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Transrepression of AP-1 by nuclear receptors in Drosophila
Mechanisms of Development 115 (2002) 91–100 www.elsevier.com/locate/modo
Transrepression of AP-1 by nuclear receptors in Drosophila Uwe Gritzan a, Carsten Weiss a,b, Julius Brennecke a,b, Dirk Bohmann a,b,* a
b
European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, NY 14642, USA Received 10 January 2002; received in revised form 22 March 2002; accepted 9 April 2002
Abstract 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 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. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Jun; Fos; Nuclear receptor; Drosophila; Gene regulation; Dorsal closure; Wing vein; Tailless; Eagle; Seven up; ERK; JNK; Basket; rolled; Transrepression; Signal transduction
1. Introduction The behavior of eukaryotic cells, particularly in multi cellular organisms is controlled by a panoply of extracellular signals. Many pathways that transduce such signals from the cell surface to the nucleus to direct changes in the gene expression program of the affected cell have been described and their constituent components have been arranged in hierarchically organized cascades by genetic and biochemical studies. Accumulating evidence indicates, however, that signaling pathways are not static linear assemblies, but that they are better understood as parts of flexible signaling networks. The response of a cell to the activation of a signaling pathway can be modulated by other signals as well as by parameters inherent to the signal-receiving cell, such as its differentiation state, its metabolic activity or other parameters that ultimately depend on the developmental history of the cell. Some signal transducers can be regulated by multiple molecular mechanisms and may thus serve as convergence points to integrate various types of biologically relevant information. This situation is exemplified by the AP-1 family of signal-regulated transcription factors and its * Corresponding author. E-mail address: [email protected] (D. Bohmann).
constituents, the Jun, Fos and ATF-2 proteins (reviewed in Karin et al., 1997; Whitmarsh and Davis, 1996; Wisdom, 1999). These proteins have been implicated in a variety of different biological processes ranging from cell proliferation to apoptosis. Consistent with their important and complex biological function, the activity of AP-1 proteins is tightly regulated. Studies in mammalian cell culture show that AP-1 activity can be controlled at different levels. First, the abundance of AP-1 components in the cell is subject to both transcriptional and post-transcriptional control (reviewed in Angel and Karin, 1991). In addition, full activation of AP-1 may require post-translational modifications, most notably phosphorylation by mitogen activated protein kinases (MAPK) (reviewed in Whitmarsh and Davis, 1996; Wisdom, 1999). Finally, interactions with other proteins can markedly influence AP-1 activity. Nuclear receptors (NRs) provide a prominent example for this mode of regulation. They can modulate AP-1 activity by a mechanism known as transrepression (Jonat et al., 1990; Schu¨le et al., 1990; Yang-Yen et al.,1990; reviewed in Herrlich, 2001; Karin, 1998). Active NRs can down-regulate AP-1-stimulated transcription. This effect does not require DNA binding by the NR and is thus distinct from conventional modes of transcription factor action. Several models have been suggested to explain the
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mechanism of transrepression (Caelles et al., 1997; Gonzalez et al., 2000; Kamei et al., 1996; Nissen and Yamamoto, 2000). However, its mechanistic basis in vivo remains poorly understood, aside from the assumption that it is mediated by protein–protein interactions. Transrepression of AP-1 is displayed by some, but not all, NRs (Saatcioglu et al., 1994). As NRs can also antagonize other transcription factors such as NF-kB in a DNA-binding-independent manner (Caldenhoven et al., 1995; Scheinman et al., 1995), the effect seems to be of general relevance. It has even been speculated that the transrepression function of NRs is evolutionarily older than the function in direct target gene activation (Karin, 1998). Genetic experiments in mice addressing the physiological relevance of the different modes of AP-1 regulation have yielded some unexpected results. Broad overexpression of c-Jun in transgenic mice has no detectable phenotypic consequences (Grigoriadis et al., 1993; reviewed in Jochum et al., 2001). This finding argues against the idea that protein levels are important determinants of AP-1 activity. Also, while the JNK phosphorylation sites Ser 63 and Ser 73 on c-Jun are essential for JNK-mediated activation of c-Jun in cell culture assays, these residues are dispensable for many c-Jun functions in the animal (Behrens et al., 1999). Thus, it is unclear how broadly this mechanism of AP-1 regulation is applied in vivo. In the case of transrepression, genetic experiments in mice support the physiological relevance of this aspect of NR biology. Replacement of the wild type form of the glucocorticoid receptor (GR) with a form that is transrepression-competent but can no longer transactivate rescues many aspects of the GR knock-out phenotype in mice (Reichardt et al., 1998). However, the relevant in vivo targets for transrepression by NRs – candidates include AP-1as well as NF-kB – have not yet been identified and the contribution of transrepression to the control of AP-1 activity in vivo remains unclear. Clearly, more work is needed to elucidate the complexities of AP-1 regulation in an intact tissue. To complement biochemical and cell culture studies on the different modes of AP-1 regulation, we addressed this issue in the genetic model organism Drosophila melanogaster. AP-1 has been implicated in a variety of processes in Drosophila development including dorsal closure, thorax closure and wing vein development (reviewed in Kockel et al., 2001). The expression of Jun and Fos in the corresponding tissues is relatively uniform and overexpression of the wildtype form of AP-1 family members has essentially no effect (Kockel et al., 1997; Zeitlinger et al., 1997; our unpublished observations). Thus, the regulation of protein abundance does not appear to contribute significantly to the control of AP-1 activity in these systems. Interestingly, in all of these processes, AP-1 is thought to function downstream of a MAPK, either JNK or ERK. Clearly, this mode of AP-1 control plays a crucial role in Drosophila development. Drosophila NRs (reviewed in Thummel, 1995) can be divided into two classes. One group is involved in mediating
responses to the steroid hormone ecdysone during metamorphosis. The second class of NRs appears not to be regulated by hormones. Eagle (Eg), Knirps (Kni) and Knirps-related (Knrl) do not possess ligand-binding domains (LBDs), but are classified as members of the NR superfamily because of sequence conservation in the zinc finger DNA-binding domain. Seven-up (Svp) and Tailless (Tll) are orphan receptors. While these proteins have LBDs, no ligands have been identified so far. As they are biologically active upon expression in a number of tissues, it is assumed that they are either ligand-independent or depend on a ubiquitous ligand. NRs of this second group have been implicated in a wide variety of developmental processes. Here, we show that transrepression of AP-1 by NRs is a conserved activity of NRs from Drosophila to man. We provide evidence that it serves to modulate AP-1 activity in at least two processes during Drosophila development. We discuss how activation by MAPKs and repression by NRs can be combined to control AP-1 activity in an intact tissue.
2. Results 2.1. Expression of NRs in the embryonic epidermis phenocopies a reduction in AP-1 function The best understood AP-1-dependent process in Drosophila development is a coordinated cell sheet movement known as dorsal closure (DC, reviewed in Noselli and Agnes, 1999). 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 homologues 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. Fig. 1B shows an embryo homozygous for kay 1, a fos null allele (Riesgo-Escovar and Hafen, 1997; Zeitlinger et al., 1997). This embryo is devoid of zygotic Fos activity and DC fails. The embryo has a large dorsal hole and the cuticle collapses. Fig. 1C shows an embryo homozygous for kay 2, a hypomorphic fos-allele (Zeitlinger et al., 1997). In this embryo, AP-1 activity is reduced but not eliminated. Correspondingly, the DC phenotype is weaker. The embryo displays a small dorsoanterior hole. To test whether Drosophila NRs can antagonize AP-1, we over-expressed a variety of them in the embryonic epidermis from appropriate transgenic constructs and scored for DC defects. Interestingly, expression of some, but not all, NRs we tested results in DC phenotypes of different strengths. Expression of Svp in the dorsal epidermis under the control of pnrGal4 (Fig. 1D) resulted in a DC phenotype reminiscent of that of kay 2 homozygotes. This finding is
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Fig. 1. Expression of Drosophila nuclear receptors causes dorsal closure phenotypes. Bright field micrographs of embryonic cuticles of the indicated genotypes are shown. Dorsal is up and anterior to the right, unless stated otherwise. Arrows indicate the anterior and posterior margins of the dorsal hole. (A) Wild type: the embryo is of normal size. The dorsal cuticle displays no holes or folds. (B) kay 1 homozygote. This panel shows a ventral view. A dorsal hole extends over the entire length of the embryo. The cuticle has collapsed and exhibits folds. The embryo is clearly smaller than a wild type embryo. (C) kay 2 homozygous embryos have variable DC phenotypes. The panel shows a relatively mild example to illustrate the consequences of a partial loss of AP-1 activity. The embryo has a small dorso-anterior hole. (D) pnrGal4/UASsvp phenocopies partial loss of Fos mutants. (E) hs tll transgenic embryo that was heat shock treated at 6 h after egg laying for 1 h at 378C. The phenotype is comparable to that of the kay 2 homozygote shown in (C). (F) pnrGal4/UASknrl. The embryo displays DC defects of an intermediate strength. (G) Summary listing the potency of Drosophila NRs in causing a dorsal open phenotype when over-expressed in the embryo. Mammalian homologs as annotated by Flybase are listed for the Drosophila NRs tested.
