dorsal-related immunity factor

Protein Interactions

The interaction of Dorsal with Cactus is be discussed at the Cactus site. Interaction of Dif with Cactus has not been studied, but Dif is known to be rapidly translocated to the nucleus upon bacterial challenge.

Given that mammalian Rel proteins have repeatedly demonstrated their ability to form heterodimers, a test of the ability of Dorsal and Dif to heterodimerize was made. Using an immunological technique, Dorsal was seen to interact with Dif (Gross, 1996).

NF-kappaB/Rel family proteins regulate genes that are critical for many cellular processes including apoptosis, inflammation, immune response, and development. NF-kappaB/Rel proteins function as homodimers or heterodimers that recognize specific DNA sequences within target promoters. The activities of different Drosophila Rel-related proteins were examined in modulating Drosophila immunity genes by expressing the Rel proteins in stably transfected cell lines. How different combinations of these transcriptional regulators control the activity of various immunity genes were also compared. The results show that Rel proteins are directly involved in regulating the Drosophila antimicrobial response. drosomycin and defensin expression is best induced by the Relish/Dif and the Relish/Dorsal heterodimers, respectively, whereas the attacin activity can be efficiently up-regulated by the Relish homodimer and heterodimers. These results illustrate how the formation of Rel protein dimers differentially regulate target gene expression (Han, 1999).

These results demonstrate that all five immunity genes tested can be regulated by Rel-related proteins. Moreover, heterodimer formation can lead to an increased potential of gene regulatory activity by these factors. At least three pathways have been proposed to be involved in the transcriptional activation of Drosophila anti-microbial gene expression. drosomycin is largely regulated by the Toll/Cactus pathway, whereas attacin and cecropin are regulated by a pathway involving imd and 18wheeler. The third pathway involving imd is employed to regulate diptericin and drosocin. Further evidence indicates that the Drosophila immune system may involve additional regulatory molecules. One example is that attacin and cecropin respond to the p38 mitogen-activated protein kinase signaling pathway. It has also been shown that Dif and Dorsal can be activated independently, implying the presence of multiple signaling components that may lead to the activation of individual Rel proteins. The present results reveal another level of complexity in the regulatory mechanism. Despite some overlapping activations by other combinations, drosomycin expression is primarily activated by Dif/Relish heterodimer; defensin expression is primarily activated by Dorsal/Relish heterodimer. The attacin expression is primarily activated by Relish homodimer, and Dif/Relish and Dorsal/Relish heterodimers can perform the function during LPS stimulation of attacin. Therefore, although the numbers of Rel proteins are limited, they can mediate a broad range of cellular processes by using different combinations of these transcription factors (Han, 1999 and references).

Since these results show that different heterodimers have different preferences with regard to target gene regulation, it is possibile that the cell may regulate Relish/Dif and Relish/Dorsal through different pathways in order to achieve specific needs. This may explain reported observations showing that in different mutant flies the activation of Dif and Dorsal can be independently regulated and that different micro-organisms induce different subsets of antimicrobial peptides. Based on these results, it is also expected that Relish has a broader effect than either Dif or Dorsal on Drosophila immunity gene expression, since Relish is a common subunit of the heterodimers. In summary, it has been established that the differential regulation of Rel proteins can lead to preferential expression of specific target genes (Han, 1999).

The possible involvement of Toll or Toll-like proteins in the Drosophila immune response was investigated by overexpressing Toll10B, a constitutively active mutant protein, in the Drosophila blood cell line mbn-2. Induction of the Cecropin A1 (CecA1) gene, coding for a bactericidal peptide, was used as an indicator for the immune response. Toll10B increases CecA1 transcription. This effect depends on the presence of a kappa B-like site in the CecA1 promoter. The endogenous Toll gene is expressed in mbn-2 cells, indicating that this gene may normally play a role in Drosophila blood cells (Rosetto, 1995).

18-Wheeler (18W), is a critical component of the humoral immune response. 18-wheeler is expressed in the larval fat body, the primary organ of antimicrobial peptide synthesis. 18W is also detected in the lymph gland, garland cells and salivary glands. Transcript levels of 18w increase after bacterial infection and four novel forms of 18w transcripts are detected in addition to the expected 5.6 kb transcript, whose expression pattern has already been characterized. A 2.3 kb transcript is more rapidly induced than the others. Four novel 18W protein forms can be detected by Western analysis. In the absence of the 18W receptor, larvae are more susceptible to bacterial infection. Nuclear translocation of the Rel protein Dorsal-like immunity factor (Dif) is inhibited, though nuclear translocation of another Rel protein, Dorsal, is unaffected. Induction of several antibacterial genes is reduced following infection, relative to wild-type: attacin is reduced by 95%, cecropin by 65% and diptericin by 12% (Williams, 1997).

The Drosophila immune response uses many of the same components as the mammalian innate immune response, including signaling pathways that activate transcription factors of the Rel/NK-kappaB family. In response to infection, two Rel proteins, Dif and Dorsal, translocate from the cytoplasm to the nuclei of larval fat-body cells. The Toll signaling pathway, which controls dorsal-ventral patterning during Drosophila embryogenesis, regulates the nuclear import of Dorsal in the immune response, but the Toll pathway is not required for nuclear import of Dif. Dif is properly translocated from fat-body cytoplasm to nuclei in response to infection in Toll and pelle mutant larvae. Cytoplasmic retention of both Dorsal and Dif depends on Cactus protein; nuclear import of Dorsal and Dif is accompanied by degradation of Cactus. Therefore the two signaling pathways that target Cactus for degradation must discriminate between Cactus-Dorsal and Cactus-Dif complexes. New genes have been identified that are required for normal induction of transcription of antibacterial peptide diptericin during the immune response. Mutations in three of these genes prevent nuclear import of Dif in response to infection, and define new components of signalling pathways involving Rel. The 18-wheeler gene, which encodes transmembrane protein that is homologous to Toll, is important for the nuclear localization of Dif during the immune response, so two of these genes may encode products that are necessary for 18-wheeler activation or cytoplasmic components that act downstream of 18-wheeler. Mutations in three other genes, constituting a second class of mutations, cause constitutive nuclear localization of Dif; these mutations may block Rel protein activity by a novel mechanism. The gene immune deficiency (imd) belongs to this second class, as both Dif and Dorsal are constitutively nuclear in imd mutants. Cactus does not appear to be found in the nucleus in class II mutants. One hypothesis that fits these observations is that the class II genes are required to allow the formation of a nuclear complex of Dif with other proteins, and that this complex is required both for activation of diptericin transcription and for turnover of the nuclear Rel proteins (Wu, 1998).

