dorsal-related immunity factor


Transcriptional Regulation

The Toll10b allele has been shown to encode a mutated form of the Toll receptor that is constitutively activated. This causes Dorsal protein, in both dorsal and ventral regions, to enter nuclei. In Toll10b/+ heterozygotes, only a fraction of the total DIF protein is localized in the nuclei of fat bodies. Toll10b/+ fat bodies display about a 5-fold increase in staining intensity, as compared with normal tissue, with a simular increase in DIF mRNA. This suggests that DIF might influence its own expression (Ip, 1993).

Targets of Activity

The upstream sequences of the Diptericin and Cecropin Al genes, which have been investigated in detail, contain, respectively, two and one sequence elements homologous to the binding site of the mammalian nuclear factor kappaB. These elements are mandatory for the immune-induced transcription of both genes. Functional studies have shown that these kappaB-related elements can be the target for the Drosophila Rel proteins Dorsal and Dif. A comparative analysis of the transactivating capacities of these proteins has been performed on reporter genes fused to either the Diptericin or the Cecropin kappaB-related motifs. The kappaB motifs of the Diptericin and Cecropin genes are not functionally equivalent and complexes formed with kappaB-Dipt and kappaB-Cec motifs have different protein compositions. Dorsal and Dif proteins manifest distinct DNA-binding characteristics. Dif can bind to any kappaB-related motif of either Zerknüllt, Diptericin or Cecropin. A single motif is sufficient for binding; double copies (2x kappaB-Dipt) yield a stronger signal. In contrast, Dorsal, which binds strongly to the motif present in the zen promoter, does not detectably bind to either the kappaB-related motif of the Cecropin or the Diptericin promoter. Dorsal can bind only to the kappaB-Dipt site if this motif is duplicated, as is the case in the native diptericin promoter. Even under these circumstances, the binding is not as marked as for Dif. Mutants containing no copies of Dorsal and a single copy of Dif retain their full capacity to express the Diptericin and Decropin genes in response to challenge (Gross, 1996).

There are a number of different controls on the expression of the antifungal polypeptide gene Drosomycin in adults: the receptor Toll, intracellular components of the dorsoventral signaling pathway (Tube, Pelle, and Cactus), and the extracellular Toll ligand, Spätzle, but not the NF-kappaB related transcription factor Dorsal. Mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. In Tl-deficient adults, the Cecropin A and, to a lesser extent, Attacin, Drosomycin and Defensin genes are only minimally inducible, in contrast with the Diptericin and Drosocin genes, which remain fully inducible in this context. The Drosomycin gene induction is not affected in mutants deficient in gastrulation defective, snake and easter, all upstream of spätzle in the dorsoventral pathway. The involvement of Spätzle in the drosomycin induction pathway is unexpected, since, in contrast with cat, pll, tub, and Tl, the spz mutant shows no striking zygotic phenotype. The partner of Cact in the Drosomycin induction pathway has not yet been identified, but it is probably a member of the Rel family, possibly Dif (Lemaitre, 1996).

Dif gene product trans-activates the Drosophila Cecropin A1 gene in co-transfection assays. The transactivation requires a 40 bp upstream element, including an insect kappa B-like motif. A dimer of the kappa B-like motif 5'-GGGGATTTTT inserted into a minimal promoter confers high levels of reporter gene expression by Dif; a multimer of several mutated versions of this motif is not activated, demonstrating the sequence specificity of Dif. Full trans-activation by Dif requires the C-terminal part of the protein. The morphogen Dorsal can also activate the Cecropin A1 promoter, but to a lesser extent and in a less sequence-specific manner than Dif. Simultaneous overexpression of Dif and DL in co-transfection assays reveals that DL possesses a dominant negative effect on Dif transactivation (Peterson, 1995).

NF-kappa B-like motifs have a regulatory role in the synthesis of cecropins, a set of anti-bacterial peptides, triggered by the presence of bacterial cell wall components in the insect blood. The upstream region of the Cecropin gene CecA1 contains elements responsible for inducible and tissue-specific expression. A trimer of kappa B-like motif confers high levels of inducible expression from a reporter gene, after transfection in a Drosophila blood cell line. As in the moth Hyalophora cecropia, stimulation with bacterial lipopolysaccharide induces a nuclear factor that specifically binds to the kappa B-like motif. These data suggest a functional and evolutionary relationship between these insect immune response factors and the mammalian NF-kappa B (Engstrom, 1993).