consistent with a suppression of AP-1 activity by Svp. Similarly, expression of Tll under the control of a heat shock promoter caused a weak dorsal open phenotype (Fig. 1E). The differentiation of ventral cells did not seem to be disturbed by Tll expression as the pattern of denticles in this part of the epidermis appeared 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 elicited stronger DC phenotypes with the dorsal hole frequently extending over several segments (Fig. 1F). Fig. 1G summarizes the results obtained with this
approach for a variety of NRs. Embryos expressing Kni under the control of pnrGal4 died early and failed to secrete a cuticle. Thus, it was not possible to determine the effect of Kni expression on DC using this assay. 2.2. Antagonistic relationship between AP-1 and NR activity in dorsal closure If, as we speculated, the DC defects caused by NR expression reflect a negative effect on AP-1, they should be sensitive to changes in Fos or Jun activity. In genetic interaction experiments, we compared the dorsal open phenotypes
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caused by NR expression in a wild-type background and in embryos with altered levels of AP-1 activity. Embryos heterozygous for kay 1 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 kay 1 than in wild type embryos. Indeed, while expression of Eg in a wild type background (Fig. 2B) mostly results DC phenotypes of intermediate strength, NR expression in embryos heterozygous for kay 1 (Fig. 2D) typically elicits complete failure of DC, indicative of a severe reduction of AP-1 activity. As embryos of both genotypes display somewhat variable phenotypes, the effect of kay 1 heterozygosity is best appreciated by quantitative analysis. We defined a clear reduction in size, a collapsed folded cuticle and a dorsal hole extending over at least half of the body length (compare Fig. 2C and D) as characteristics of a strong DC phenotype. Embryos with a smaller dorsoanterior hole and normal body size (compare Fig. 2A and B) were scored as showing weak DC phenotypes. We determined what percentage of embryos has strong DC defects for each genotype (Fig. 2G). This analysis confirms that Eg expression has more severe phenotypic consequences in kay 1 heterozygotes than in wild type embryos and supports the suggestion that NRs cause defective DC by suppressing AP-1 activity. In a complementary experiment, we examined the effect of Eg expression 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 (Martin-Blanco et al., 1998). Thus, in contrast to kay 1 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 were 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 (Fig. 2F,G). Expression of the NR Knrl in the various backgrounds yields essentially identical results (Fig. 2A,C,E,G). These data support the hypothesis that several Drosophila NRs can antagonize AP-1 as has been shown for mammalian NRs. 2.3. Drosophila NRs can antagonize AP-1 in reporter gene assays 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, we sought to monitor the effect of Drosophila NRs on bona fide AP-1 target genes. However, direct AP-1 target
Fig. 2. Embryonic defects caused by ectopic NR expression are modulated by changes in JNK signaling. Cuticles are presented as in Fig. 1. DC defects caused by NR expression in different genetic backgrounds. (A) pnrGal4/ UASknrl. The embryo shows DC defects of intermediate strength and a large dorsal hole. The cuticle is bent but has not collapsed. (B) pnrGal4/ UASeg. The phenotype is similar to the one in (A). (C) pnrGal4 kay 1/ UASknrl. The embryo has strong DC defects. It has a large dorsal hole and is reduced in size. The cuticle has collapsed. (D) pnrGal4 kay 1/ UASeg. The phenotype is similar to the one in (C). (E) pnrGal4 pucE69/ UASknrl. The embryo has a dorsal hole and is bent. The phenotype is somewhat weaker than that in (A). (F) pnrGal4 pucE69/UASeg. The phenotype is comparable to that in (E). It is somewhat weaker than that in (B). (G) Quantitation of the distribution of phenotypic strengths in the individual genotypes. A clear reduction in size, a large dorsal hole and a collapsed cuticle were defined as characteristics of a strong DC phenotype. One hundred embryos of each genotype were scored and the percentage of embryos falling into this phenotypic class was determined.
genes have not yet been clearly defined in Drosophila. While it is known that dpp and puc expression in DC requires AP-1 (Kockel et al., 1997), it cannot be excluded that this effect is indirect. To circumvent this problem, we turned to a mammalian cell culture system. Transrepression of AP-1 by mammalian NRs was first described in the
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context of collagenase transcription (Jonat et al., 1990; Schu¨ le et al., 1990; Yang-Yen et al., 1990). Extensive studies of the collagenase promoter have identified AP-1 as one of its primary regulators (Angel et al., 1987). Activation of the GR down-regulates AP-1-mediated transcription of collagenase. We 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, we found that both Eg and Tll efficiently antagonized AP-1 activity in a dose-dependent manner (Fig. 3). The observed effects are quantitatively comparable to those reported for the GR (Jonat et al., 1990). As a control, we co-transfected a reporter in which renilla luciferase transcription is controlled by the human ubiquitin C enhancer. This reporter is largely unaffected by the presence of NRs. Thus, the down-regulation of the collagenase reporter by Drosophila NRs cannot be attributed to a general effect on transcription or cell viability, but is specific for AP-1-dependent genes. 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. 2.4. The orphan receptor Seven up modulates AP-1 activity during DC After showing that over-expression of NRs can antagonize AP-1 function in the embryo, we sought to determine whether this effect reflects a developmental function of
Fig. 3. Drosophila NRs antagonize AP-1 in reporter gene assays. AP-1 reporter gene assays were performed in 293 cells in the absence or presence of the Drosophila NRs Eg or Tll. The amount of the respective expression vector transfected is indicated. An AP-1 independent ubiquitin C reporter construct was co-transfected as a control. Normalized luciferase activity is the ratio of firefly luciferase counts (measuring activity of the AP-1 dependent collagenase 3 enhancer) and Renilla luciferase counts (measuring the activity of the constitutive ubiquitin C enhancer). Each experiment was done three times. Error bars indicate standard deviation. The average ratio in the absence of NRs (2) was defined as 100%.
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endogenous NRs. Thus, we asked whether loss of NR function could affect DC. No NRs have previously been implicated in DC. However, the analysis of homozygous NR mutants in an otherwise wild type background could have failed to reveal a potential involvement in DC because of redundancies among NRs. We used dominant genetic interaction experiments to analyze the function of endogenous NRs in DC. In contrast to kay 1 homozygous embryos, which invariably have strong DC defects, the hetero-allelic combination kay 1/kay 2 shows diverse DC phenotypes. Whereas some embryos of this genotype display defects as strong as those of kay 1 embryos, others have much milder phenotypes. These observations indicate that the hetero-allelic combination represents a sensitized experimental system that is well suited for genetic interaction experiments. If endogenous NRs transrepress AP-1 during DC, lowering their activity in a kay 1/kay 2 background should suppress the dorsal open phenotype. Reducing the gene dosage of kni or eg in this background does not alter the strength of the DC defects (Fig. 4D). Thus, these NRs do not appear to be prominently involved in AP-1 regulation in DC, possibly because their expression in the embryo is too low or absent. Strikingly, however, inactivating one copy of the svp gene in kay 1/kay 2 embryos strongly suppresses the DC defects (compare Fig. 4A,B, see also Fig. 4D). The dorsal hole is smaller and the overall morphology of double mutant embryos is more normal than in embryos with two wild type copies of the svp gene. This result implies that svp is involved in the regulation of AP-1 activity in DC. As the relevant genotypes show some phenotypic variability, we again tested our interpretations by quantitating the distribution of phenotypic strength for each genotype (using the criteria described above). Quantitative analysis confirms that mutating one copy of the svp gene strongly supresses the DC defects of kay 1/kay 2 embryos. These results prompted us to quantitate the viability of kay 2 homozygotes in various backgrounds. While under the experimental conditions employed here, kay 2 homozygosity is strictly lethal, flies homozygous for kay 2 and heterozygous for svp 1 are recovered at almost Mendelian ratios. Some of these flies display mild thorax closure phenotypes (data not shown) indicating that AP-1 activity is still compromised. The finding that reducing svp gene dosage by half rescues the lethality of kay 2 homozygotes is another clear indication of an antagonistic relationship between the two gene products. 2.5. Svp, Eg and Tll antagonize Fos in wing vein differentiation We next wanted to determine whether modulation of AP1 activity by NRs occurs only in situations where AP-1 is regulated by JNK or whether this type of regulation also operates in different contexts. We have recently demonstrated a function for Fos downstream of ERK in the differ-
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Fig. 4. Loss of svp suppresses the DC defects of kay mutants. DC defects of kay 1/kay 2 transheterozygotes in different genetic backgrounds. Cuticles are presented as in Fig. 1. (A) kay 1/kay 2 transheterozygote. The embryo carries a dorsal hole spanning several segments and the cuticle exhibits folds. (B) kay 1/kay 2 svp 1. The embryo has only a small dorso-anterior hole. (C) svp 1 homozygote. The embryo does not display any DC defects. Thus, modulation of AP-1 activity by zygotic Svp is not required for DC in a wild type background. (D) Quantitation of the distribution of phenotypic strengths, scoring criteria as for Fig. 2G. Embryos (3 £ 30) were scored for each genotype. Error bars indicate standard deviation.
