leucine zipper (bZIP) transcription factors Jun and Fos. Much less is known about morphogenesis of the fly abdomen, which involves replacement of larval epidermal cells (LECs) with adult histoblasts that divide, migrate and finally fuse to form the adult epidermis during metamorphosis. This study implicates Drosophila Activating transcription factor 3 (Atf3), the single ortholog of human ATF3 and JDP2 bZIP proteins, in abdominal morphogenesis. During the process of the epithelial cell replacement, transcription of the atf3 gene declines. When this downregulation is experimentally prevented, the affected LECs accumulate cell-adhesion proteins and their extrusion and replacement with histoblasts are blocked. The abnormally adhering LECs consequently obstruct the closure of the adult abdominal epithelium. This closure defect can be either mimicked and further enhanced by knockdown of the small GTPase Rho1 or, conversely, alleviated by stimulating ecdysone steroid hormone signaling. Both Rho and ecdysone pathways have been previously identified as effectors of the LEC replacement. To elicit the gain-of-function effect, Atf3 specifically requires its binding partner Jun. These data thus identify Atf3 as a new functional partner of Drosophila Jun during development (Sekyrova, 2010).

Metamorphosis of Drosophila larvae into pupae and adult flies provides remarkable examples of morphogenetic changes that involve replacement of entire cell populations. Epithelia that had served larval function undergo programmed cell death while imaginal cells proliferate and differentiate to take their position. The Drosophila abdomen is an attractive system for studying the developmental replacement of one epithelial cell population with another. Unlike the adult head and thorax with appendages, all forming from pre-patterned imaginal discs, the adult abdomen derives from histoblasts that reside in each abdominal segment. Soon after the onset of metamorphosis, the diploid histoblasts undergo an initial phase of synchronized cell divisions; later the histoblasts expand while proliferating and replace the old polyploid larval epidermal cells (LECs) that cover the surface of the abdomen. To free space for the histoblasts, LECs are extruded from the epithelial monolayer. In order to maintain integrity of the epithelia, changes in cell adhesion and cell migration must be precisely orchestrated during this tissue remodeling (Sekyrova, 2010).

Rho kinase signaling, which stimulates constriction of the apical actomyosin cytoskeleton through myosin phosphorylation, is necessary for the extrusion and the ensuing apoptosis of LECs. Perturbed myosin phosphorylation leaves the process of the epithelial exchange incomplete, with residual LECs obstructing closure of the adult abdominal epidermis at the dorsal midline. A similar defect results from compromised function of the ecdysone receptor (EcR), which is required for both the initial phase of histoblast proliferation and for the removal of LECs. Other factors besides Rho signaling and EcR that regulate the epithelial cell replacement are unknown (Sekyrova, 2010).

This study implicates Atf3 (A3-3 -- FlyBase), the single Drosophila ortholog of the vertebrate Activating transcription factor 3 (ATF3) and Jun dimerization protein 2 (JDP2) in abdominal development. ATF3 and JDP2 belong among basic region-leucine zipper (bZIP) proteins, some of which play important roles in epithelial morphogenesis. Particularly the functions of Jun and Fos bZIP proteins in epithelial closure events during development are well understood owing to genetic studies in Drosophila. By contrast, no morphogenetic function has yet been reported for Atf3 in Drosophila (Sekyrova, 2010).

Mammalian ATF3 and JDP2 form homodimers but preferentially dimerize with members of the Jun subfamily (Aronheim, 1997; Hai, 1989; Hsu, 1991), functioning either as transcriptional activators (ATF3-Jun) or repressors (JDP2-Jun). Based mainly on cell-culture studies, multiple roles in cell proliferation, differentiation and apoptosis have been ascribed to ATF3 and JDP2. Atf3-/- mice are viable but suffer from altered glucose and immune homeostasis. Also Jdp2-/- mice survive but produce extra fat in their brown adipose tissue. In vivo significance of the interaction between the ATF3 or JDP2 proteins and Jun remains unclear (Sekyrova, 2010).

This study shows that Atf3 interacts biochemically and genetically with Jun in Drosophila. Temporal downregulation of atf3 transcription during metamorphosis is crucial, since sustained atf3 expression alters adhesive properties of LECs, thus preventing their extrusion and replacement by the adult epidermis. This effect of Atf3 requires the presence of Jun (Sekyrova, 2010).