Neither Toll nor cactus are strictly maternal-effect genes. While null mutations in Toll result in partial lethality, strong allelefat bodys of cactus are completely lethal at the end of the third instar larval stage. Both Dominant Toll and viable cactus mutations show a constitutive nuclear localization of Dorsal in the fat body but they also exhibit a melanotic tumor phenotype. Melanotic tumors are non-invasive and are generally thought to represent an immune-like response to invasive foreign or altered self substances. The melanotic tumor phenotype results from abnormalities in the differentiation of hemocytes. In normal larvae the hemocyte population consists almost entirely of small rounded cells referred to as plasmatocytes, which in the wild-type differentiate into large flat lamellocytes shortly after puparium formation. Lamellocytes play a centrol role in the encapsulation process of foreign bodies or of cell debris derived from tissue remodeling during metamorphosis. In Toll-Dominant mutants, up to 50% of all hemocytes are lamellocytes, whereas in the hemolymph of wild-type larvae these lamellocytes are not observed (Lemaitre, 1995b)

In Drosophila embryos, dorsal-ventral polarity is defined by a signal transduction pathway that regulates nuclear import of the Dorsal protein. Dorsal protein's ability to act as a transcriptional activator of some zygotic genes and a repressor of others defines structure along the dorsal-ventral axis. Dorsal is a member of a group of proteins, the Rel-homologous proteins, whose activity is regulated at the level of nuclear localization. Dif, a more recently identified Drosophila Rel-homolog, has been proposed to act as a mediator of the immune response in Drosophila. In an effort to understand the function and regulation of Rel-homologous proteins in Drosophila, Dif protein was expressed in Drosophila embryos derived from dorsal mutant mothers. Dif protein is capable of restoring embryonic dorsal-ventral pattern elements and is able to define polarity correctly with respect to the orientation of the egg shell. This, together with the observation that the ability of Dif to restore a dorsal-ventral axis depends on the signal transduction pathway that normally regulates Dorsal, suggests that Dif protein forms a nuclear concentration gradient similar to that seen for Dorsal. By studying the expression of Dorsal target genes it has been found that Dif can activate the zygotic genes that Dorsal activates and repress the genes repressed by Dorsal. Differences in the expression of these target genes, as well as the results from interaction studies carried out in yeast, suggest that Dif is not capable of synergizing with the basic helix-loop-helix transcription factors with which Dorsal normally interacts, and thereby lacks an important component of Dorsal-mediated pattern formation. Unlike Dorsal, Dif does not cooperate with Twist (Stein, 1998).

It is proposed that the Dorsal and Dif proteins differ in their abilities to physically interact with bHLH proteins in the control of transcription. Dorsal has been shown to be capable of interacting directly with the bHLH proteins Twist and Scute. Studies of the transcription of various Dorsal target genes indicate that synergy between Dorsal protein and bHLH proteins occurs in two distinct modes. In one mode, sharp on/off patterns of transcription in the mesoderm do not require that the Dorsal and bHLH binding sites on the promoter be adjacent. In such promoters (e.g. sna), synergy is thought to occur when Dorsal and Twist interact with different components of the transcriptional machinery. Although this mode of synergy would not require a physical interaction between Dorsal and Twist, direct interactions between Dorsal and Twist have been observed in vitro, and it is demonstrated here that Dorsal and Twist are capable of associating in yeast cells. Moreover, the same domains of Dorsal and Twist that mediate protein-protein interactions also cooperate in synergistic activation of transcription in transfected tissue culture cells and in a cell-free in vitro transcription system; this synergistic activation of transcription does not seem to involve cooperative binding to target DNA. Thus, while the mechanism by which the Dorsal/Twist interaction yields transcriptional activation is unclear, the data presented here, as well as that of others, indicate that this synergistic interaction requires direct protein-protein contact (Stein, 1998 and references).

The second mode of Dorsal/bHLH interaction requires that the Dorsal and bHLH binding sites be linked. In such cases, represented by the rho (and perhaps sim) promoter, cooperative binding of the promoter by Dorsal and bHLH proteins such as Scute results in promoter occupancy by Dorsal, even in lateral regions of the embryo where nuclear concentrations of Dorsal are low. The proposal that Dif is incapable of appropriately interacting with bHLH proteins provides an explanation for the ventral shift in rho and sim expression that is observed in Dif-expressing embryos. In the absence of cooperative interactions facilitating promoter occupancy by Dif in lateral regions of the embryo, expression would be limited to more ventral regions where there are sufficient concentrations of Dif to allow binding to low affinity sites. Taken together, these data suggest that one level of specificity in Rel homology protein action is defined by the protein-protein contacts that they are capable of forming with other transcription factors (Stein, 1998 and references).

The ability of Dif to restore a range of pattern elements along the DV axis, and to define an appropriately polarized gastrulation pattern, suggests that the Dif protein responds correctly to the signals that normally act to regulate Dorsal activity. Therefore, it is likely that in these embryos Dif, like Dorsal, forms a nuclear concentration gradient along the DV axis. Dorsal activity is exquisitely sensitive to the dosage of Cactus, which suggests that it might be possible to observe differences in the phenotypic rescue of dl - embryos expressing one copy of dif by reducing the cact gene copy number. Removing one copy of cact results in an overall relative ventralization. The majority of embryos from females heterozygous for cact exhibit denticle belts, while females carrying two copies of wild-type cact produced a minority of embryos with this level of cuticular rescue. These observations suggest that Cactus regulates the nuclear localization of Dif. The expression pattern of sim also provides molecular evidence for the increased ventralization of these embryos. Embryos from cact + females with only one copy of the dif transgene express sim only rarely and in patches. However, in embryos from females heterozygous for cact, sim expression is often observed in a continuous or discontinuous ventral stripe. Reduction of the cact gene dosage does not lead to detectable differences in the patterns of expression of the other genes examined. The presence of kappaB-type binding sites in the promoter regions of the cecropin and diptericin genes has led to the suggestion that controlled nuclear localization of an RH protein is involved in the activation of these genes. Its expression in the Drosophila fat body makes Dif a likely candidate to carry out this function. For this reason, an examination was carried out to determine whether maternally expressed Dif is capable of activating immune response genes in the embryo. cec-lacZ and dipt-lacZ reporter genes were crossed into the background of embryos expressing Dif. These transgenes carry the cecropin and diptericin promoters driving lacZ. They are expressed in the fat body in response to bacterial injection of larvae, presumably through Dif activation. If Dif alone is sufficient to activate these promoters in the embryo, one would expect to see a lacZ expression pattern similar to that seen for twi. In fact, no expression of lacZ from these reporter genes is observed in dl - embryos expressing Dif. Therefore, if Dif is involved in activating these promoters in vivo, additional factors that are not expressed in embryos may also be required (Stein, 1998).

