Gene name - Dorsal-related immunity factor
Cytological map position - 36C2
Function - Transcription factor
Keywords - immune response - Dorsal and DIF act downstream of the Toll pathway
Symbol - Dif
Genetic map position - 2-
Classification - rel homolog
Cellular location - cytoplasmic and nuclear
|Recent literature||Khor, S. and Cai, D. (2020). Control of lifespan and survival by Drosophila NF-κB signaling through neuroendocrine cells and neuroblasts. Aging (Albany NY) 12(24): 24604-24622. PubMed ID: 33232282
This paper reports a comparative analysis of the effects of immune activation in the fly nervous system using genetic activation models to target Drosophila NF-κB within Toll versus Imd pathways. Genetic gain-of-function models for either pathway pan-neuronally, as well as in discrete subsets of neural cells including neuroendocrine insulin-producing cells (IPCs) or neuroblasts, reduce fly lifespan, however, these phenotypes in IPCs and neuroblasts are stronger with Toll activation than Imd activation. Of note, while aging is influenced more by Toll/NF-κB activation in IPCs during adulthood, neuroblasts influence aging more substantially during development. The study then focused on Toll/NF-κB inhibition, revealing that IPCs or neuroblasts are important for the effects of lifespan and healthspan extension but in a life stage-dependent manner while some of these effects display sexual dimorphism. Importantly, co-inhibition of Toll/NF-κB pathway in IPCs and neuroblasts increased fly lifespan greater than either cell population, suggesting that independent mechanisms might exist. Toll/NF-κB inhibition in IPCs was also sufficient to enhance survival under various fatal stresses, supporting the additional benefits to fly healthspan. In conclusion, IPCs and neuroblasts are important for Drosophila NF-κB for controlling lifespan.
|Huang, C., Xu, R., Liegeois, S., Chen, D., Li, Z. and Ferrandon, D. (2020). Differential Requirements for Mediator Complex Subunits in Drosophila melanogaster Host Defense Against Fungal and Bacterial Pathogens. Front Immunol 11: 478958. PubMed ID: 33746938
The humoral immune response to bacterial or fungal infections in Drosophila relies largely on a transcriptional response mediated by the Toll and Immune deficiency NF-κB pathways. Antimicrobial peptides are potent effectors of these pathways and allow the organism to attack invading pathogens. Dorsal-related Immune Factor (DIF), a transcription factor regulated by the Toll pathway, is required in the host defense against fungal and some Gram-positive bacterial infections. The Mediator complex is involved in the initiation of transcription of most RNA polymerase B (PolB)-dependent genes by forming a functional bridge between transcription factors bound to enhancer regions and the gene promoter region and then recruiting the PolB pre-initiation complex. Mediator is formed by several modules that each comprises several subunits. The Med17 subunit of the head module of Mediator has been shown to be required for the expression of Drosomycin, which encodes a potent antifungal peptide, by binding to DIF. Thus, Mediator is expected to mediate the host defense against pathogens controlled by the Toll pathway-dependent innate immune response. This study first focused on the Med31 subunit of the middle module of Mediator and find that it is required in host defense against Aspergillus fumigatus, Enterococcus faecalis, and injected but not topically-applied Metarhizium robertsii. Thus, host defense against M. robertsii requires Dif but not necessarily Med31 in the two distinct infection models. The induction of some Toll-pathway-dependent genes is decreased after a challenge of Med31 RNAi-silenced flies with either A. fumigatus or E. faecalis, while these flies exhibit normal phagocytosis and melanization. This study further tested most Mediator subunits using RNAi by monitoring their survival after challenges to several other microbial infections known to be fought off through DIF. The host defense against specific pathogens involves a distinct set of Mediator subunits with only one subunit for C. glabrata or Erwinia carotovora carotovora, at least one for M. robertsii or a somewhat extended repertoire for A. fumigatus (at least eight subunits) and E. faecalis (eight subunits), with two subunits, Med6 and Med11 being required only against A. fumigatus. Med31 but not Med17 is required in fighting off injected M. robertsii conidia. Thus, the involvement of Mediator in Drosophila innate immunity is more complex than expected.