Among Drosophila bZIP proteins, the predicted product of the CG11405 gene (also referred to as a3-3), located on the X chromosome, shows the closest similarity to the mammalian ATF3 and JDP2 proteins. The DNA-binding/dimerization bZIP domains of the human ATF3 and Drosophila Atf3 proteins are identical in 60% of their amino acids; there is 58% identity between Atf3 and JDP2 in this region (Sekyrova, 2010).

Dimerization between Atf3 and Jun in Drosophila has been theoretically predicted and confirmed by a yeast two-hybrid screen. To demonstrate direct binding, co-immunoprecipitation experiments were conducted. The endogenous Jun protein from Drosophila S2 cells co-precipitates with a transiently expressed Atf3 whereas Fos did not. A DNA mobility-shift assay with recombinant bZIP domains of Atf3, Jun and Fos was conducted to test for their DNA-binding properties. Atf3 specifically bound an ATF/CRE consensus element but not the AP-1 site, which was recognized by the Jun-Fos (AP-1) complex. Although Atf3 bound DNA by itself, presumably as a homodimer, the binding was enhanced in the presence of Jun. Fos did not synergize with Atf3 in DNA binding. Excess unlabeled DNA bearing the ATF/CRE binding site competed for the Atf3 bandshift activity whereas the AP-1 binding element did not. These results have shown that, like ATF3 or JDP2 in mammals, Atf3 in Drosophila selectively dimerizes with Jun, with which it cooperatively and specifically binds the ATF/CRE DNA element (Sekyrova, 2010).

To test whether Atf3 and Jun interact in vivo, experiments were conducted in the Drosophila compound eye, the precise structure of which sensitively reflects genetic interactions. Overexpression of atf3 under the GMR-Gal4 driver disrupted the ommatidial arrangement, resulting in smaller eyes with a glossy appearance. This atf3 misexpression phenotype could be completely suppressed by simultaneous RNAi-mediated knockdown of jun but not of fos. Conversely, the phenotype was exacerbated when jun was overexpressed in the eye together with atf3, suggesting that it is Atf3 in a complex with Jun that derails the normal eye development. Neither RNAi nor overexpression of jun alone had any effect on eye morphology. Interestingly, like depletion of Jun, co-expression of fos under the GMR-Gal4 driver completely averted the atf3 misexpression phenotype, restoring the normal appearance of the eye. Expression of fos or its mutant versions alone had no effect. These data can be explained by the ability of the surplus Fos to bind Jun and thus reduce its availability for interaction with the Atf3 protein. This interpretation is further supported by experiments showing that expression of the truncated bZIP domain of Fos is sufficient to suppress the Atf3 gain-of-function phenotype, whereas its transcription activation domain or phosphorylation sites are dispensable (Sekyrova, 2010).

Taken together, these results show that Atf3 cooperates with Jun, as Jun is specifically required for an effect caused by overexpression of Atf3 in the developing eye. Given the capacity of both Atf3 and Fos to bind Jun, and based on the ability of Jun to enhance and of Fos to suppress the Atf3 gain-of-function phenotype, it is suggested that Atf3 and Fos compete for their common partner Jun in vivo (Sekyrova, 2010).

To find out whether Atf3 is required for Drosophila development and whether its absence might resemble a phenotype caused by loss of its partner Jun, atf3 mutant flies were generated. The longest deletion (line atf376) obtained by imprecise excision of a P element, removed the entire bZIP domain of Atf3, and atf376 hemizygous (male) larvae lacked detectable atf3 mRNA. Thus, atf376 probably represents a null allele. Most atf376 larvae die soon after hatching and during all three larval stages. Only a few (approximately 2%) reach the third instar but die before metamorphosis as defective pseudopuparia. Expression of atf3 cDNA under the ubiquitous armadillo (arm-Gal4) driver rescued some atf376 hemizygotes to adults, confirming that loss of atf3 was the cause of the lethal phenotype. Interestingly, the moribund atf376 larvae abnormally enlarged lipid droplets in their fat body, thus displaying a phenotype reminiscent of that in mice lacking one of the Atf3 orthologs, JDP2. However, in contrast to viable Jdp2 or Atf3 knockout mice, atf3 is an essential gene in Drosophila (Sekyrova, 2010).