In Drosophila, three Cecropin genes have been characterized: CecA1, CecA2, and CecB, all in a dense cluster at 99E on the third chromosome. From the same locus, a fourth member of the cecropin gene family, CecC, has been isolated; it is mainly expressed at the early pupal stage. CecC is induced in the anterior end of the larval hindgut and in other larval tissues that are undergoing histolysis. Within these other tissues it is often expressed in distinct foci that may correspond to hemocytes. A similar pattern of expression in the metamorphosing pupa is also observed for the CecA and CecB genes. Comparing the DNA sequences of the cecropin genes, a conserved region is observed about 30 bp upstream of the TATA box. It consists of three shorter motifs, two of which are reminiscent of a putative promoter element in immune protein genes from the cecropia moth (Tryselius, 1992).

The GATA motif is a well known positive cis-regulatory element in vertebrates. Experimental evidence is provided for the direct participation of a GATA motif in the expression of the Drosophila antibacterial peptide gene Cecropin A1. A kappaB-like site is necessary for Cecropin A1 gene expression. The Drosophila Rel protein which binds to the kappaB-like site, requires an intact GATA site for maximal Dif-mediated transactivation of the Cecropin A1 gene. A Drosophila blood cell line contains factors binding specifically to the GATA motif of the Cecropin A1 gene. The GATA binding activity is likely to include member(s) of the GATA family of transcriptional regulators. The promoters of several inducible insect immune genes possess GATA sites 0-12 base pairs away from kappaB-like sites in functionally important promoter regions. The serpent gene is expressed both in fat body and hemocytes, and embryos mutatnt for srp lack mature fat body and hemocytes. Like the srp gene, the Cec genes are also expressed in fat body and hemocytes. The overlapping expression pattern of srp and Cec genes makes serpent an interesting candidate for the GATA-binding activity. Clusters of GATA and kappaB sites are also observed in the promoters of two important mammalian immune genes: IL6 and IL3. The consistent proximity of GATA and kappaB sites appears to be a common theme in the immune gene expression of insects and mammals (Kadalayil, 1997).

The Drosophila cell line mbn-2 is of blood cell origin, derived from larval hemocytes of the mutant lethal (2) malignant blood neoplasm (l[2]mbn). The mbn-2 cells respond to microbial substances by the activation of cecropin genes, coding for bactericidal peptides. The response here is stronger than that found in other Drosophila cell lines, including four that were totally unresponsive. Bacterial lipopolysaccharide, algal laminarin (a beta-1,3-glucan), and bacterial flagellin are strong inducers, while bacterial peptidoglycan fragments give a weaker response. Experiments with different drugs indicate that the response may be mediated by a G protein, but not by protein kinase C or eicosanoids, and that it requires a protein factor with a high rate of turnover (Samakovlis, 1992).

DNaseI footprinting experiments were performed on the Diptericin gene combined with gel-shift assays in two inducible systems: the larval fat body and a tumorous Drosophila blood cell line. These results confirm the importance of kappa B-like elements in the immune response of insects and reveal for the first time the involvement of other regions containing sequences homologous to mammalian acute-phase response elements (Georgel, 1993 and Hoffmann, 1993).

Diptericins are 9 kDa inducible antibacterial peptides initially isolated from immune hemolymph of Phormia (Diptera). The Diptericin gene is inducible by injection of live bacteria or complete Freund's adjuvant and respects the tissue specific expression pattern of the resident diptericin gene. There are at least four distinct phases in the regulation of this gene: young larvae, late third instar larvae, pupae and adults. This complexity may be related to the presence in the upstream sequences of multiple copies of response elements previously characterized in genes encoding acute phase response proteins in mammals (e.g. NK-kappa B, NF-kappa B related, NF-IL6 response elements) (Reichhart, 1992).

Insect defensins are a family of 4-kDa, cationic, inducible antibacterial peptides which bear six cysteine residues engaged in three intramolecular disulfide bridges. They owe their name to certain sequence similarities with defensins from mammalian neutrophiles and macrophages. A novel Defensin isoform in Drosophila encoding a preprodefensin is intronless and present in a single copy/haploid genome; it maps at position 46CD on the right arm of the second chromosome. The upstream region of the gene contains multiple putative cis-regulatory sequences similar to mammalian regulatory motifs of acute-phase-response genes. Transcriptional profiles indicate that the Drosophila Defensin gene is induced by bacterial challenge with acute-phase kinetics. It is also expressed in the absence of immune challenge during metamorphosis. It is likely that insect and mammalian defensins have evolved independently (Dimarcq, 1994).