entiation of wing veins (Ciapponi et al., 2001; reviewed in De Celis, 1998). 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 (Oellers and Hafen, 1996). 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 (Ciapponi et al., 2001). 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 (compare Fig. 5A,B). Reducing fos gene dosage in this system strongly suppresses the vein phenotype (Fig. 5C, Ciapponi et al., 2001), consistent with Fos’ proposed role 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, we removed one copy of kni, eg, tll or svp in 32B Gal4, UAS Sem flies. Reducing kni function does not influence the vein phenotype (Fig. 5G). However, heterozygosity for any of the other three receptors tested reproducibly led to a mild enhancement of the ectopic vein differentiation (see Fig. 5D,E,F). As an unambiguously scoreable criterion to statistically evaluate phenotypic effects, we chose the presence of ectopic vein material posterior to L5. 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 was suppressed by reducing fos activity, it was enhanced by a reduction of eg, svp or tll function (Fig. 5G). These data suggest that all three NRs antagonize AP-1 activity in wing vein differentiation, conceivably in a redundant manner. If indeed, the NRs act in a redundant manner to counteract AP-1 activity during wing development, severely impairing NR function might result in over-activation of AP-1 and consequentially the formation of ectopic vein material even in a wild type background. To test this idea, we generated clones of wing cells devoid of svp or tll function or doubly mutant for svp and tll, both genes with the potential to transrepress AP-1 as indicated by the previous experiments. svp as well as tll single mutant clones were recovered efficiently but showed no extra vein material (data not shown). Strikingly however, tll, svp double mutant clones exhibit significant formation of extra vein tissue (Fig. 5H, see arrowheads), a phenotype that is diagnostic of elevated AP-1 activity, at close to 100% penetrance. As expected, this phenotype is strictly cell autonomous, i.e. formation of extra vein material was only observed within the clone. The clonal analysis provides strong evidence that antagonism of AP-1 by NRs is essential for proper vein formation. Several NRs appear to act in concert to execute this function. 2.6. D-Fos can antagonize endogenous NR activity In mammalian cell culture systems, transrepression has been shown to be mutual. Not only can NRs repress AP-1mediated transcription, but conversely AP-1 can also repress NR function (Go¨ ttlicher et al., 1998; Jonat et al.,
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Fig. 5. Interplay between ERK signaling and NRs during wing vein patterning. Adult wing vein phenotypes caused by manipulations of AP-1 and NR activity are displayed. Arrows indicate ectopic vein material posterior to vein L5 in B–F. (A) Wild type. The wing shows the normal venation pattern. (B) 32B Gal4, UAS Sem. The wing displays ectopic vein material. (C) 32B Gal4, UAS Sem/kay 2. The amount of ectopic vein material is reduced compared to panel B. (D–F) Reducing NR gene dosage leads to an increase in ectopic vein formation compared to panel B (most clearly seen posterior to L5, see arrows). (G) Quantitation of the distribution of phenotypic strengths. Error bars indicate standard deviation. (H) svp 1 tll 1 double mutant clones. Patches of svp tll double mutant tissue display formation of ectopic vein tissue (see arrowheads, blowup).
1990; Saatcioglu et al., 1994; Schu¨ le et al., 1990; Yang-Yen et al., 1990). We wanted to determine whether transrepression in Drosophila is mutual as well. Viable combinations of eg alleles frequently exhibit a held-out wing phenotype (hence the name of the gene). As a consequence of the reduction of eg activity, the wings are not positioned parallel to the anterior–posterior body axis but are held at an angle (compare Fig. 6C to A). In the course of our studies of vein differentiation, we noticed that flies heterozygous for the eg null allele eg 18B and expressing Rl Sem (genotype eg18B/32B Gal4 UAS Sem) have held-out wings (Fig. 6E). This phenotype is not
displayed by either eg 18B heterozygotes (Fig. 6B) or 32B Gal4 UAS Sem flies (Fig. 6D). One potential explanation for this observation is that elevated ERK levels lead to an increase in AP-1 activity, which in turn reduces Eg activity below a critical threshold. To test this idea, we examined the effects of expressing full length Drosophila Fos under the control of the ubiquitous tubGal4 driver in eg heterozygotes. While tubGal4/UASfos flies are semi-viable at 18 and 258C, the combination tubGal4/ eg18B UASfos causes lethality at both temperatures. Thus, reducing eg function renders flies more sensitive to elevated AP-1 levels, consistent with the antagonistic relationship between eg and AP-1.
Fig. 6. Inhibitory interactions between Fos and Eagle. Held-out wing phenotypes in adult flies caused by manipulation of eg activity. (A) Wild type. The wings are aligned along the anterior–posterior body axis. (B) Flies heterozygous for the null allele eg 18B show normal wing posture. (C) Flies homozygous for eg MZ360 (a partial loss-of-function allele) display a classical held-out wing phenotype. (D) 32B Gal4 UAS Sem/1. Increasing ERK activity does not affect wing posture. (E) 32B Gal4 UAS Sem/eg 18B. A simultaneous increase in ERK activity and reduction of eg activity leads to a clear held-out wing phenotype. (F) tub Gal4 UASfosNterm/eg 18B Expressing the Fos N-terminal domain in flies with compromised eg function results in a mild held-out wing phenotype.