Regulated Nuclear Import of Dif

Many developmental and physiological responses rely on the selective translocation of transcriptional regulators in and out of the nucleus through the nuclear pores. The Drosophila gene members only (mbo) encodes a nucleoporin homologous to the mammalian Nup88. mbo is associated with an enhancer trap insertion l(3)5043, identified in a screen for P-element strains that express the lacZ marker in distinct subsets of cells of the Drosophila tracheal system. The phenotypes of mbo mutants and mbo expression during development are cell specific, indicating that the nuclear import capacity of cells is differentially regulated. Using inducible assays for nucleocytoplasmic trafficking, it has been shown that mRNA export and classic NLS-mediated protein import are unaffected in mbo mutants. Instead, mbo is selectively required for the nuclear import of the yeast transcription factor GAL4 in a subset of the larval tissues. The first endogenous targets of the mbo nuclear import pathway have been found in the Rel proteins Dorsal and Dif. In mbo mutants the upstream signaling events leading to the degradation of the IB homolog Cactus are functional, but Dorsal and Dif remain cytoplasmic and the larval immune response is not activated in response to infection. These results demonstrate that distinct nuclear import events require different nucleoporins in vivo and suggest a regulatory role for mbo in signal transduction (Uv, 2000).

Homozygous embryos from the l(3)5043 P-element strain show sporadic but distinct defects in the connecting branches of the tracheal network. In this strain the transposon is inserted into the 5'-untranslated part of mbo, causing pupal lethality in homozygous animals. To investigate the function of mbo in the trachea, revertants and strong loss-of-function P-element excision mutants were generated and characterized. Several of the strains established are homozygous viable, and five strains, including mbo-1 and mbo-2, are homozygous lethal and fail to complement either one another, the original P-element mutation or a chromosomal deletion for the region (Uv, 2000).

During embryogenesis, 100 of the ~1600 cells of the tracheal epithelium mediate the connection of 20 individual metameres to a network that facilitates respiration during larval life. Every branch fusion event involves two cells, each located at the tip of the fusing branches. The fusion cells extend elaborate cytoplasmic processes, form an intracellular lumen, contact each other, and finally form a continuous bicellular anastomosis connecting the two branches. These cells selectively express a set of fusion marker genes, including l(3)5043 (mbo-lacZ). In mbo-1 embryos 20% of the dorsal anastomoses failed to form, and in mbo larvae the dorsal branches remain unconnected. The rest of the trachea, including the terminal branches that derive from cells not expressing the mbo-lacZ marker and that grow parallel to the fusion sprouts, are unaffected in mbo mutants. In addition, the expression of the early fusion cell marker esg-lacZ and other tracheal cell-specific markers is not altered in mbo mutant embryos, suggesting that mbo is required in the fusion cells subsequent to their cell fate specification. The fusion cells have another important role in mediating the breakage and release of old tracheal cuticle at each larval molt. The mbo-lacZ marker is expressed in the fusion cells throughout larval life, and all mbo larvae show discontinuities in the tracheal cuticle in positions corresponding to the fusion junctions of the dorsal trunks. Thus, the cell-specific zygotic expression of mbo-lacZ in the trachea reflects cell-specific requirements in the fusion cells. In the embryo, mbo-lacZ expression is also prominent in a subset of the cells of the developing CNS, in dynamic stripes of epidermal cells, in the lymph glands, and in the intestinal tract including the proventriculus and foregut. Because of the abundant distribution of maternally derived gene products during embryogenesis, however, significant differences in the abundance of RNA or protein could not be detected in these tissues (Uv, 2000).

In the larva, mbo-lacZ is expressed in the fat body, the trachea, the CNS, and the imaginal tissues. Abundant mbo RNA expression can be detected in the proliferating parts of the larval nerve cord, in the optic lobes of the brain, and in the imaginal disks. Accordingly, the size of the CNS and imaginal disks of third instar mbo mutant larvae is severely reduced. Because the antiserum against the carboxy-terminal part of Dnup88 is not sensitive enough to detect the protein by in situ staining in larvae, a second rabbit antiserum was generated against the amino-terminal part of Dnup88 and used to describe the distribution of larval Dnup88. In situ staining of wild-type larvae with this antiserum shows that the distribution of larval Dnup88 is tissue specific. Nuclear Dnup88 staining could be detected in the fat body, trachea, CNS, and imaginal disks but not in the epidermis, muscles, and gut. This tissue-specific staining is absent in mbo mutant larvae (Uv, 2000).

To identify endogenous targets of the Dnup88 import pathway, the translocation of Drosophila proteins that enter the nucleus upon signaling during developmental and physiological responses was examined. During early embryogenesis, the Drosophila NF-kappaB protein Dorsal is released from the IkappaB homolog Cactus in response to signaling from the Toll receptor and becomes nuclear on the ventral side of the embryo to activate transcription. The mbo gene product is maternally provided, since early embryos (0-90 min after egg laying) contain both Mbo mRNA and protein. To study the embryonic function of mbo in animals devoid of the maternal product, attempts were made to generate embryos from mbo homozygous germ-line clones. Such germ-line clones were, however, unable to produce eggs; and dissection of mosaic ovaries followed by DAPI staining revealed an early mbo function in oogenesis, because mutant ovarioles do not form an oocyte. In addition, eggs from mothers homozygous for two hypomorphic mbo alleles contain an increased amount of dorsal appendage material, resembling the mutant phenotypes of genes involved in determining the dorsoventral polarity of the oocyte. Because of the difficulty in generating embryos lacking maternal Dnup88, the functional analysis focussed on early third instar larvae that lack the zygotic mbo gene product and contain only small residual amounts of maternal Dnup88. The Dorsal signaling cascade is part of the activation of the larval immune response in the fat body and is induced upon challenging the larvae with a bacterial infection. The subcellular localization of Dorsal was examined in fat bodies of homozygous mbo larvae and their heterozygous siblings. When mbo heterozygous larvae are infected with a needle dipped in a bacterial culture, Dorsal becomes translocated into the nuclei of fat body cells within 45 min. In mbo mutants that receive the same treatment, Dorsal remains cytoplasmic. Dorsal enters the nuclei of the larval hematopoietic organ, the lymph gland, upon infection of wild-type animals. Also in this tissue, the nuclear translocation of Dorsal is severely impaired in mbo mutants (Uv, 2000).