Fly embryos lacking the function of Jun or Fos die because of the failed dorsal closure. However, atf376 embryos develop normally, without the dorsal open defect, even when derived from atf3-deficient germline clones induced in atf376/ovoD1 mothers. Thus, unlike its partner Jun, Atf3 is not required for dorsal closure, suggesting that dorsal closure is regulated by Jun-Fos dimers and that the Atf3-Jun complex has another function later in development (Sekyrova, 2010).

Consistent with the vital requirement for Atf3 during larval stages, atf3 mRNA was expressed in embryos and larvae. Expression then sharply declines by the late-third larval instar, and no atf3 mRNA was detected by northern blot hybridization in wandering larvae and during metamorphosis from the time of puparium formation until the second day of pupal development. Detailed RT-PCR analysis showed that atf3 downregulation coincided with the cessation of feeding and the onset of metamorphosis [0 hours after puparium formation (APF)]. A pulse of expression occurred at 6 hours APF. RT-PCR from isolated fat body and abdominal integuments, together with in situ hybridization performed on puparia at this stage, showed that atf3 mRNA was primarily present in the larval epidermis (LECs) during the expression peak at 6 hours APF. From the time of head eversion (12 hours APF) the mRNA level remained low until the second day of pupal development, and then it grew steadily during morphogenesis of the adult. Quantitative RT-PCR revealed a 4.3-fold difference in atf3 mRNA abundance between 0 and 72 hours APF. In contrast to the tight regulation of atf3, the mRNAs of fos and jun fluctuated little during the examined period. Therefore, unlike Jun or Fos, Atf3 was dynamically regulated during metamorphosis at the level of transcription (Sekyrova, 2010).

The precise temporal control of atf3 expression suggested that the rise and subsequent fall of Atf3 during metamorphosis might be critical for the complex morphogenesis occurring in fly pupae. This possibility was tested by means of sustained expression of the full-length Atf3 protein using the UAS-Gal4 system with various drivers. A striking, fully penetrant metamorphic defect was observed with the pumpless (ppl) Gal4 driver. Although ppl>atf3 animals developed normally until the pupal stage, they failed to complete fusion of the adult abdominal epidermis. A dorsal cleft in the abdomen remained that could not be covered with the adult cuticle, and consequently 86% of the flies died inside the puparium. All of the ppl>atf3 adults that did eclose showed abdominal lesions filled with the old pupal cuticle lacking adult pigmentation and bristles, often with a clot covering a bleeding wound. Adults with the same abdominal cleft (but otherwise normal) also emerged when atf3 was moderately and ubiquitously misexpressed under the arm-Gal4 driver, suggesting that abdominal morphogenesis was the process most sensitive to ectopic Atf3 (Sekyrova, 2010).

The adult fly abdomen derives from histoblasts that proliferate, replace LECs and finally differentiate, giving rise to the adult cuticle. Therefore, the observed abdominal defect suggested a compromised function of the epidermis, either LECs, histoblasts or both cell types. To distinguish between these possibilities, expression of the ppl-Gal4 driver was first examined in the epidermis. It was found that ppl-Gal4 was active in LECs but not in histoblasts. Second, another driver, Eip71CD-Gal4, which was inactive in histoblasts but strongly expressed in LECs, was examined. Eip71CD-Gal4-driven misexpression of atf3 mostly produced lethal pupae lacking adult cuticle, but it occasionally yielded adults with a dorsal abdominal cleft. In addition to being active in LECs, both ppl-Gal4 and Eip71CD-Gal4 (data not shown) were also expressed in the fat body. However, no abdominal defects occurred when atf3 was misexpressed under either of three fat-body-specific Gal4 drivers, Lsp2, Cg or C7. Third, to rule out the possibility that ectopic Atf3 affected the imaginal epidermis, its expression was directed to histoblasts by using the escargot (esg) and T155 Gal4 drivers; in neither case the fusion of the adult abdominal epidermis was affected (Sekyrova, 2010).

To finally confirm that abdominal morphogenesis was disrupted by sustained atf3 activity in LECs, atf3 was induced by using the flp-out technique. Owing to the timing of heat-shock induction to the mid-third instar, this method triggers expression in the polyploid larval cells but not in the diploid histoblasts . Misexpression of atf3 under the actin promoter following the flp-out event invariantly led to an abdominal cleft. The lesions were often more severe than those observed in ppl>atf3 animals, affecting also lateral and ventral parts of the abdomen. Together, the above data demonstrate that the sustained expression of atf3 prevents fusion of the adult abdominal epidermis by acting upon LECs, suggesting that the replacement of these obsolete larval cells by adult histoblasts requires the developmental downregulation of atf3 expression (Sekyrova, 2010).

