DJun

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).

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 puc^E69, 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 puc^E69 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 rolled^Sevenmaker (rl^Sem), encodes a form of ERK with increased resistance to inactivation by dephosphorylation. Expression of a dominant-negative form of Fos in the wings of rl^Sem flies results in loss of ectopic vein material. Conversely, overexpression of Fos enhances the extra-vein phenotype caused by rl^Sem (Gritzan, 2002).

32B Gal4, UAS Sem flies express the Rl^Sem 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).

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