|Zhou, H., Li, S., Wu, S., Jin, P. and Ma, F. (2021). LncRNA-CR11538 Decoys Dif/Dorsal to Reduce Antimicrobial Peptide Products for Restoring Drosophila Toll Immunity Homeostasis. Int J Mol Sci 22(18). PubMed ID: 34576280
Avoiding excessive or insufficient immune responses and maintaining homeostasis are critical for animal survival. Although many positive or negative modulators involved in immune responses have been identified, little has been reported to date concerning whether the long non-coding RNA (lncRNA) can regulate Drosophila immunity response. Firstly, this study discovered that the overexpression of lncRNA-CR11538 can inhibit the expressions of antimicrobial peptides Drosomycin (Drs) and Metchnikowin (Mtk) in vivo, thereby suppressing the Toll signaling pathway. Secondly, the results demonstrate that lncRNA-CR11538 can interact with transcription factors Dif/Dorsal in the nucleus based on both subcellular localization and RIP analyses. Thirdly, the findings reveal that lncRNA-CR11538 can decoy Dif/Dorsal away from the promoters of Drs and Mtk to repress their transcriptions by ChIP-qPCR and dual luciferase report experiments. Fourthly, the dynamic expression changes of Drs, Dif, Dorsal and lncRNA-CR11538 in wild-type flies (w(1118)) at different time points after M. luteus stimulation disclose that lncRNA-CR11538 can help Drosophila restore immune homeostasis in the later period of immune response. Overall, this study reveals a novel mechanism by which lncRNA-CR11538 serves as a Dif/Dorsal decoy to downregulate antimicrobial peptide expressions for restoring Drosophila Toll immunity homeostasis, and provides a new insight into further studying the complex regulatory mechanism of animal innate immunity.
|Kanoh, H., Iwashita, S., Kuraishi, T., Goto, A., Fuse, N., Ueno, H., Nimura, M., Oyama, T., Tang, C., Watanabe, R., Hori, A., Momiuchi, Y., Ishikawa, H., Suzuki, H., Nabe, K., Takagaki, T., Fukuzaki, M., Tong, L. L., Yamada, S., Oshima, Y., Aigaki, T., Dow, J. A. T., Davies, S. A. and Kurata, S. (2021). cGMP signaling pathway that modulates NF-κB activation in innate immune responses. iScience 24(12): 103473. PubMed ID: 34988396
The nuclear factor-kappa B (NF-κB) pathway is an evolutionarily conserved signaling pathway that plays a central role in immune responses and inflammation. This study shows that Drosophila NF-κB signaling is activated via a pathway in parallel with the Toll receptor by receptor-type guanylate cyclase, Gyc76C. Gyc76C produces cyclic guanosine monophosphate (cGMP) and modulates NF-κB signaling through the downstream Toll receptor components dMyd88, Pelle, Tube, and Dif/Dorsal (NF-κB). The cGMP signaling pathway comprises a membrane-localized cGMP-dependent protein kinase (cGK) called DG2 and protein phosphatase 2A (PP2A) and is crucial for host survival against Gram-positive bacterial infections in Drosophila. A membrane-bound cGK, PRKG2, also modulates NF-κB activation via PP2A in human cells, indicating that modulation of NF-κB activation in innate immunity by the cGMP signaling pathway is evolutionarily conserved.
|Wijesekera, T. P., Wu, Z., Stephens, N. P., Godula, R., Lew, L. K. and Atkinson, N. S. (2022). A non-nuclear NF-kappaB modulates alcohol sensitivity but not immunity. J Neurosci. PubMed ID: 35273084
NF-κB proteins are well known as transcription factors important in immune system activation. In this highly conserved role, they contribute to changes in behavior in response to infection and in response to a variety of other insults and experiences. In some mammalian neurons, NF-κBs can be found at the synapse and translocate to the nucleus to alter gene expression when activated by synaptic activity. This study demonstrates that, in Drosophila melanogaster, NFκB action is important both inside and outside the nucleus and that the Dif gene has segregated nuclear and non-nuclear NFκB action into different protein isoforms. The DifA isoform is a canonical nuclear-acting NFκB protein that enters the nucleus and is important for combating infection. The DifB variant, but not the DifA variant, is found in the central nervous system (mushroom bodies and antennal lobes). DifB does not enter the nucleus and co-localizes with a synaptic protein. In males and females, a DifB mutant alters alcohol behavioral sensitivity without an obvious effect on combating infection, whereas a DifA mutant does not affect alcohol sensitivity but compromises the immune response. These data are evidence that the non-nuclear DifB variant contributes to alcohol behavioral sensitivity by a nongenomic mechanism that diverges from the NF-κB transcriptional effects used in the peripheral immune system. Enrichment of DifB in brain regions rich in synapses and biochemical enrichment of DifB in the synaptoneurosome fraction indicates that the protein may act locally at the synapse (Wijesekera, 2022).