Because the basic transport machinery across the nuclear pore appears functional in mbo mutants it was anticipated that Dnup88 might participate in a protein complex that facilitates nuclear translocation of Dorsal. Indeed, Dnup88 is bound by Dorsal, but the fraction of Dorsal protein found in complex with Dnup88 is smaller than the amount of Dorsal bound to Cactus. Thus, Dnup88 appears to participate directly in Dorsal nuclear import (Uv, 2000).

In response to a bacterial injection in wild-type larvae, nuclear translocation of the Rel proteins Dorsal and Dif is followed by the rapid transcriptional activation of genes encodingn antimicrobial peptides. Whether the inducible nuclear entry of Dif might also require mbo was examined. Like Dorsal, Dif is translocated into the fat body nuclei of mbo heterozygous larvae upon infection, and this translocation is impaired in mbo larvae. Accordingly, a reporter construct, composed of the inducible cecropin A promoter coupled to lacZ (cecA1-lacZ), is strongly induced in heterozygous larvae upon infection, whereas it is nonresponsive in mbo mutants). An analysis of the inducible expression of the genes for antimicrobial peptides Drosomycin and Diptericin by Northern blot hybridizations reveals that their induction is also severely impaired in mbo larvae. These results indicate that at least two of the identified fly Rel transcription factors require mbo for their nuclear entry and the concomitant activation of their target genes during the larval immune response (Uv, 2000).

The Drosophila atypical protein kinase C-ref(2)p complex constitutes a conserved module for signaling in the toll pathway

Recent results have demonstrated the critical role of the mammalian p62-atypical protein kinase C (aPKC) complex in the activation of NF-kappaB in response to different stimuli. Using the RNA interference technique on Schneider cells it has been shown that Drosophila aPKC (DaPKC) is required for the stimulation of the Toll-signaling pathway, which activates the NF-kappaB homologs Dif and Dorsal. However, DaPKC does not appear to be important for the other Drosophila NF-kappaB signaling cascade, which activates the NF-kappaB homolog Relish in response to lipopolysaccharides. Interestingly, DaPKC functions downstream of the nuclear translocation of Dorsal or Dif, controlling the transcriptional activity of the Drosomycin promoter. The Drosophila Ref(2)P protein is the homolog of mammalian p62, since it binds to DaPKC: its overexpression is sufficient to activate the Drosomycin but not the Attacin promoter, and its depletion severely impairs Toll signaling. Collectively, these results demonstrate the conservation of the p62-aPKC complex for the control of innate immunity signal transduction in Drosophila melanogaster (Avila, 2002).

Drosophila represents an ideal system in which to determine the primary role of the aPKCs in NF-kappaB signal transduction because it encodes only one aPKC isoform. According to the data presented in this study, aPKC is selectively required for the innate immune Toll-signaling pathway, acting downstream of the translocation of Dorsal and Dif and playing a critical role in the induction (a typical NF-kappaB-dependent process) of the antimicrobial peptide gene for Drosomycin. Therefore, it can be argued that the primary role of the aPKCs, particularly that of zetaPKC in higher eukaryotic cells, is to somehow control the transcriptional activity of NF-kappaB through a still not completely understood mechanism that most likely involves the direct phosphorylation of RelA and Dif. Interestingly, in Drosophila it is well documented that the phosphorylation of Dorsal is required not only for its transcriptional activity but also for its nuclear translocation. In Drosophila aPKC-depleted cells, a strong inhibition of Dorsal or Dif nuclear translocation is not observed, suggesting that the role of Drosophila aPKC is independent of the previously characterized role for Dorsal phosphorylation in regulating nuclear translocation. Based on experiments in mammalian systems, which demonstrate that p65 transcriptional activity must be stimulated by phosphorylation, it is possible that the residues that control the transcriptional activities of both Dorsal and Dif are different from those controlling the nuclear import of the protein. It is also possible that Drosophila aPKC-mediated phosphorylation has a subtle, yet important, role in the nuclear translocation of Dif and/or Dorsal. Future studies will address this important issue (Avila, 2002).

These studies also demonstrate that Ref(2)P is most likely the functional homolog of p62 in Drosophila. Like p62, Ref(2)P interacts physically with the aPKCs. Therefore, it appears that the p62-aPKC signaling module, like the Par/aPKC complex, is highly conserved. Importantly, a functional role of Ref(2)P in Toll signaling is demonstrated. Thus, the ectopic expression of Ref(2)P is capable by itself of activating the Drosomycin promoter. More interestingly, its depletion severely impairs the Toll pathway (Drosomycin induction) but not the LPS pathway (Attacin induction). Thus, the Ref(2)P/DaPKC complex is critical for Toll signaling (Avila, 2002).

The results presented here also demonstrate that, similar to the p62-TRAF6 connection in mammals, Ref(2)P and Drosophila TRAF2 physically and functionally interact. Together with the results demonstrating that Drosophila aPKC and Ref(2)P are essential for a downstream event in the Toll-signaling pathway, this suggests that a putative Ref(2)P/aPKC/TRAF2 complex might function in the signal-induced stimulation of Dif or Dorsal transcriptional activity. In this regard, it is noteworthy that recent results suggest that TRAF6, in addition to its role in IKK recruitment and activation, may also be involved in the control of RelA transcriptional activity. However, the role of Drosophila TRAF2 in Toll signaling requires further investigation, since the effect of inhibiting (or mutating) TRAF2 has not yet been reported. Further studies will also address the precise mechanism whereby aPKC controls the Toll pathway. The data presented here clearly establish the conserved role of the homolog of the p62/aPKC cassette in NF-kappaB signaling in Drosophila (Avila, 2002).