To understand the cellular events underlying the incomplete epithelial closure in ppl>atf3 animals, cell membranes were visualized by antibody staining of the septate junction component, Discs large 1 (Dlg1), or used a transgenic DE-cadherin::GFP fusion protein (shg::gfp). In wild-type animals 24 hours APF, LECs covering the surface of the abdomen gave way to the rapidly expanding nests of histoblasts that began to fuse laterally and ventrally. In ppl>atf3 pupae the histoblast nests also spread, and at least at 16 hours APF, before their fusion, they comprised normal numbers of histoblasts. By 48 hours APF a control abdomen was fully covered with adult epidermis consisting exclusively of histoblasts, now forming sensory bristles. Histoblasts in ppl>atf3 abdomens also differentiated the adult cuticle with sensory bristles, although polarity of the bristles near the dorsal cleft was altered. However, in contrast to the control, a large population of LECs remained in the dorsal abdomen of ppl>atf3 animals at 48 hours APF. The membranes of the persisting LECs accumulated the Dlg protein, and although these cells became severely deformed they survived throughout metamorphosis to the adult stage. When visualized in live ppl>atf3 pupae, the apical junctions of the remaining LECs displayed interdigitation and accumulation of DE-cadherin::GFP. Another adherens junction component, the Drosophila β-catenin Armadillo, was also enriched in atf3-expressing LECs (Sekyrova, 2010).

Cooperation between adherens junctions and the apical ring of actomyosin cytoskeleton is required for basal extrusion of LECs. The altered pattern of DE-cadherin and β-catenin therefore suggests that excessive Atf3 might prevent LEC extrusion through stabilization of the cell-cell adhesion complex. To examine the effect of Atf3 on LECs in further detail, the flp-out technique, which allows comparisons of atf3-misexpressing and control LECs within one tissue, was employed. Membrane interdigitation occurred between atf3-positive LECs already at 18 and 24 hours APF, even in areas where the LECs had no contact with histoblasts. At 48 hours APF only LECs expressing atf3 persisted, apparently being squeezed by the expanding histoblasts. The membrane-associated DE-cadherin::GFP signal was stronger in adjacent atf3-positive LECs compared with non-induced LECs, and quantitative analysis of confocal images acquired at 18 hours APF and at 24 hours APF both revealed a statistically significant 1.4-fold increase of the DE-cadherin::GFP signal intensity upon atf3 induction. Enrichment of DE-cadherin on apical membranes of atf3-expressing LECs was further confirmed on confocal cross sections (Sekyrova, 2010).

Although some atf3-positive LECs began the extrusion process, they could not detach from the apical surface even when entirely surrounded by histoblasts, possibly being tethered to it by the excessive adhesion protein. By contrast, control LECs did completely separate from the epithelium. In addition, LECs overexpressing atf3 displayed apical enrichment of moesin, an actin-binding protein of the ERM (ezrin, radixin, moesin) family, which links transmembrane proteins to cortical actin filaments. Interestingly, prominent accumulation of DE-cadherin was also observed in atf3-expressing clones of epithelial cells within the hinge region of wing discs that form the adult thorax, indicating that the effect of Atf3 on cell adhesion components may not be limited to larval epithelia (Sekyrova, 2010).

In summary, these results show that deregulation of atf3 expression causes marked changes of cell membranes, including interdigitation and accumulation of cell adhesion molecules, suggesting that LEC adhesiveness might be increased. Although some of the affected LECs initiate extrusion, this process stays incomplete. Consequently, the adhering LECs present a physical barrier for the migrating histoblasts (Sekyrova, 2010).

Rho kinase (Rok)-dependent phosphorylation of myosin regulatory light chain was shown to be required for LEC extrusion. To examine a possible relationship between the Rok-dependent cytoskeletal regulation and Atf3, the function of the GTPase Rho1 (also called RhoA), which acts immediately upstream of Rok, was disrupted. RNAi silencing of Rho1 using the ppl-Gal4 driver produced a phenocopy of atf3 misexpression, causing a dorsal abdominal cleft in 100% of ppl>Rho1(RNAi) adults, of which most died in the puparium and about 12% eclosed, similar to ppl>atf3 animals. However, when Rho1 RNAi and misexpression of atf3 in LECs were combined, the abdominal defect became more severe, not allowing any pharate adults to eclose. Conversely, co-expression of a dominantly active Rho1V14 protein suppressed the otherwise fully penetrant abdominal defect in some ppl-atf3 flies. Surprisingly, it was found that the endogenous Rho1 protein was mislocalized in atf3-misexpressing LECs, showing a diffuse cytoplasmic signal, compared with membrane localization in control LECs. These results suggest a genetic interaction between Rho signaling and atf3, and support the idea that excess Atf3 prevents extrusion of LECs by altering their cell adhesion properties (Sekyrova, 2010).