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However, because of the lethality, this combination cannot be used to ask whether fos can antagonize eg function in the control of wing posture. To circumvent this problem, we expressed a transactivation-incompetent form of Fos in eg heterozygotes. For a mammalian cell culture system, it has been reported that the N-terminal transactivation domain of c-Fos can antagonize NR-dependent transcription (Lucibello et al., 1990). We used an analogous approach in the fly. Expression of the N-terminal 150 amino acids of Fos (excluding the DNA-binding domain) with the tubGal4 driver has no effect on viability. However, expression of this construct in flies heterozygous for eg results in a mild but clear held-out wing phenotype and the affected individuals are unable to fly (Fig. 6F). Taken together, these experiments suggest that AP-1 can antagonize endogenous eg function. Importantly, the equilibrium of AP-1 and NR activities can be influenced by a transactivation-incompetent form of Fos. Thus, the antagonism is not mediated by the activation of opposing genetic programs but appears to be based on protein–protein interactions. 3. Discussion 3.1. Transrepression of AP-1 by NRs is conserved from Drosophila to mammals This paper presents several lines of evidence indicating that the unorthodox mechanism of AP-1 transrepression by NRs, as originally described in mammals, also exists in Drosophila. Using both, gain- and loss-of-function approaches, we demonstrated that Drosophila NRs can specifically antagonize AP-1 in two unrelated processes during Drosophila development and in mammalian cell culture reporter gene assays. Importantly, in all three experimental settings, several NRs with distinct DNA-binding sequences (Hoch et al., 1992) display the same activity. This observation strongly suggests that AP-1 antagonism is DNA-binding independent. Furthermore, the experiments on the control of wing posture show that a transactivation-incompetent form of Fos can suppress NR activity, implying protein–protein interactions as opposed to a process involving Fos target gene expression as the underlying mechanism. Taken together, these data show that AP-1 transrepression is a conserved activity of NRs from Drosophila to mammals. Thus, studying the mutual antagonism between AP-1 and NRs in Drosophila can help to improve our understanding of both the molecular mechanism of transrepression and the physiological relevance of this mode of AP-1 regulation (see below). 3.2. The physiological relevance of AP-1 transrepression by NRs in Drosophila While transrepression by the GR has been implicated in AP-1 regulation in a mouse skin carcinogenesis model (Tuckermann et al., 1999), the relevance of transrepres-
sional control of AP-1 under physiological conditions has not previously been demonstrated. Here, we present evidence that transrepression by NRs serves to modulate AP-1 activity during Drosophila development. Loss-offunction experiments demonstrate that Svp antagonizes AP-1 in DC. In wing vein differentiation, Fos activity is down-regulated by Svp, Tll and Eg. Our data suggest that the NRs exert these effects by transrepression. It has previously been shown that AP-1 activity is regulated by JNK in DC and by ERK in wing vein differentiation. Thus, our findings also provide strong support for the idea that several mechanisms act in parallel to control AP-1 activity in these developmental processes. 3.3. Combinatorial control of AP-1 activity in vivo The finding that MAPKs and NRs act in parallel to control AP-1 activity raises the question of what types of information are conveyed by the distinct mechanisms to ensure proper activation of AP-1. In the wing, the venation pattern is prefigured by the pattern of active EGF ligand (De Celis, 1998). Localized EGF pathway activation generates a pattern of ERK activation. Presumably, this pattern is translated into spatial differences of AP-1 activity, which in turn, govern vein positioning. Cells in which ERK activates AP-1 differentiate into vein tissue whereas cells in which ERK activity is not high enough to activate AP-1 adopt an intervein fate. Clearly, the regulation of AP-1 by ERK conveys external information. We speculate that the modulation of AP-1 activity by NRs contributes to what has recently been termed signal consolidation (Jordan et al., 2000). 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. We propose 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. The identification of Drosophila NRs as modulators of MAPK to AP-1 signaling assigns a novel developmental role to these proteins. This aspect of Svp or Tll function and a contribution of these proteins to DC or wing vein patterning had not previously been discovered. A possible reason for this could be that the role as AP-1 antagonist
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appears to shared by several NRs that act redundantly (at least in the wing). This is not the case for the better known functions of Tll, Svp and Eg, which presumably depend on their direct transcriptional targets e.g. in photoreceptor differentiation or embryo development. A multi-pronged approach based on Drosophila genetics in combination with tissue culture studies and biochemistry may shed light on the developmental function and the so far elusive mechanism of the phenomenon of NR-mediated transrepression of AP-1.
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senting the ratio of firefly luciferase activities and renilla luciferase activities were calculated. 4.3. Wing vein analysis Wings were dissected and mounted according to standard procedures. For quantitation purposes, 40 male wings of each genotype were scored. For the induction of clones in the wing flies of the genotype y w f hsflp; FRT82 svp 1/ FRT82; y w f hsflp; FRT82 tll 1/FRT82 or y w hsflp; FRT82 svp 1tll 1/FRT82 m were heat shock treated at 84 ^ 12 h after egg laying for 1 h at 388C.
4. Materials and methods Most fly strains used in this study were described previously: kay 1, kay 2 (Zeitlinger et al., 1997); hs-tll (Steingrimsson et al., 1991); tll 1 (Pignoni et al., 1990); svp 1 (Mlodzik et al., 1990); UASsvp (Kramer et al., 1995); kni 2 (Gerwin et al., 1994); UASkni, UASknrl (Lunde et al., 1998); hs-EcR (Lam and Thummel, 2000); hs-HR38 (Kozlova et al., 1998); hs-HR78 (Fisk and Thummel, 1998); hs-usp (Oro et al., 1992); eg 18B, eg MZ360, UASeg (Dittrich et al., 1997); tub Gal4 (Lee and Luo, 1999); pnr Gal4 (Calleja et al., 1996). UAS Sem and UAS fosNterm were constructed by cloning the corresponding coding sequences (the N-terminal 150 amino acids in the case of UAS fosNterm) into pCaSpeR3 and generating transgenic lines. 4.1. Cuticle preparations Embryos were collected for 24 h at 258C on apple juice agar plates unless stated otherwise. Then embryos were aged for 24 h or, if including exclusively embryonic lethals in the preparation was crucial, for approximately 32 h. Larvae were removed from the plates by scraping off the yeast and cuticle preparations were performed according to a standard protocol (van der Meer, 1977). For quantitation purposes, approximately 100 embryos of each genotype were scored. 4.2. Cell culture reporter gene assays HEK293 cells were transfected at 50–70% confluency by the calcium phosphate method (Gorman et al., 1982) with 1 mg of reporter plasmid–517 collagenase-Luc (kindly provided by A. Cato) together with either increasing amounts of CMV-tailless, CMV-eagle (the corresponding coding sequences were cloned into pcDNA3.1, Invitrogen) or empty expression vector. Fourteen to 16 h after transfection, cells were incubated with 100 ng/ml TPA (Sigma) or 0.1% dimethyl sulfoxide (DMSO) as a solvent control. Transfection efficiency was controlled by co-transfection with 10 ng ubiquitin promoter-driven Renilla luciferase reporter construct (kindly provided by M. Go¨ ttlicher). Twenty fours after treatment, cells were washed twice with phosphate-buffered saline (PBS) and lysed using passive lysis buffer (Promega). Normalized values repre-
4.4. Documentation of held-out wing phenotypes Held-out wing phenotypes are best scored in live flies. Anesthesia frequently causes abnormal wing positions even in wild type flies. For documentation purposes, flies were briefly anesthetized with CO2 and photographed upon ‘waking up’.
Acknowledgements We thank J. Lengyel, M. Mlodzik, Y. Hiromi, E. Bier, C. Thummel, B. Hall, J. Urban, G. Rubin, A. Cato, M. Go¨ ttlicher and the Umea and Bloomington Stock Centers for reagents. P. Rørth, F. Stewart, S. Cohen, M. Noble and members of the Bohmann lab have contributed helpful comments to the manuscript.