Traf2 functions upstream of Dif in the immune response

Two Drosophila tumor necrosis factor receptor-associated factors (TRAFs), Traf1 and Traf2, are proposed to have similar functions with their mammalian counterparts as a signal mediator of cell surface receptors. However, as yet their in vivo functions and related signaling pathways are not fully understood. Traf1 is shown to be an in vivo regulator of c-Jun N-terminal kinase (JNK) pathway in Drosophila. Ectopic expression of Traf1 in the developing eye induces apoptosis, thereby causing a rough-eye phenotype. Further genetic interaction analyses reveal that the apoptosis in the eye imaginal disc and the abnormal eye morphogenesis induced by Traf1 are dependent on JNK and its upstream kinases, Hep and TGF-ß activated kinase 1. In support of these results, the Traf1-null mutant shows a remarkable reduction in JNK activity with an impaired development of imaginal discs< and a defective formation of photosensory neuron arrays. In contrast, Traf2 was demonstrated as an upstream activator of nuclear factor-kappaB (NF-kappaB). Ectopic expression of Traf2 induces nuclear translocation of two Drosophila NF-kappaBs, Dif and Relish, consequently activating the transcription of the antimicrobial peptide genes diptericin, diptericin-like protein, and drosomycin. Consistently, the null mutant of Traf2 shows immune deficiencies in which NF-kappaB nuclear translocation and antimicrobial gene transcription against microbial infection were severely impaired. Collectively, these findings demonstrate that Traf1 and Traf2 play pivotal roles in Drosophila development and innate immunity by differentially regulating the JNK- and the NF-kappaB-dependent signaling pathway, respectively (Cha, 2003).

Microbial infection studies have demonstrated the ability of Drosophila to detect pathogens and activate specific signaling pathways, Toll or Imd pathways, which lead to adapted immune responses. In recent years, several families of antimicrobial peptides and their coding genes have been successfully identified: cecropins, attacins, diptericin, defensin, drosomycin, drosocin, and diptericin-like protein (dptlp). Understanding the molecular mechanisms underlying how microbial infection induces expression of these antimicrobial peptides has been the main question to answer in this field. Meanwhile, Traf2 have been identified as a downstream adaptor for Toll receptor (Shen, 2001) and Toll activation leads to immune responses. Therefore, it was suspected that DTRAFs would be involved in this defense mechanism (Cha, 2003).

Three representative antimicrobial genes, diptericin, dptlp, and drosomycin, were chosen as probes to determine the activity of the antimicrobial defense system. To examine whether Drosophila Traf1 and Traf2 have the ability to induce the transcription of diptericin, dptlp, and drosomycin, Traf1 or Traf2 was ectopically expressed in third-instar larvae by using hs-GAL4 driver, and the expression levels of diptericin, dptlp, and drosomycin were monitored by Northern blot analyses (Cha, 2003).

The transcription of diptericin, dptlp, and drosomycin was increased by ectopic expression of Traf2 in the absence of microbial infection. However, the expression levels of diptericin, dptlp, and drosomycin were not altered by Traf1 overexpression. In addition, Traf2-induced expression of diptericin and dptlp is completely inhibited in a relish (rel, Drosophila NF-kappaB)-null mutant background, whereas drosomycin expression is partially inhibited by the same mutation. The partial inhibition of the drosomycin expression by rel mutation suggests that the involvement of another Drosophila NF-kappaB, such as Dif, in antimicrobial response gene transcription. These results strongly suggest that Traf2, but not Traf1, functions downstream of microbial sensory receptors, Toll or Imd, and upstream of the NF-kappaBs to regulate Drosophila immune responses (Cha, 2003).

To further confirm the results, transgenic fly lines that have a GFP or a LacZ reporter gene fused to the drosomycin or the diptericin promoter, respectively, were used, allowing observation of the reporter gene activity, which reflects the drosomycin or diptericin gene expression level. The drosomycin-GFP reporter activity is dramatically increased in the microbe-infected larva compared to the uninfected control. As expected, Traf2 overexpression alone in the absence of microbial infection strongly induces drosomycin-GFP reporter gene activity. Further dissection analyses show that drosomycin-GFP and diptericin-LacZ reporter activities are highly induced in the fat body, which is a representative target tissue for immune responses in Drosophila. However, Traf1 overexpression fails to induce the reporter activities in both whole larvae and their fat bodies, further confirming the noninvolvement of Traf1 in the immune responses of Drosophila (Cha, 2003).

In order to confirm that the Traf2-induced immune responses are mediated by Dif and Relish, which are Drosophila NF-kappaBs specifically activated by Toll and Imd pathways, respectively, the subcellular localization of Dif and Relish was determened by using their specific antibodies (Cha, 2003).

Dif and Relish are dispersed in the cytoplasm of fat body cells in the absence of microbial infection. In contrast to this, either the microbial infection or overexpression of Traf2 fully induces the nuclear translocation of both Dif and Relish, demonstrating that both Dif and Relish participate in the Traf2-mediated immune responses. However, the subcellular localization of Dif and Relish is not altered by Traf1 induction, further confirming that Traf1 is not involved in the NF-kappaB signaling pathway. These data clearly demonstrated that Traf2, but not Traf1, has the capability to induce transcriptional activation of immune response genes by specifically activating NF-kappaBs (Cha, 2003).

The Traf2-null mutant, Traf2^ex1, was generated by P-element excision method. RT-PCR analysis shows that the homozygous Traf2^ex1 mutant fails to produce Traf2 mRNA. Intriguingly, the mutant flies manage to develop into adults and show no morphological defects. To determine whether the Traf2^ex1 mutant shows a deficiency in immune responses, the transcriptional induction level of diptericin and drosomycin was examined after microbial infection. The null mutation of Traf2 drastically disrupts the transcriptional induction of diptericin and drosomycin when compared to the wild-type control. However, Traf1-null mutation (Traf1^ex1) has no effect on the induction of diptericin and drosomycin gene expression after microbial infection. The nuclear translocation of Dif and Relish was examined in the Traf2-null mutant. Consistent with Northern blot analysis, the nuclear translocation of Dif and Relish induced by microbial infections is impaired in the Traf2^ex1 mutant. These results support the position that Traf2, but not Traf1, is critical for the NF-kappaB-mediated Drosophila innate immune responses (Cha, 2003).