Disturbed function of the ecdysone receptor (EcR) has been shown to prevent extrusion of LECs, causing a dorsal abdominal cleft that closely resembles the Atf3 gain-of-function phenotype. Therefore whether stimulating EcR-dependent signaling by addition of the natural agonist 20E might overcome the defect caused by sustained atf3 expression was examined. Indeed, supplying third-instar ppl>atf3 larvae with dietary 20E increased the number of eclosing adults, the abdominal scars of which were in 22% of the cases partially or completely sealed with normal adult cuticle (Sekyrova, 2010).

Atf3 interacts with Jun to form a DNA-binding complex and genetically when overexpressed in the developing compound eye. To see if this interaction is biologically relevant during abdominal morphogenesis, whether Atf3 relies on the presence of Jun to cause the dorsal cleft phenotype was tested. First, it was confirmed that Jun is indeed expressed in LECs during metamorphosis. RNAi-mediated depletion of Jun in animals that misexpressed atf3 under the ppl-Gal4 driver restored viability of adults from 14% (atf3 alone) to 100%. Strikingly, 87% of the ppl>atf3, jun(RNAi) adults eclosed with a completely normal abdomen. By contrast, RNAi knockdown of Fos in ppl>atf3 background did not improve the abdominal defect. RNAi silencing of either jun or fos alone under the ppl-Gal4 driver had no effect on the abdomen. These results demonstrate that Atf3 requires its partner Jun but not Fos to disrupt abdominal morphogenesis. Similar to the situation in the compound eye, the effect of misexpressed atf3 can be neutralized by simultaneously expressing Fos or its truncated bZIP domain under the ppl-Gal4 driver. Therefore, the model in which Atf3 and Fos compete for their common partner Jun may be extended to the developing abdomen (Sekyrova, 2010).

This study has identified Atf3 as a new partner of Jun in Drosophila. Previously, Jun has only been known to dimerize with itself and with the Drosophila homolog of Fos. Functional analysis of Atf3 has not yet been reported. These biochemical data show that, similar to mammalian ATF3 and JDP2, the Atf3 protein selectively binds Jun but not Fos. Also consistent with the properties of ATF3 and JDP2 is the ability of Atf3 to bind the ATF/CRE response element alone or synergistically with Jun. In contrast to its mammalian counterparts, however, neither Atf3 alone nor in complex with Jun bound to the AP-1 element under the same conditions. The selective interactions of Atf3 point to distinct biological roles for the Atf3-Jun and the Fos-Jun dimers, respectively (Sekyrova, 2010).

This study has shown a genetic interaction between Atf3 and Jun. The evidence is based on the ability of ectopic Atf3 to disturb morphogenesis of the adult abdomen and the compound eye, which strictly depends on the availability of Jun. Importantly, none of the Atf3 gain-of-function phenotypes could be induced by misexpression of the truncated bZIP domain of Atf3, suggesting that the functional Atf3 protein in complex with Jun is required. Based on the selectivity of Atf3 in a DNA-binding assay, it is predicted that the Atf3-Jun complex regulates specific target genes distinct from those targeted by Fos-Jun dimers (Sekyrova, 2010).

The data also reflect a relationship between the AP-1 and Atf3-Jun complexes. Although Fos does not dimerize or bind DNA with Atf3, its ability to suppress the Atf3 misexpression phenotype in the eye suggests that Fos and Atf3 compete in vivo for their common partner Jun. The fact that the same suppression can be achieved by overexpressing either the truncated Fos bZIP domain or Fos lacking phosphorylation sites indicates that the suppression does not rely on a transcriptional function of Fos but probably occurs through sequestering of Jun, even by a transcriptionally inactive Fos protein. Early in vitro studies have proposed a competition model for the AP-1 and Atf3 proteins to explain a temporal regulation of gene expression in the regenerating liver. However, to date such a relationship among Fos, Jun and Atf3 has not been supported with direct genetic evidence (Sekyrova, 2010).