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Lunde, K., Biehs, B., Nauber, U., Bier, E., 1998. The knirps and knirpsrelated genes organize development of the second wing vein in Drosophila. Development 125, 4145–4154. Martin-Blanco, E., Gampel, A., Ring, J., Virdee, K., Kirov, N., Tolkovsky, A.M., Martinez-Arias, A., 1998. puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila. Genes Dev. 12, 557–570. van der Meer, S., 1977. Optical clean and permanent whole mount preparation for phase contrast microscopy of cuticular structures of insect larvae. Dros. Inf. Serv. 52, 160–161. Mlodzik, M., Hiromi, Y., Weber, U., Goodman, C.S., Rubin, G.M., 1990. The Drosophila seven-up gene, a member of the steroid receptor gene superfamily, controls photoreceptor cell fates. Cell 60, 211–224. Nissen, R.M., Yamamoto, K.R., 2000. The glucocorticoid receptor inhibits NFkappaB by interfering with serine-2 phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes Dev. 14, 2314–2329. Noselli, S., Agnes, F., 1999. Roles of the JNK signaling pathway in Drosophila morphogenesis. Curr. Opin. Genet. Dev. 9, 466–472. Oellers, N., Hafen, E., 1996. Biochemical characterization of rolledSem, an activated form of Drosophila mitogen-activated protein kinase. J. Biol. Chem. 271, 24939–24944. Oro, A.E., McKeown, M., Evans, R.M., 1992. The Drosophila retinoid X receptor homolog ultraspiracle functions in both female reproduction and eye morphogenesis. Development 115, 449–462. Pignoni, F., Baldarelli, R.M., Steingrimsson, E., Diaz, R.J., Patapoutian, A., Merriam, J.R., Lengyel, J.A., 1990. The Drosophila gene tailless is expressed at the embryonic termini and is a member of the steroid receptor superfamily. Cell 62, 151–163. Reichardt, H.M., Kaestner, K.H., Tuckermann, J., Kretz, O., Wessely, O., Bock, R., Gass, P., Schmid, W., Herrlich, P., Angel, P., Schu¨ tz, G., 1998. DNA binding of the glucocorticoid receptor is not essential for survival. Cell 93, 531–541. Riesgo-Escovar, J.R., Hafen, E., 1997. Common and distinct roles of DFos and DJun during Drosophila development. Science 278, 669–672. Saatcioglu, F., Claret, F.X., Karin, M., 1994. Negative transcriptional regulation by nuclear receptors. Semin. Cancer Biol. 5, 347–359. Scheinman, R.I., Gualberto, A., Jewell, C.M., Cidlowski, J.A., Baldwin Jr, A.S., 1995. Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors. Mol. Cell Biol. 15, 943–953. Schu¨ le, R., Rangarajan, P., Kliewer, S., Ransone, L.J., Bolado, J., Yang, N., Verma, I.M., Evans, R.M., 1990. Functional antagonism between oncoprotein c-Jun and the glucocorticoid receptor. Cell 62, 1217–1226. Steingrimsson, E., Pignoni, F., Liaw, G.J., Lengyel, J.A., 1991. Dual role of the Drosophila pattern gene tailless in embryonic termini. Science 254, 418–421. Thummel, C.S., 1995. From embryogenesis to metamorphosis: the regulation and function of Drosophila nuclear receptor superfamily members. Cell 83, 871–877. Tuckermann, J.P., Reichardt, H.M., Arribas, R., Richter, K.H., Schutz, G., Angel, P., 1999. The DNA binding-independent function of the glucocorticoid receptor mediates repression of AP-1-dependent genes in skin. J. Cell Biol. 147, 1365–1370. Whitmarsh, A.J., Davis, R.J., 1996. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J. Mol. Med. 74, 589–607. Wisdom, R., 1999. AP-1: one switch for many signals. Exp. Cell Res. 253, 180–185. Yang-Yen, H.F., Chambard, J.C., Sun, Y.L., Smeal, T., Schmidt, T.J., Drouin, J., Karin, M., 1990. Transcriptional interference between cJun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein–protein interaction. Cell 62, 1205–1215. Zeitlinger, J., Kockel, L., Peverali, F.A., Jackson, D.B., Mlodzik, M., Bohmann, D., 1997. Defective dorsal closure and loss of epidermal decapentaplegic expression in Drosophila fos mutants. EMBO J. 16, 7393–7401.
Transrepression of AP-1 by nuclear receptors in Drosophila Uwe Gritzan a, Carsten Weiss a,b, Julius Brennecke a,b, Dirk Bohmann a,b,* a
b
European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, NY 14642, USA Received 10 January 2002; received in revised form 22 March 2002; accepted 9 April 2002
Abstract 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 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. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Jun; Fos; Nuclear receptor; Drosophila; Gene regulation; Dorsal closure; Wing vein; Tailless; Eagle; Seven up; ERK; JNK; Basket; rolled; Transrepression; Signal transduction
1. Introduction The behavior of eukaryotic cells, particularly in multi cellular organisms is controlled by a panoply of extracellular signals. Many pathways that transduce such signals from the cell surface to the nucleus to direct changes in the gene expression program of the affected cell have been described and their constituent components have been arranged in hierarchically organized cascades by genetic and biochemical studies. Accumulating evidence indicates, however, that signaling pathways are not static linear assemblies, but that they are better understood as parts of flexible signaling networks. The response of a cell to the activation of a signaling pathway can be modulated by other signals as well as by parameters inherent to the signal-receiving cell, such as its differentiation state, its metabolic activity or other parameters that ultimately depend on the developmental history of the cell. Some signal transducers can be regulated by multiple molecular mechanisms and may thus serve as convergence points to integrate various types of biologically relevant information. This situation is exemplified by the AP-1 family of signal-regulated transcription factors and its * Corresponding author. E-mail address: [email protected] (D. Bohmann).
constituents, the Jun, Fos and ATF-2 proteins (reviewed in Karin et al., 1997; Whitmarsh and Davis, 1996; Wisdom, 1999). These proteins have been implicated in a variety of different biological processes ranging from cell proliferation to apoptosis. Consistent with their important and complex biological function, the activity of AP-1 proteins is tightly regulated. Studies in mammalian cell culture show that AP-1 activity can be controlled at different levels. First, the abundance of AP-1 components in the cell is subject to both transcriptional and post-transcriptional control (reviewed in Angel and Karin, 1991). In addition, full activation of AP-1 may require post-translational modifications, most notably phosphorylation by mitogen activated protein kinases (MAPK) (reviewed in Whitmarsh and Davis, 1996; Wisdom, 1999). Finally, interactions with other proteins can markedly influence AP-1 activity. Nuclear receptors (NRs) provide a prominent example for this mode of regulation. They can modulate AP-1 activity by a mechanism known as transrepression (Jonat et al., 1990; Schu¨le et al., 1990; Yang-Yen et al.,1990; reviewed in Herrlich, 2001; Karin, 1998). Active NRs can down-regulate AP-1-stimulated transcription. This effect does not require DNA binding by the NR and is thus distinct from conventional modes of transcription factor action. Several models have been suggested to explain the
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00116-8
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mechanism of transrepression (Caelles et al., 1997; Gonzalez et al., 2000; Kamei et al., 1996; Nissen and Yamamoto, 2000). However, its mechanistic basis in vivo remains poorly understood, aside from the assumption that it is mediated by protein–protein interactions. Transrepression of AP-1 is displayed by some, but not all, NRs (Saatcioglu et al., 1994). As NRs can also antagonize other transcription factors such as NF-kB in a DNA-binding-independent manner (Caldenhoven et al., 1995; Scheinman et al., 1995), the effect seems to be of general relevance. It has even been speculated that the transrepression function of NRs is evolutionarily older than the function in direct target gene activation (Karin, 1998). Genetic experiments in mice addressing the physiological relevance of the different modes of AP-1 regulation have yielded some unexpected results. Broad overexpression of c-Jun in transgenic mice has no detectable phenotypic consequences (Grigoriadis et al., 1993; reviewed in Jochum et al., 2001). This finding argues against the idea that protein levels are important determinants of AP-1 activity. Also, while the JNK phosphorylation sites Ser 63 and Ser 73 on c-Jun are essential for JNK-mediated activation of c-Jun in cell culture assays, these residues are dispensable for many c-Jun functions in the animal (Behrens et al., 1999). Thus, it is unclear how broadly this mechanism of AP-1 regulation is applied in vivo. In the case of transrepression, genetic experiments in mice support the physiological relevance of this aspect of NR biology. Replacement of the wild type form of the glucocorticoid receptor (GR) with a form that is transrepression-competent but can no longer transactivate rescues many aspects of the GR knock-out phenotype in mice (Reichardt et al., 1998). However, the relevant in vivo targets for transrepression by NRs – candidates include AP-1as well as NF-kB – have not yet been identified and the contribution of transrepression to the control of AP-1 activity in vivo remains unclear. Clearly, more work is needed to elucidate the complexities of AP-1 regulation in an intact tissue. To complement biochemical and cell culture studies on the different modes of AP-1 regulation, we addressed this issue in the genetic model organism Drosophila melanogaster. AP-1 has been implicated in a variety of processes in Drosophila development including dorsal closure, thorax closure and wing vein development (reviewed in Kockel et al., 2001). The expression of Jun and Fos in the corresponding tissues is relatively uniform and overexpression of the wildtype form of AP-1 family members has essentially no effect (Kockel et al., 1997; Zeitlinger et al., 1997; our unpublished observations). Thus, the regulation of protein abundance does not appear to contribute significantly to the control of AP-1 activity in these systems. Interestingly, in all of these processes, AP-1 is thought to function downstream of a MAPK, either JNK or ERK. Clearly, this mode of AP-1 control plays a crucial role in Drosophila development. Drosophila NRs (reviewed in Thummel, 1995) can be divided into two classes. One group is involved in mediating
responses to the steroid hormone ecdysone during metamorphosis. The second class of NRs appears not to be regulated by hormones. Eagle (Eg), Knirps (Kni) and Knirps-related (Knrl) do not possess ligand-binding domains (LBDs), but are classified as members of the NR superfamily because of sequence conservation in the zinc finger DNA-binding domain. Seven-up (Svp) and Tailless (Tll) are orphan receptors. While these proteins have LBDs, no ligands have been identified so far. As they are biologically active upon expression in a number of tissues, it is assumed that they are either ligand-independent or depend on a ubiquitous ligand. NRs of this second group have been implicated in a wide variety of developmental processes. Here, we show that transrepression of AP-1 by NRs is a conserved activity of NRs from Drosophila to man. We provide evidence that it serves to modulate AP-1 activity in at least two processes during Drosophila development. We discuss how activation by MAPKs and repression by NRs can be combined to control AP-1 activity in an intact tissue.