In the mammalian system, when interleukin-1 (IL-1) receptor, a Toll-like receptor, is stimulated by binding of its ligand, IL-1 receptor associated kinase (IRAK) is recruited to the IL-1 receptor complex and phosphorylated. Consequently, the receptor associated IRAK (Drosophila homolog: Pelle) binds to TRAF6, which evokes a strong activation of the NF-kappaB signaling pathway. The importance of TRAF6 in the activation of this pathway has been confirmed by various experiments. For example, overexpression of TRAF6 can lead to NF-kappaB activation, and a dominant-negative mutant of TRAF6 inhibits IL-1-induced NF-kappaB activation (Cha, 2003).

Between the two Drosophila homologs of mammalian TRAFs, the TRAF domain of Traf2 is most closely related to that of mammalian TRAF6. Based on this structural similarity, there have been reports that Traf2 contributes to dorsal activation and immune responses by activating NF-kappaB in a cell culture system (Kopp, 1999; Shen, 2001). Thus, in agreement with these results, it has been demonstrated that Traf2 can activate Drosophila NF-kappaBs and their downstream target genes diptericin, dptlp and drosomycin. Also, it has been suggested that Traf1 is involved in the NF-kappaB-mediated immune response (Zapata, 2000). However, in the present study, Traf1 does not induce NF-kappaB activation and the consequent NF-kappaB-dependent immune responses in vivo. These data suggest that Traf2 is a highly specific signal mediator activating the NF-kappaB signaling pathway (Cha, 2003).

Although overexpression of Traf2 is sufficient to activate the NF-kappaB signaling pathway and induce innate immune responses, the Traf2-null mutation could not completely block the processes. This suggests the presence of other signaling pathway(s) that bypass Traf2 to transmit the exogenous microbial signals to NF-kappaBs. Further studies with the Traf2-null mutant are required to elucidate the unknown signaling mechanism (Cha, 2003).

Inhibitor kappaB-like proteins from a polydnavirus inhibit NF-kappaB activation and suppress the insect immune response

Complex signaling pathways regulate the innate immune system of insects, with NF-kappaB transcription factors playing a central role in the activation of antimicrobial peptides and other immune genes. Although numerous studies have characterized the immune responses of insects to pathogens, comparatively little is known about the counter-strategies pathogens have evolved to circumvent host defenses. Among the most potent immunosuppressive pathogens of insects are polydnaviruses that are symbiotically associated with parasitoid wasps. This study reports that the Microplitis demolitor bracovirus encodes a family of genes with homology to inhibitor kappaB (IkappaB) proteins from insects and mammals. Functional analysis of two of these genes, H4 and N5, were conducted in Drosophila S2 cells. Recombinant H4 and N5 greatly reduce the expression of drosomycin and attacin reporter constructs, which are under NF-kappaB regulation through the Toll and Imd pathways. Coimmunoprecipitation experiments indicated that H4 and N5 bind to the Rel proteins Dif and Relish, and N5 also weakly binds to Dorsal. H4 and N5 also inhibit binding of Dif and Relish to kappaB sites in the promoters of the drosomycin and cecropin A1 genes. Collectively, these results indicate that H4 and N5 function as IkappaBs and, circumstantially, suggest that other IkappaB-like gene family members are involved in the suppression of the insect immune system (Thoetkiattikul, 2005).

Dorsal interacting protein 3 potentiates activation by Drosophila Rel homology domain proteins

Dorsal interacting protein 3 (Dip3) contains a MADF DNA-binding domain and a BESS protein interaction domain. The Dip3 BESS domain was previously shown to bind to the Dorsal (DL) Rel homology domain. This study shows that Dip3 also binds to the Relish Rel homology domain and enhances Rel family transcription factor function in both dorsoventral patterning and the immune response. While Dip3 is not essential, Dip3 mutations enhance the embryonic patterning defects that result from dorsal haplo-insufficiency, indicating that Dip3 may render dorsoventral patterning more robust. Dip3 is also required for optimal resistance to immune challenge since Dip3 mutant adults and larvae infected with bacteria have shortened lifetimes relative to infected wild-type flies. Furthermore, the mutant larvae exhibit significantly reduced expression of antimicrobial defense genes. Chromatin immunoprecipitation experiments in S2 cells indicate the presence of Dip3 at the promoters of these genes, and this binding requires the presence of Rel proteins at these promoters (Ratnaparkhi, 2009).

The Drosophila genome encodes three rel homology domain (RHD) containing proteins, Dorsal (Dl), Dorsal-related immunity factor (Dif), and Relish (Rel). The RHD, which is also found in the human NFκB family of transcriptional activators, mediates dimerization and sequence-specific DNA binding. Rel/NFκB family proteins in vertebrates and invertebrates play central roles in the innate immune response by triggering the expression of antimicrobial defense genes in response to signals transduced by Toll and the Immune deficiency (Imd) signal transduction pathways. In Drosophila, Dl also directs dorsoventral (D/V) patterning of the embryo. Specifically, the regulated nuclear localization of maternally expressed Dl in response to Toll signaling in the embryo leads to the formation of a ventral-to-dorsal nuclear concentration gradient of Dl and to the spatially restricted regulation of a large number of genes, including twist (twi), snail (sna), and rhomboid (rho), which are activated by Dl, and zerknullt and decapentaplegic, which are repressed by Dl. This serves to subdivide the embryo into multiple developmental domains along its D/V axis (Ratnaparkhi, 2009).

Unlike Dl, Dif and Rel are not required for D/V patterning. Instead, these two rel-family proteins function along with Dl in the innate immune response. Toll signaling in the immune system leads to the translocation of Dl and Dif to the nucleus and the consequent activation of a subset of anti-microbial defense genes, including drosomycin (drs) and Immune induced molecule 1. Dl and Dif are believed to have redundant roles in this process and thus either one alone is sufficient for the induction of drs. Activation of the Imd signal transduction pathway, leads to proteolytic cleavage of Rel. The N-terminal region of Rel, which contains the RHD, then translocates into the nucleus where it activates expression of anti-bacterial genes, such as diptericin (dipt), cecropin-A1 (cec-A), and attacin-A. Dl, Dif, and Rel homo- and hetero-dimerize to activate different subsets of the anti-microbial defense genes in response to signals from the Toll and Imd pathways (Ratnaparkhi, 2009).