Insects resist bacterial infections through the induction of both cellular and humoral immune responses. The cellular response involves the mobilization of hemocytes, whereas the humoralbresponse utilizes antibacterial peptides that are synthesized in the fat bodies and secreted into the circulating hemolymph. To better understand the fly's immune responses, some familiarity with the proteins involved in these responses is useful. In Drosophila, these proteins are coded for by two functionally distinct classes of inducible antimicrobial immunity genes: those that code for antibacterial peptides, namely Cecropins (Kylsten, 1990 and Tryselius, 1992), Diptericin (Wicker, 1990), Drosocin (Bulet, 1993), Attacin (Asling, 1995), and insect Defensin (Dimarcq, 1994), and a gene that codes for an antifungal peptide, Drosomycin (Fehlbaum, 1994)
Recent studies suggest that the induction of the humoral response involves two regulatory proteins, Dif and Dorsal, that are related to mammalian NF-kappa B. Both Dorsal and Dif are expressed in immuno-responsive tissues, and both proteins are translocated into the nuclei after bacterial challenge. These regulatory proteins function as sequence-specific transcription factors that induce the expression of immunity genes (Ip, 1994). It is clear that the cell membrane and intracellular components of the Dorsal group (Toll, Cactus and Dorsal), as well as Dif, control the inducible immune response.
In addition to the dorsoventral pathway, several other pathways are implicated in the immune response. A recessive mutation, immune deficiency (imd), has been described that impairs the inducibility of all genes encoding antibacterial peptides during the immune respone. The antifungal peptide Drosomycin remains fully inducible in imd mutants, pointing to the existence of different pathways leading to expression of the antifungal and antibacterial peptides genes. The imd gene has not yet been cloned (Lemaitre, 1995a).
Another pathway, not yet determined to be distinct from that in which imd participates, involves Jun amino terminal kinase (JNK), coded for by basket. basket is activated by endotoxic lipopolysaccharide (LPS). LPS is a component of bacterial cell walls, and is known to be a stimulant for the immune response in both insects and mammals. Addition of LPS to cultured cell lines causes marked induction of cecropin and diptercin genes. basket/JNK is activated within 5 minutes of LPS addition. The activation of DJun by DJNK in LPS-treated cells may lead to increased AP-1 (a heterodimer of Fos-related antigen and Jun related antigen) transcriptional activity. Targets of Drosophila AP-1 may include the JRA promoter (Sluss, 1996).
Along with the sequences in imd and basket, there is another sequence, in the diptericin promoter that regulates its activity and is homologous to mammalian interferon consensus response element. All these sequences bind a polypeptide that cross-reacts with an antiserum directed against mammalian interferon regulatory factor-I, known to bind to the promoter of interferon-inducible genes (Georgel, 1995).
What is known about the role of Dif in the Drosophia immune response, and how may the relative importance of Dorsal and Dif to one another best be characterized? Dorsal and Dif both rapidly translocate into the nucleus after bacterial challenge. But nuclear localization requires only tissue injury; the simple act of tearing tissue is enough to induce nuclear localization. Nuclear localization is regulated by the transmembrane receptor Toll, which initiates an intracellular regulatory cascade that leads to the dissociation of Cactus (the I-kappaB homolog) from the Dorsal protein. Toll homologs include the mammalian Interleukin 1 gene, but also IL-1 related genes in invertebrates and plants. It is clear that the role of IL-1 in the immune response is conserved in some fashion throughout metazoa. Although interaction of Dif with Cactus has not been studied, it is presumed that like Dorsal, Dif too is regulated by an interaction with Cactus.
Dif binds to promoter motifs whose sequences are evolutionarily conserved. These sequences, called kappaB motifs, are similar to those targeted by NFkappaB, the vertebrate homolog of Dorsal and Dif. The promoters of immunity genes such as cecropin and diptericin have been analyzed for their interaction with both Dorsal and Dif. Dif has a substantially higher binding affiinity for the CecA1 kappaB sequence than does Dorsal. In addition, induction of the immune response gives rise to a nuclear binding activity that recognizes the CeCA1 kappaB motif. This complex is specifically disrupted by pretreating nuclear extracts with anti-Dif antibodies. These results provide strong evidence that the induction of the immune response activates Dif (Ip, 1993).