2. Results 2.1. Expression of NRs in the embryonic epidermis phenocopies a reduction in AP-1 function The best understood AP-1-dependent process in Drosophila development is a coordinated cell sheet movement known as dorsal closure (DC, reviewed in Noselli and Agnes, 1999). 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 homologues 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. Fig. 1B shows an embryo homozygous for kay 1, a fos null allele (Riesgo-Escovar and Hafen, 1997; Zeitlinger et al., 1997). This embryo is devoid of zygotic Fos activity and DC fails. The embryo has a large dorsal hole and the cuticle collapses. Fig. 1C shows an embryo homozygous for kay 2, a hypomorphic fos-allele (Zeitlinger et al., 1997). In this embryo, AP-1 activity is reduced but not eliminated. Correspondingly, the DC phenotype is weaker. The embryo displays a small dorsoanterior hole. To test whether Drosophila NRs can antagonize AP-1, we over-expressed a variety of them in the embryonic epidermis from appropriate transgenic constructs and scored for DC defects. Interestingly, expression of some, but not all, NRs we tested results in DC phenotypes of different strengths. Expression of Svp in the dorsal epidermis under the control of pnrGal4 (Fig. 1D) resulted in a DC phenotype reminiscent of that of kay 2 homozygotes. This finding is
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Fig. 1. Expression of Drosophila nuclear receptors causes dorsal closure phenotypes. Bright field micrographs of embryonic cuticles of the indicated genotypes are shown. Dorsal is up and anterior to the right, unless stated otherwise. Arrows indicate the anterior and posterior margins of the dorsal hole. (A) Wild type: the embryo is of normal size. The dorsal cuticle displays no holes or folds. (B) kay 1 homozygote. This panel shows a ventral view. A dorsal hole extends over the entire length of the embryo. The cuticle has collapsed and exhibits folds. The embryo is clearly smaller than a wild type embryo. (C) kay 2 homozygous embryos have variable DC phenotypes. The panel shows a relatively mild example to illustrate the consequences of a partial loss of AP-1 activity. The embryo has a small dorso-anterior hole. (D) pnrGal4/UASsvp phenocopies partial loss of Fos mutants. (E) hs tll transgenic embryo that was heat shock treated at 6 h after egg laying for 1 h at 378C. The phenotype is comparable to that of the kay 2 homozygote shown in (C). (F) pnrGal4/UASknrl. The embryo displays DC defects of an intermediate strength. (G) Summary listing the potency of Drosophila NRs in causing a dorsal open phenotype when over-expressed in the embryo. Mammalian homologs as annotated by Flybase are listed for the Drosophila NRs tested.
consistent with a suppression of AP-1 activity by Svp. Similarly, expression of Tll under the control of a heat shock promoter caused a weak dorsal open phenotype (Fig. 1E). The differentiation of ventral cells did not seem to be disturbed by Tll expression as the pattern of denticles in this part of the epidermis appeared 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 elicited stronger DC phenotypes with the dorsal hole frequently extending over several segments (Fig. 1F). Fig. 1G summarizes the results obtained with this
approach for a variety of NRs. Embryos expressing Kni under the control of pnrGal4 died early and failed to secrete a cuticle. Thus, it was not possible to determine the effect of Kni expression on DC using this assay. 2.2. Antagonistic relationship between AP-1 and NR activity in dorsal closure If, as we speculated, the DC defects caused by NR expression reflect a negative effect on AP-1, they should be sensitive to changes in Fos or Jun activity. In genetic interaction experiments, we compared the dorsal open phenotypes
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caused by NR expression in a wild-type background and in embryos with altered levels of AP-1 activity. Embryos heterozygous for kay 1 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 kay 1 than in wild type embryos. Indeed, while expression of Eg in a wild type background (Fig. 2B) mostly results DC phenotypes of intermediate strength, NR expression in embryos heterozygous for kay 1 (Fig. 2D) typically elicits complete failure of DC, indicative of a severe reduction of AP-1 activity. As embryos of both genotypes display somewhat variable phenotypes, the effect of kay 1 heterozygosity is best appreciated by quantitative analysis. We defined a clear reduction in size, a collapsed folded cuticle and a dorsal hole extending over at least half of the body length (compare Fig. 2C and D) as characteristics of a strong DC phenotype. Embryos with a smaller dorsoanterior hole and normal body size (compare Fig. 2A and B) were scored as showing weak DC phenotypes. We determined what percentage of embryos has strong DC defects for each genotype (Fig. 2G). This analysis confirms that Eg expression has more severe phenotypic consequences in kay 1 heterozygotes than in wild type embryos and supports the suggestion that NRs cause defective DC by suppressing AP-1 activity. In a complementary experiment, we examined the effect of Eg expression 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 (Martin-Blanco et al., 1998). Thus, in contrast to kay 1 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 were 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 (Fig. 2F,G). Expression of the NR Knrl in the various backgrounds yields essentially identical results (Fig. 2A,C,E,G). These data support the hypothesis that several Drosophila NRs can antagonize AP-1 as has been shown for mammalian NRs. 2.3. Drosophila NRs can antagonize AP-1 in reporter gene assays 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, we sought to monitor the effect of Drosophila NRs on bona fide AP-1 target genes. However, direct AP-1 target
Fig. 2. Embryonic defects caused by ectopic NR expression are modulated by changes in JNK signaling. Cuticles are presented as in Fig. 1. DC defects caused by NR expression in different genetic backgrounds. (A) pnrGal4/ UASknrl. The embryo shows DC defects of intermediate strength and a large dorsal hole. The cuticle is bent but has not collapsed. (B) pnrGal4/ UASeg. The phenotype is similar to the one in (A). (C) pnrGal4 kay 1/ UASknrl. The embryo has strong DC defects. It has a large dorsal hole and is reduced in size. The cuticle has collapsed. (D) pnrGal4 kay 1/ UASeg. The phenotype is similar to the one in (C). (E) pnrGal4 pucE69/ UASknrl. The embryo has a dorsal hole and is bent. The phenotype is somewhat weaker than that in (A). (F) pnrGal4 pucE69/UASeg. The phenotype is comparable to that in (E). It is somewhat weaker than that in (B). (G) Quantitation of the distribution of phenotypic strengths in the individual genotypes. A clear reduction in size, a large dorsal hole and a collapsed cuticle were defined as characteristics of a strong DC phenotype. One hundred embryos of each genotype were scored and the percentage of embryos falling into this phenotypic class was determined.
genes have not yet been clearly defined in Drosophila. While it is known that dpp and puc expression in DC requires AP-1 (Kockel et al., 1997), it cannot be excluded that this effect is indirect. To circumvent this problem, we turned to a mammalian cell culture system. Transrepression of AP-1 by mammalian NRs was first described in the
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context of collagenase transcription (Jonat et al., 1990; Schu¨ le et al., 1990; Yang-Yen et al., 1990). Extensive studies of the collagenase promoter have identified AP-1 as one of its primary regulators (Angel et al., 1987). Activation of the GR down-regulates AP-1-mediated transcription of collagenase. We 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, we found that both Eg and Tll efficiently antagonized AP-1 activity in a dose-dependent manner (Fig. 3). The observed effects are quantitatively comparable to those reported for the GR (Jonat et al., 1990). As a control, we co-transfected a reporter in which renilla luciferase transcription is controlled by the human ubiquitin C enhancer. This reporter is largely unaffected by the presence of NRs. Thus, the down-regulation of the collagenase reporter by Drosophila NRs cannot be attributed to a general effect on transcription or cell viability, but is specific for AP-1-dependent genes. 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. 2.4. The orphan receptor Seven up modulates AP-1 activity during DC After showing that over-expression of NRs can antagonize AP-1 function in the embryo, we sought to determine whether this effect reflects a developmental function of
Fig. 3. Drosophila NRs antagonize AP-1 in reporter gene assays. AP-1 reporter gene assays were performed in 293 cells in the absence or presence of the Drosophila NRs Eg or Tll. The amount of the respective expression vector transfected is indicated. An AP-1 independent ubiquitin C reporter construct was co-transfected as a control. Normalized luciferase activity is the ratio of firefly luciferase counts (measuring activity of the AP-1 dependent collagenase 3 enhancer) and Renilla luciferase counts (measuring the activity of the constitutive ubiquitin C enhancer). Each experiment was done three times. Error bars indicate standard deviation. The average ratio in the absence of NRs (2) was defined as 100%.