Very little is known about the identity of factors that assist the RHD proteins in the activation of the anti-microbial defense genes. Proteins that modulate expression of these genes include transcription factors such as the GATA factor Serpent (Srp), Hox factors, Helicase89B, and an unknown protein that binds region 1 (R1), a regulatory module in cec-A and other anti-microbial defense genes. In addition, a recent screen identified several POU domain proteins as potential regulators of anti-microbial defense genes (Ratnaparkhi, 2009).

To date, about a dozen proteins that interact directly with Dl and modulate its regulatory functions have been identified by genetic and biochemical means. For example, an interaction between Dl and Twist (Twi) enhances the activation of Dl target genes, while an interaction between Dl and Groucho (Gro) is essential for Dl-mediated repression. A yeast two-hybrid screen to identify Dl interacting proteins yielded, in addition to the well characterized Dl-interactors Twi and Cactus, four novel Dl-interactors (Dip1, Dip2, Dip3, and Dip4/Ubc9). Conjugation of SUMO to Dl by Ubc9 was subsequently shown to result in more potent activation by Dl (Ratnaparkhi, 2009).

Dip3 belongs to a family of proteins that contain both MADF (for Myb/SANT-like in ADF) and BESS (for BEAF, Stonewall, SuVar(3)7-like) domains. While MADF-BESS domain proteins are found in both insects and vertebrates, only a few have been characterized and their functions are largely unknown. The Drosophila genome encodes 14 MADF-BESS domain factors. In addition to Dip3, these include Adf-1, which was initially found as an activator of Alcohol dehydrogenase, and Stonewall, which is required for oogenesis. The Dip3 MADF domain mediates sequence specific binding to DNA, while the Dip3 BESS domain mediates binding to a subset of TATA binding protein associated factors as well as to the Dl RHD and to Twi. In addition to functioning as an activator, Dip3 can function as a coactivator to stimulate synergistic activation by Dl and Twi in S2 cells (Ratnaparkhi, 2009).

This study shows that Dip3 assists RHD proteins during both embryonic development and the innate immune response. By stimulating the expression of antimicrobial defense genes, Dip3 improves survival of both larvae and adults following septic injury. The presence of Dip3 near the promoters of antimicrobial defense genes depends upon Rel family proteins suggesting that Dip3 functions as a coactivator at these promoters (Ratnaparkhi, 2009).

It has been shown that Dip3, which binds both Dl and Twi via its BESS domain, synergistically enhances the activation of a luciferase reporter with multiple Dl and Twi binding sites upstream of the promoter. In addition, Dip3 has been implicated as the 'mystery protein' which binds to sites adjacent to Dl and Twi binding sites in a subset of Dl target genes. Therefore the ability of Dip3 to enhance the expression of the Dl target promoters twi, sna, and rho in S2 cell transient transfection assays was examined. All three promoters require both Dl and Twi for full activity. Dip3 was found to synergize with Dl and Twi in the activation of the sna and twi promoters, but not in the activation of the rho promoter (Ratnaparkhi, 2009).

A polyclonal antibody against recombinant Dip3 was generated, and used to determine where and when Dip3 is present in the embryo. Maternally expressed Dip3 is observed in all nuclei as early as nuclear cycle 7. It was detected in subsequent nuclear cycles during formation of the Dl nuclear concentration gradient. In interphase embryonic as well as S2 cell nuclei, Dip3 localizes to nuclear speckles of unknown identity. During mitosis Dip3 is enriched on chromosomes. It associates with the centrosome proximal portion of the anaphase chromatids and the inside ring of the polar body rosette suggesting a predominant pericentromeric location at this stage of the cell cycle and hinting at a possible role of Dip3 in centromeric function. Confirming the specificity of the antibodies, the immunoreactivity is absent from Dip3¹ embryos in which the Dip3 transcriptional and translational start sites as well as a large segment of the Dip3 coding region have been deleted. Weak Dip3 expression is also detected in the fat body (Ratnaparkhi, 2009).

Homozygous Dip3¹ flies are viable and fertile, indicating that Dip3 cannot have an essential role in embryonic D/V pattern formation. However, a small proportion (7±4%) of the embryos fail to hatch and exhibit D/V patterning defects. Embryos produced by females transheterozygous for Dip3¹ and a deficiency that removes a portion of the second chromosome containing the Dip3 gene (Df(PC4) exhibit similar embryonic lethality (10%) and D/V patterning defects. Also, maternal overexpression of Dip3 using the Gal4-UAS system leads to 54±9 % embryonic lethality with cuticles of the dead embryos showing both anteroposterior and D/V patterning defects, indicating that Dip3 may have a role in embryonic pattern formation (Ratnaparkhi, 2009).

Consistent with a non-essential role for Dip3 in D/V patterning, a Dip3 mutation enhances the temperature sensitive dl haploinsufficieny phenotype. The degree of dorsalization is often quantified by categorizing embryos on a scale from D0 (completely dorsalized, lacking all dorsoventral pattern elements other than dorsal epidermis) to D3 (inviable, but with little or no apparent defect in the cuticular pattern). At 29°, about half the dead embryos produced by dl¹/+ females exhibit detectable D/V patterning defects and the majority of these fall into the D2 category (moderately dorsalized, exhibiting mildly expanded ventral denticle belts and a twisted germ band). Removal of maternal Dip3 increases the proportion of dorsalized embryos to about 75% with most of the increase being due to an increase in the number of D2 embryos. The effect seems to be strictly maternal as the paternal genotype does not modulate the dl haploinsufficiency phenotype (Ratnaparkhi, 2009).