However, Dif appears to be less efficient as a transactivator than Dorsal when tested using a diptericin reporter gene. The protein complexes produced on kappaB reporter sequences by Dorsal and Dif appear to be different, suggesting that the two proteins do not have identical partners in the activation of promoter sequences by protein complexes. Dif and Dorsal interact differently with different constructs of kappaB sites, suggesting that their DNA binding characteristics are not identical. Mutants containing no copies of dorsal and a single copy of Dif retain their full capacities to express the diptericin and cecropin genes in response to immune challenge. Thus it is clear that Dorsal and Dif are likely to carry out distinct functions, and as yet uncharacterized differences, in target gene activation (Gross, 1996).
It is commonly thought the immune response in Drosophila is related to the acute-phase response in vertebrates. In vertebrates inflammation or injury is accompanied by significant alterations in the serum levels of several plasma proteins, known as acute-phase proteins. Many of the acute-phase proteins act as antiproteinases, opsonins, or blood-clotting and wound-healing factors, which may protect against the generalized tissue destruction associated with inflammation (Baumann, 1994). This analogy only goes so far. Regulation of many of the important accessory proteins of the vertebrate immune response are regulated by the same gene systems involved in the immune response in Drosophila. Making the reasonable assumption that the vertebrate immune response evolved from a non-immunoglobulin based system similar to that in Drosophila, it can be argued that the immune response in Drosophila represents the evolutionary core of the immune response in invertebrates, lacking only an inducible immunoglobulin system. This argument is developed more fully in the rolled gene site.
Two Drosophila lines were isolated that carry point mutations in the gene coding for the NF-kappaB-like factor Dif. Like mutants of the Toll pathway, Dif mutant flies are susceptible to fungal but not to bacterial infections. Genetic epistasis experiments demonstrate that Dif mediates the Toll-dependent control of the inducibility of the antifungal peptide gene Drosomycin. Strikingly, Dif alone is required for the antifungal response in adults, but is redundant in larvae with Dorsal, another Rel family member. In Drosophila, Dif appears to be dedicated to the antifungal defense elicited by fungi and gram-positive bacteria. The possibility is discussed that NF-kappaB1/p50 might be required more specifically in the innate immune response against gram-positive bacteria in mammals (Rutschmann, 2000).
Transcription factors of the Rel family have long been suspected to play an important role in the control of the expression of insect antimicrobial peptides. To date, three members of this family have been reported in Drosophila, namely, Dorsal, DIF, and Relish. In this study, two mutations in the gene encoding Dif were generated and show that this Rel protein plays a critical role in the control of the antifungal response in Drosophila (Rutschmann, 2000).
The Dif1 mutation is a strong mutation, since no significant Drosomycin induction is observed in flies subjected to a natural infection by the fungus B. bassiana in contrast to wild-type flies, where this infection triggers a strong and sustained expression of the antifungal gene. Similar results have been obtained upon injury with either gram-positive bacteria or fungi. Furthermore, in response to a fungal or gram-positive bacterial infection, Dif1 homozygous and hemizygous flies transcribe the Drosomycin gene at the same low, residual level and show similar survival curves to fungal infections. These data indicate that the Dif1 mutation is genetically close to a null mutation. It is likely that the Dif2 mutation is also a strong mutation, since the induction of Drosomycin by immune challenge and the survival to fungal infections were similar in Dif1 and Dif2 homozygous, hemizygous, or transheterozygous Dif1/Dif2 flies (Rutschmann, 2000).
The two mutations isolated in this study were induced in the Rel homology domain (RHD). Rel proteins bind as dimers to DNA with an unusually high affinity through the conserved RHD. The structure of this domain has been determined by X-ray crystallography in several NF-κB members over the last years. Its main features have been remarkably conserved throughout evolution. The dimer wraps around the DNA, giving the appearance of a butterfly to the complex. Each monomer consists of two immunoglobulin (Ig)-like domains connected by a short linker. The N-terminal Ig-like domain consists of a nine-stranded β barrel and contains a recognition loop (L1) that makes specific contacts with the DNA binding site. This domain also contacts the binding site through a second loop (L2) that clamps the DNA at the central minor groove via basic residues. The C-terminal Ig-like domain consists of a seven-stranded β barrel, which contacts the DNA backbone through two loops (L4 and L5) and mediates homo- or heterodimerization of NF-κB subunits. Together with the nuclear localization signal located right at the C-terminal end of the RHD, the C-terminal Ig-like domain also binds to the six ankyrin repeats of the I-κB inhibitor. Finally, a basic amino acid located in the linker (loop L3) that joins the N-terminal to the C-terminal Ig-like domains makes a specific contact with a nucleotide of the DNA binding site at position ± 2 (the dyad axis of symmetry passes usually through the nucleotide in position 0) (Rutschmann, 2000).