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endogenous NRs. Thus, we asked whether loss of NR function could affect DC. No NRs have previously been implicated in DC. However, the analysis of homozygous NR mutants in an otherwise wild type background could have failed to reveal a potential involvement in DC because of redundancies among NRs. We used dominant genetic interaction experiments to analyze the function of endogenous NRs in DC. In contrast to kay 1 homozygous embryos, which invariably have strong DC defects, the hetero-allelic combination kay 1/kay 2 shows diverse DC phenotypes. Whereas some embryos of this genotype display defects as strong as those of kay 1 embryos, others have much milder phenotypes. These observations indicate that the hetero-allelic combination represents a sensitized experimental system that is well suited for genetic interaction experiments. If endogenous NRs transrepress AP-1 during DC, lowering their activity in a kay 1/kay 2 background should suppress the dorsal open phenotype. Reducing the gene dosage of kni or eg in this background does not alter the strength of the DC defects (Fig. 4D). Thus, these NRs do not appear to be prominently involved in AP-1 regulation in DC, possibly because their expression in the embryo is too low or absent. Strikingly, however, inactivating one copy of the svp gene in kay 1/kay 2 embryos strongly suppresses the DC defects (compare Fig. 4A,B, see also Fig. 4D). The dorsal hole is smaller and the overall morphology of double mutant embryos is more normal than in embryos with two wild type copies of the svp gene. This result implies that svp is involved in the regulation of AP-1 activity in DC. As the relevant genotypes show some phenotypic variability, we again tested our interpretations by quantitating the distribution of phenotypic strength for each genotype (using the criteria described above). Quantitative analysis confirms that mutating one copy of the svp gene strongly supresses the DC defects of kay 1/kay 2 embryos. These results prompted us to quantitate the viability of kay 2 homozygotes in various backgrounds. While under the experimental conditions employed here, kay 2 homozygosity is strictly lethal, flies homozygous for kay 2 and heterozygous for svp 1 are recovered at almost Mendelian ratios. Some of these flies display mild thorax closure phenotypes (data not shown) indicating that AP-1 activity is still compromised. The finding that reducing svp gene dosage by half rescues the lethality of kay 2 homozygotes is another clear indication of an antagonistic relationship between the two gene products. 2.5. Svp, Eg and Tll antagonize Fos in wing vein differentiation We next wanted to determine whether modulation of AP1 activity by NRs occurs only in situations where AP-1 is regulated by JNK or whether this type of regulation also operates in different contexts. We have recently demonstrated a function for Fos downstream of ERK in the differ-
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Fig. 4. Loss of svp suppresses the DC defects of kay mutants. DC defects of kay 1/kay 2 transheterozygotes in different genetic backgrounds. Cuticles are presented as in Fig. 1. (A) kay 1/kay 2 transheterozygote. The embryo carries a dorsal hole spanning several segments and the cuticle exhibits folds. (B) kay 1/kay 2 svp 1. The embryo has only a small dorso-anterior hole. (C) svp 1 homozygote. The embryo does not display any DC defects. Thus, modulation of AP-1 activity by zygotic Svp is not required for DC in a wild type background. (D) Quantitation of the distribution of phenotypic strengths, scoring criteria as for Fig. 2G. Embryos (3 £ 30) were scored for each genotype. Error bars indicate standard deviation.
entiation of wing veins (Ciapponi et al., 2001; reviewed in De Celis, 1998). 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 (Oellers and Hafen, 1996). 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 (Ciapponi et al., 2001). 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 (compare Fig. 5A,B). Reducing fos gene dosage in this system strongly suppresses the vein phenotype (Fig. 5C, Ciapponi et al., 2001), consistent with Fos’ proposed role 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, we removed one copy of kni, eg, tll or svp in 32B Gal4, UAS Sem flies. Reducing kni function does not influence the vein phenotype (Fig. 5G). However, heterozygosity for any of the other three receptors tested reproducibly led to a mild enhancement of the ectopic vein differentiation (see Fig. 5D,E,F). As an unambiguously scoreable criterion to statistically evaluate phenotypic effects, we chose the presence of ectopic vein material posterior to L5. 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 was suppressed by reducing fos activity, it was enhanced by a reduction of eg, svp or tll function (Fig. 5G). These data suggest that all three NRs antagonize AP-1 activity in wing vein differentiation, conceivably in a redundant manner. If indeed, the NRs act in a redundant manner to counteract AP-1 activity during wing development, severely impairing NR function might result in over-activation of AP-1 and consequentially the formation of ectopic vein material even in a wild type background. To test this idea, we generated clones of wing cells devoid of svp or tll function or doubly mutant for svp and tll, both genes with the potential to transrepress AP-1 as indicated by the previous experiments. svp as well as tll single mutant clones were recovered efficiently but showed no extra vein material (data not shown). Strikingly however, tll, svp double mutant clones exhibit significant formation of extra vein tissue (Fig. 5H, see arrowheads), a phenotype that is diagnostic of elevated AP-1 activity, at close to 100% penetrance. As expected, this phenotype is strictly cell autonomous, i.e. formation of extra vein material was only observed within the clone. The clonal analysis provides strong evidence that antagonism of AP-1 by NRs is essential for proper vein formation. Several NRs appear to act in concert to execute this function. 2.6. D-Fos can antagonize endogenous NR activity In mammalian cell culture systems, transrepression has been shown to be mutual. Not only can NRs repress AP-1mediated transcription, but conversely AP-1 can also repress NR function (Go¨ ttlicher et al., 1998; Jonat et al.,
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Fig. 5. Interplay between ERK signaling and NRs during wing vein patterning. Adult wing vein phenotypes caused by manipulations of AP-1 and NR activity are displayed. Arrows indicate ectopic vein material posterior to vein L5 in B–F. (A) Wild type. The wing shows the normal venation pattern. (B) 32B Gal4, UAS Sem. The wing displays ectopic vein material. (C) 32B Gal4, UAS Sem/kay 2. The amount of ectopic vein material is reduced compared to panel B. (D–F) Reducing NR gene dosage leads to an increase in ectopic vein formation compared to panel B (most clearly seen posterior to L5, see arrows). (G) Quantitation of the distribution of phenotypic strengths. Error bars indicate standard deviation. (H) svp 1 tll 1 double mutant clones. Patches of svp tll double mutant tissue display formation of ectopic vein tissue (see arrowheads, blowup).
1990; Saatcioglu et al., 1994; Schu¨ le et al., 1990; Yang-Yen et al., 1990). We wanted to determine whether transrepression in Drosophila is mutual as well. Viable combinations of eg alleles frequently exhibit a held-out wing phenotype (hence the name of the gene). As a consequence of the reduction of eg activity, the wings are not positioned parallel to the anterior–posterior body axis but are held at an angle (compare Fig. 6C to A). In the course of our studies of vein differentiation, we noticed that flies heterozygous for the eg null allele eg 18B and expressing Rl Sem (genotype eg18B/32B Gal4 UAS Sem) have held-out wings (Fig. 6E). This phenotype is not
displayed by either eg 18B heterozygotes (Fig. 6B) or 32B Gal4 UAS Sem flies (Fig. 6D). One potential explanation for this observation is that elevated ERK levels lead to an increase in AP-1 activity, which in turn reduces Eg activity below a critical threshold. To test this idea, we examined the effects of expressing full length Drosophila Fos under the control of the ubiquitous tubGal4 driver in eg heterozygotes. While tubGal4/UASfos flies are semi-viable at 18 and 258C, the combination tubGal4/ eg18B UASfos causes lethality at both temperatures. Thus, reducing eg function renders flies more sensitive to elevated AP-1 levels, consistent with the antagonistic relationship between eg and AP-1.