Dip3 is present in the fat body, the organ in which RHD factors activate antimicrobial defense genes in response to infection. Since Dip3 binds the Dl RHD, the role of Dip3 in the innate immune response was examined by assessing the sensitivity of Dip3¹ flies to bacterial and fungal infection. Wild-type and Dip3¹ adults and larvae were injected with gram positive bacteria (M. luteus), gram negative bacteria (E. coli), and fungi (B. brassiana). For comparison, flies were infected that contained mutations in known components of the Toll (spz^rm7) and Imd (Rel^E20) pathways. Wild-type, Rel^E20, spz^rm7, and Dip3¹ adults showed little lethality (<15%) 30 days after mock infection. However, the Dip3¹ adult flies exhibited 55% lethality one month after injection with a 1:1 mixture of M. luteus and E. coli, compared to 10% lethality after 30 days for wild-type flies and 98% after 30 days for Rel^E20 flies. In contrast, wild-type and Dip3¹ adults were equally sensitive to fungal infection, both showing 55-70% lethality after 30 days compared to 100% lethality after 22 days for Rel^E20 adults and 100% lethality after 7 days for spz^rm7 adults. Similar results were seen in larvae in which Dip3¹, Rel^E20 and spz^rm7 mutations resulted in reduced rates of eclosion following septic injury compared to wild-type. The effectiveness of the immune challenge was further verified by an experiment showing that septic injury leads to translocation of Dl into the nucleus (Ratnaparkhi, 2009).

To determine if the sensitivity of Dip3¹ flies to infection results from reduced induction of antimicrobial peptides, the expression of dipt, drs and cec-A was monitored as a function of time following septic injury. Relative to uninfected flies, the levels of expression of drs and dipt were reduced by the Dip3¹ mutation, especially at the 2 and 4 hr time points, while the levels of cec-A expression were not significantly altered. Thus, some, but not all, antimicrobial defense genes that are regulated by RHD family proteins exhibit dependence on Dip3. At the 4 hr time point, relative to infected, wild type flies, the spz^rm7 mutation reduced drs expression to basal levels while the Rel^E20 mutation reduced dipt expression ten fold (Ratnaparkhi, 2009).

Dip3 was over expressed in the larvae using the Cg-Gal4 driver to examine the effect of increasing levels of Dip3 on the expression of antimicrobial defense genes in the fat body. Cec-A and drs levels were unaffected, while dipt levels increased two-fold in infected flies. Thus, both loss-of-function and over expression data are consistent with the conclusion that Dip3 makes the immune response more robust by elevating the expression of a subset of antimicrobial defense genes (Ratnaparkhi, 2009).

Radiolabeled Dip3 interacts with FLAG-tagged Dl and Rel immobilized on anti-FLAG beads. Similarly, immobilized FLAG-Dip3 binds Dl (Bhaskar, 2002) and Rel (Residues 1-600). Dip3 binds to DNA via its MADF domain and to the RHD via its BESS domain, and can thus function either as an activator or as a coactivator (Bhaskar, 2002). To determine if Dip3 is present at the promoters of antimicrobial defense genes, ChIP assays were carried out in S2 cells transfected with FLAG-Dip3. FLAG antibody was used to immunoprecipitate Dip3 crosslinked to chromatin. Compared both to mock-transfected cells and to the transcribed region of a ribosomal protein-encoding gene (rp49), Dip3 was highly enriched at the drs, dipt and cecA promoters. As expected, dsRNA directed against Dip3 eliminated the ChIP signal verifying antibody specificity. The association of Dip3 with the promoters of the anti-microbial defense genes depended on Rel family proteins, since knockdown of these proteins by dsRNAi significantly reduced association of Dip3 with the promoters. Similar results were observed with an anti-GFP antibody and cells expressing a Dip3-GFP fusion protein (Ratnaparkhi, 2009).

These results suggest that Dip3 may synergize with RHD proteins in multiple developmental contexts possibly through contact with the Dl rel homology domain. Dip3 is expressed maternally and present in cleavage stage nuclei at the time that Dl is functioning to pattern the D/V axis. Furthermore, Dip3 can potentiate Dl-mediated activation of the twist and snail promoters in S2 cells. These observations suggest that Dip3 might have a role in D/V patterning. Consistent with this possibility, it was found that removal of maternal Dip3 results in occasional D/V patterning defects and significantly enhances the dl haploinsufficiency phenotype suggesting the Dip3 renders D/V patterning more robust perhaps by assisting in Dl-mediated activation (Ratnaparkhi, 2009).

An important aspect of the immune response is activation in the fat body of genes encoding antimicrobial peptides by the Rel family transcription factors Dl, Dif, and Rel. This study found that synergistic killing of flies by a mixture of E.coli and M. luteus is enhanced in Dip3¹ flies. This suggests roles for Dip3 in the Imd and/or Toll pathways, which mediate the response to microbial infection. In accord with this idea, it was found that activation of the Imd pathway target dipt and the Toll pathway target drs are compromised in Dip3 mutant larvae (Ratnaparkhi, 2009).

To determine if the role of Dip3 at antimicrobial defense gene promoters is direct, ChIP assays were carried out demonstrating that this factor associates directly with the drs, dipt, and cec-A promoters in S2 cells. Since Dip3 contains a DNA binding domain, it is possible that it binds to these promoters through a direct interaction with DNA. However, with one exception in the drs promoter, these promoters lack matches for the consensus Dip3 binding sites. Thus, Dip3 may be acting as a coactivator at these promoters consistent with its ability to bind the rel homology domain. In support of this idea, it was found that simultaneous knockdown of all three rel family proteins significantly reduced recruitment of Dip3 to the promoters (Ratnaparkhi, 2009).

The mechanism of Dip3 co-activation remains unclear. The finding that the Dip3 BESS domain binds TAFs (Bhaskar, 2002) suggests a role for Dip3 in the recruitment of the basal machinery. In addition, the MADF domain is closely related to the SANT domain, which binds histone tails and may have a role in interpreting the histone code. While analysis of RHD targets suggests roles for Dip3 in activation, Dip3 also associates with pericentromeric heterochromatin during mitosis, consistent with a possible role in silencing. Other heterochromatic proteins including a suppressor of position effect variegation (Su(Var)3-7) also contain BESS domains. However, the loss of Dip3 does not appear to modify position effect variegation (Ratnaparkhi, 2009).

In flies, additional roles for RHD-mediated activation have been demonstrated in haematopoesis, neural fate specification, and glutamate receptor expression. Antimicrobial defense genes are also expressed constitutively in barrier epithelia and in the male and female reproductive tracts. It will be interesting to determine if Dip3 is involved in rel protein-dependent and independent gene activation in some or all of these tissues. One tissue in which Dip3 appears to have clear rel-independent functions is in the developing compound eye, where Dip3 overexpression results in conversion of eye to antenna, while Dip3 loss-of-function leads to mispatterning of the retina (Ratnaparkhi, 2009 and references therein).

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