In Drosophila, a septic wound induces the rapid appearance in the hemolymph of a battery of antibacterial peptides that includes the Cecropins, Drosocin, insect Defensin, Metchnikowin, Attacin and one major antifungal peptide, Drosomycin. These peptides are synthesized mostly in the fat body, a functional equivalent of the liver, and secreted into the hemolymph. This reaction constitutes a systemic antimicrobial response. Since experimental wounds are restricted to a single point of entry and since all of the disseminated fat body is responding to the attack, it is thought that a signal is transmitted to the fat body through the hemolymph from the entry site of microorganisms, where non-self recognition presumably occurs. This study asked whether antimicrobial peptides are also expressed in barrier epithelia in Drosophila, independent of a systemic response. This question was specifically addressed regarding the expression of the antifungal peptide Drosomycin in both larvae and adults. Using a drosomycin-green fluorescent protein (GFP) reporter gene, it was shown that in addition to the fat body, a variety of epithelial tissues that are in direct contact with the external environment, including those of the respiratory, digestive and reproductive tracts, can all express the antifungal peptide, suggesting a local response to infections affecting these barrier tissues. As is the case for vertebrate epithelia, insect epithelia appear to be more than passive physical barriers and are likely to constitute an active component of innate immunity. In contrast to the systemic antifungal response, this local immune response is independent of the Toll pathway (Ferrandon, 1998).

An in vitro study of NF-kappaB factors cooperatively in regulation of Drosophila melanogaster antimicrobial peptide genes

An important innate immune response in Drosophila melanogaster is the production of antimicrobial peptides (AMPs). Expression of AMP genes is mediated by the Toll and immune deficiency (IMD) pathways via NF-kappaB transcription factors Dorsal, DIF and Relish. Dorsal and DIF act downstream of the Toll pathway, whereas Relish acts in the IMD pathway. Dorsal and DIF are held inactive in the cytoplasm by the IkappaB protein Cactus, while Relish contains an IkappaB-like inhibitory domain at the C-terminus. NF-kappaB factors normally form homodimers and heterodimers to regulate gene expression, but formation of heterodimers between Relish and DIF or Dorsal and the specificity and activity of the three NF-kappaB homodimers and heterodimers are not well understood. This study compared the activity of Rel homology domains (RHDs) of Dorsal, DIF and Relish in activation of Drosophila AMP gene promoters, demonstrated that Relish-RHD (Rel-RHD) interacted with both Dorsal-RHD and DIF-RHD, Relish-N interacted with DIF and Dorsal, and overexpression of individual RHD and co-expression of any two RHDs activated the activity of AMP gene promoters to various levels, suggesting formation of homodimers and heterodimers among Dorsal, DIF and Relish. Rel-RHD homodimers were stronger activators than heterodimers of Rel-RHD with either DIF-RHD or Dorsal-RHD, while DIF-RHD-Dorsal-RHD heterodimers were stronger activators than either DIF-RHD or Dorsal-RHD homodimers in activation of AMP gene promoters. The nucleotides at the 6th and 8th positions of the 3' half-sites of the kappaB motifs were identified that are important for the specificity and activity of NF-kappaB transcription factors (Chowdhury, 2019).

The miR-317 functions as a negative regulator of Toll immune response and influences Drosophila survival

The miR-317 has been revealed to involve in the reproductive response and the larval ovary morphogenesis of Drosophila. However, whether the miR-317 can also regulate Drosophila innate immune responses, which remains unclear to date. This study verified that miR-317 can directly target the 3'UTR of Dif-Rc to down-regulate the expression levels of AMP Drs to negatively control Drosophila Toll immune response in vivo and vitro. Specially, the Dif is an important transcription factor of Toll pathway with four transcripts (Dif-Ra, Dif-Rb, Dif-Rc and Dif-Rd). The results show that miR-317 only targets to Dif-Rc, but not Dif-Ra/b/d. Furthermore, it was demonstrated that the miR-317 sponge can restore the expression levels of Drs and Dif-Rc at mRNA and protein levels. Remarkably, during Gram-positive bacterial infection, the overexpressed miR-317 flies have poor survival outcome, whereas the knockout miR-317 flies have favorable survival compared to the control group, respectively, suggesting that the miR-317 might play a key role in Drosophila survival. Taken together, this work not only reveal an innate immune function and a novel regulation pattern of miR-317, but also provide a new insight into the underlying molecular mechanisms of immunity disorder influencing on Drosophila survival (Li, 2019).

dorsal-related immunity factor: Biological Overview | Evolutionary Homologs | Protein Interactions | Effects of Mutation | References

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