Fig. 6. Inhibitory interactions between Fos and Eagle. Held-out wing phenotypes in adult flies caused by manipulation of eg activity. (A) Wild type. The wings are aligned along the anterior–posterior body axis. (B) Flies heterozygous for the null allele eg 18B show normal wing posture. (C) Flies homozygous for eg MZ360 (a partial loss-of-function allele) display a classical held-out wing phenotype. (D) 32B Gal4 UAS Sem/1. Increasing ERK activity does not affect wing posture. (E) 32B Gal4 UAS Sem/eg 18B. A simultaneous increase in ERK activity and reduction of eg activity leads to a clear held-out wing phenotype. (F) tub Gal4 UASfosNterm/eg 18B Expressing the Fos N-terminal domain in flies with compromised eg function results in a mild held-out wing phenotype.
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However, because of the lethality, this combination cannot be used to ask whether fos can antagonize eg function in the control of wing posture. To circumvent this problem, we expressed a transactivation-incompetent form of Fos in eg heterozygotes. For a mammalian cell culture system, it has been reported that the N-terminal transactivation domain of c-Fos can antagonize NR-dependent transcription (Lucibello et al., 1990). We used an analogous approach in the fly. Expression of the N-terminal 150 amino acids of Fos (excluding the DNA-binding domain) with the tubGal4 driver has no effect on viability. However, expression of this construct in flies heterozygous for eg results in a mild but clear held-out wing phenotype and the affected individuals are unable to fly (Fig. 6F). Taken together, these experiments suggest that AP-1 can antagonize endogenous eg function. Importantly, the equilibrium of AP-1 and NR activities can be influenced by a transactivation-incompetent form of Fos. Thus, the antagonism is not mediated by the activation of opposing genetic programs but appears to be based on protein–protein interactions. 3. Discussion 3.1. Transrepression of AP-1 by NRs is conserved from Drosophila to mammals This paper presents several lines of evidence indicating that the unorthodox mechanism of AP-1 transrepression by NRs, as originally described in mammals, also exists in Drosophila. Using both, gain- and loss-of-function approaches, we demonstrated that Drosophila NRs can specifically antagonize AP-1 in two unrelated processes during Drosophila development and in mammalian cell culture reporter gene assays. Importantly, in all three experimental settings, several NRs with distinct DNA-binding sequences (Hoch et al., 1992) display the same activity. This observation strongly suggests that AP-1 antagonism is DNA-binding independent. Furthermore, the experiments on the control of wing posture show that a transactivation-incompetent form of Fos can suppress NR activity, implying protein–protein interactions as opposed to a process involving Fos target gene expression as the underlying mechanism. Taken together, these data show that AP-1 transrepression is a conserved activity of NRs from Drosophila to mammals. Thus, studying the mutual antagonism between AP-1 and NRs in Drosophila can help to improve our understanding of both the molecular mechanism of transrepression and the physiological relevance of this mode of AP-1 regulation (see below). 3.2. The physiological relevance of AP-1 transrepression by NRs in Drosophila While transrepression by the GR has been implicated in AP-1 regulation in a mouse skin carcinogenesis model (Tuckermann et al., 1999), the relevance of transrepres-
sional control of AP-1 under physiological conditions has not previously been demonstrated. Here, we present evidence that transrepression by NRs serves to modulate AP-1 activity during Drosophila development. Loss-offunction experiments demonstrate that Svp antagonizes AP-1 in DC. In wing vein differentiation, Fos activity is down-regulated by Svp, Tll and Eg. Our data suggest that the NRs exert these effects by transrepression. It has previously been shown that AP-1 activity is regulated by JNK in DC and by ERK in wing vein differentiation. Thus, our findings also provide strong support for the idea that several mechanisms act in parallel to control AP-1 activity in these developmental processes. 3.3. Combinatorial control of AP-1 activity in vivo The finding that MAPKs and NRs act in parallel to control AP-1 activity raises the question of what types of information are conveyed by the distinct mechanisms to ensure proper activation of AP-1. In the wing, the venation pattern is prefigured by the pattern of active EGF ligand (De Celis, 1998). Localized EGF pathway activation generates a pattern of ERK activation. Presumably, this pattern is translated into spatial differences of AP-1 activity, which in turn, govern vein positioning. Cells in which ERK activates AP-1 differentiate into vein tissue whereas cells in which ERK activity is not high enough to activate AP-1 adopt an intervein fate. Clearly, the regulation of AP-1 by ERK conveys external information. We speculate that the modulation of AP-1 activity by NRs contributes to what has recently been termed signal consolidation (Jordan et al., 2000). 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. We propose 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. The identification of Drosophila NRs as modulators of MAPK to AP-1 signaling assigns a novel developmental role to these proteins. This aspect of Svp or Tll function and a contribution of these proteins to DC or wing vein patterning had not previously been discovered. A possible reason for this could be that the role as AP-1 antagonist
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appears to shared by several NRs that act redundantly (at least in the wing). This is not the case for the better known functions of Tll, Svp and Eg, which presumably depend on their direct transcriptional targets e.g. in photoreceptor differentiation or embryo development. A multi-pronged approach based on Drosophila genetics in combination with tissue culture studies and biochemistry may shed light on the developmental function and the so far elusive mechanism of the phenomenon of NR-mediated transrepression of AP-1.
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senting the ratio of firefly luciferase activities and renilla luciferase activities were calculated. 4.3. Wing vein analysis Wings were dissected and mounted according to standard procedures. For quantitation purposes, 40 male wings of each genotype were scored. For the induction of clones in the wing flies of the genotype y w f hsflp; FRT82 svp 1/ FRT82; y w f hsflp; FRT82 tll 1/FRT82 or y w hsflp; FRT82 svp 1tll 1/FRT82 m were heat shock treated at 84 ^ 12 h after egg laying for 1 h at 388C.
4. Materials and methods Most fly strains used in this study were described previously: kay 1, kay 2 (Zeitlinger et al., 1997); hs-tll (Steingrimsson et al., 1991); tll 1 (Pignoni et al., 1990); svp 1 (Mlodzik et al., 1990); UASsvp (Kramer et al., 1995); kni 2 (Gerwin et al., 1994); UASkni, UASknrl (Lunde et al., 1998); hs-EcR (Lam and Thummel, 2000); hs-HR38 (Kozlova et al., 1998); hs-HR78 (Fisk and Thummel, 1998); hs-usp (Oro et al., 1992); eg 18B, eg MZ360, UASeg (Dittrich et al., 1997); tub Gal4 (Lee and Luo, 1999); pnr Gal4 (Calleja et al., 1996). UAS Sem and UAS fosNterm were constructed by cloning the corresponding coding sequences (the N-terminal 150 amino acids in the case of UAS fosNterm) into pCaSpeR3 and generating transgenic lines. 4.1. Cuticle preparations Embryos were collected for 24 h at 258C on apple juice agar plates unless stated otherwise. Then embryos were aged for 24 h or, if including exclusively embryonic lethals in the preparation was crucial, for approximately 32 h. Larvae were removed from the plates by scraping off the yeast and cuticle preparations were performed according to a standard protocol (van der Meer, 1977). For quantitation purposes, approximately 100 embryos of each genotype were scored. 4.2. Cell culture reporter gene assays HEK293 cells were transfected at 50–70% confluency by the calcium phosphate method (Gorman et al., 1982) with 1 mg of reporter plasmid–517 collagenase-Luc (kindly provided by A. Cato) together with either increasing amounts of CMV-tailless, CMV-eagle (the corresponding coding sequences were cloned into pcDNA3.1, Invitrogen) or empty expression vector. Fourteen to 16 h after transfection, cells were incubated with 100 ng/ml TPA (Sigma) or 0.1% dimethyl sulfoxide (DMSO) as a solvent control. Transfection efficiency was controlled by co-transfection with 10 ng ubiquitin promoter-driven Renilla luciferase reporter construct (kindly provided by M. Go¨ ttlicher). Twenty fours after treatment, cells were washed twice with phosphate-buffered saline (PBS) and lysed using passive lysis buffer (Promega). Normalized values repre-
4.4. Documentation of held-out wing phenotypes Held-out wing phenotypes are best scored in live flies. Anesthesia frequently causes abnormal wing positions even in wild type flies. For documentation purposes, flies were briefly anesthetized with CO2 and photographed upon ‘waking up’.
Acknowledgements We thank J. Lengyel, M. Mlodzik, Y. Hiromi, E. Bier, C. Thummel, B. Hall, J. Urban, G. Rubin, A. Cato, M. Go¨ ttlicher and the Umea and Bloomington Stock Centers for reagents. P. Rørth, F. Stewart, S. Cohen, M. Noble and members of the Bohmann lab have contributed helpful comments to the manuscript.
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