Removal of LECs is normally complete by 36 hours APF, at which time the sheets of histoblasts reach the dorsal midline. The data strongly support the argument that the temporal downregulation of atf3 expression during abdominal morphogenesis is necessary for LECs to be replaced by the adult epidermis. When experimentally sustained, atf3 activity in LECs interfered with this exchange by blocking extrusion and death of the LECs. This was evident as the atf3-expressing LECs survived within the epithelial layer for days after their scheduled destruction (Sekyrova, 2010).

Interdigitation of cell membranes and accumulation of adherens junction proteins in LECs suggested that ectopic Atf3 caused adjacent LECs to reinforce their mutual contacts. This probably resulted from altered distribution of the proteins, as levels of the shg (DE-cadherin) mRNA remained unchanged in LECs of ppl>atf3 animals. By contrast, junctions between atf3-expressing LECs and their normal neighbors or histoblasts were smooth and presumably less rigid. DE-cadherin was similarly enriched in clones of imaginal disc cells. These observations suggested that differential adhesion of atf3-expressing cells might have led to their sorting out from the surrounding epithelium. Even modest differences in cadherin levels have been shown to cause segregation of cells within a population by altering their adhesiveness (Sekyrova, 2010).

Recent live imaging data have revealed that migrating histoblasts push the LECs ahead of themselves towards the dorsal midline, where histoblasts fuse last. The atf3-expressing LECs that adhered to each other were probably moved and pressed by the expanding histoblasts to the dorsal side, whereas non-induced LECs were eliminated. This explains why the abdominal lesions primarily occurred at the dorsal midline, although flp-out experiments showed that atf3 misexpression could affect LECs in other areas as well. Strengthened contacts among persisting LECs probably blocked invasion of histoblasts in between them and inhibited LEC extrusion, eventually causing gaps in the adult epidermis (Sekyrova, 2010).

In accord with the notion that extrusion from the epithelium is a prerequisite for LECs to undergo apoptosis, it is assumed that sustained presence of Atf3 primarily enhanced adhesiveness of LECs, which only consequently prevented their death. This view is supported by the observation that membranes of atf3-expressing LECs interdigitated and accumulated DE-cadherin as early as 18-24 hours APF, even in areas of the larval epidermis that were far from histoblasts and where control LECs did not yet extrude. In addition, the Atf3 gain-of-function phenotype was stronger than abdominal closure defects caused by caspase mutation or inhibition. When the anti-apoptotic proteins p35 or DIAP1 (Thread — FlyBase) was misexpressed under the ppl-Gal4 driver, the resulting dorsal lesions were not lethal and were clearly milder than the broad, mostly fatal scars in ppl>atf3 animals. Compared with the large contiguous populations of persisting LECs in ppl>atf3 pupae, inhibiting apoptosis with p35 only allowed small islands of LECs to survive (Sekyrova, 2010).

Ecdysone signaling promotes replacement of the abdominal epithelia by stimulating both the early histoblast proliferation and the extrusion of LECs. As atf3 misexpression affected LECs but did not impair early histoblast proliferation, the latter possibility remains, that added 20E counteracted the effect of ectopic Atf3 by facilitating the extrusion process. Since normal 20E titers was detected in ppl>atf3 larvae or prepupae, the failure of LEC extrusion was not a result of steroid deficiency. Also, 20E had no effect on atf3 mRNA levels, at least in Drosophila S2 cells or third-instar larvae. Atf3 and ecdysone signaling therefore probably influence LEC extrusion by acting independently (Sekyrova, 2010).

Although the mechanism through which ecdysone contributes to LEC removal is unknown, one attractive possibility is that it might cooperate with Rho signaling, which is required for LEC extrusion as well. It has been demonstrated that genetic interaction between the 20E-response gene broad and components of the Rho pathway including RhoGEF2, Rho1 and myosin II is important for ecdysone-dependent epithelial cell elongation during Drosophila leg morphogenesis. The current data show that Rho1 becomes mislocalized in LECs upon atf3 misexpression and that Rho1 silencing enhances the abdominal gain-of-function phenotype of atf3. The exact relationship between Atf3, Rho1 and ecdysone remains to be determined. However, Atf3 clearly represents a new intrinsic regulator of epithelial cell replacement during Drosophila metamorphosis (Sekyrova, 2010).

DJun Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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