The positions of the two mutations in the Dif structure were visualized by modeling a putative Dif based on the crystallographic data from the mosquito Rel protein Gambif, which is 37% homologous to Dif over the RHD. Glycine 181 is located in the middle of β strand E′ on the inside of the N-terminal domain, close to the DNA clamping loop L2. This glycine has been conserved in all RHDs known to date and presumably plays an important structural role. Its replacement by a bulky, negatively charged amino acid, in close vicinity to the DNA helix, certainly perturbs the local structure and prevents loop L2 from binding to DNA. Thus, it is anticipated that the Dif1 mutation severely affects the high-affinity DNA binding of Dif. The C-terminal dimerization domain of the RHD that contacts I-κBs is not affected by the mutation and nuclear import of the protein is not altered, as illustrated by nuclear localization of Dif1 following an immune challenge (Rutschmann, 2000).
The Dif2 mutation replaces a serine by a phenylalanine residue. This serine is unlikely to be the target of a kinase, since its alcoholic function is not accessible to solvents. The same holds true for serines at the same position in the other Rel structures that have been determined. It is buried on the inside of the protein where the oxygen of the alcoholic function makes a hydrogen bond with the amide function of leucine 225 in the modeled Dif structure. The minimal hypothesis is favored that the Dif2 mutation induces a strong local perturbation of the structure of the N-terminal Ig-like domain. Indeed, serine 245 is part of β strand G that connects β strand F, in particular through its alcoholic function. It is likely that this contact stabilizes the orientation of loop L3, which is highly structured when bound to DNA. It usually makes a specific contact with a central DNA base, probably through lysine 251 in Dif. As for Dif1, it is expected that the Dif2 mutation affects DNA binding, although the overall stability of the protein might also be affected. It is therefore likely that both mutations prevent the recognition of κB binding sites by Dif and that they do not impair protein–protein interactions with putative cofactors (Rutschmann, 2000).
Two lines of evidence indicate that Dif plays a pivotal role in the antifungal response in adult Drosophila. First, Dif flies are more susceptible to fungal infections than wild-type flies. Second, immune induction of Drosomycin, the predominant antifungal peptide gene of Drosophila, is severely reduced, if not abolished in those flies. Furthermore, Defensin induction is significantly decreased in Dif mutants. As regards Cecropin, Dif and dl appear to participate, at least partially, in the regulation of its immune-induced expression. Defensin and Cecropin display some antifungal properties in addition to their antibacterial activities. Even though the decreased expression of antifungal peptides certainly contributes to susceptibility to fungal infection, the possibility that Dif also activates other antifungal defense mechanisms and, namely, cellular responses, cannot be excluded (Rutschmann, 2000).
The Dif phenotype reported in this study is strikingly similar to that of mutants that affect the spz/Tl/tub/pll/cac gene cassette (Lemaitre, 1996). The epistatic relationship between Tl and Dif on one hand and cact and Dif on the other hand provides a clear demonstration that the control of Drosomycin inducibility is mediated through Dif in response to activation of the Tl pathway. Since fungal infections seem to have similar lethal effects on Dif and spz mutants, it is proposed that Dif controls the various aspects of the Tl-mediated antifungal response in adult Drosophila (Rutschmann, 2000).
As regards the induction of Cecropin, Dif and dorsal appear to be functionally redundant in adults. Indeed, immune induction of Cecropin is unaffected in Dif mutants and is significantly reduced in Dif/TW119 hemizygous flies. Since the Dif mutations are strong and are likely to affect DNA binding of Dif rather than putative interactions with cofactors, this effect cannot be ascribed to a hypomorphic effect of the Dif mutations on the Cecropin promoter. Rather, it is likely that the removal of a copy of dl in TW119 combined with the total lack of Dif is responsible for this phenotype in the Dif hemizygous background. It thus appears that the single dose of dl left in this genetic context is not sufficient to mediate the contribution of the Tl pathway to the regulation of Cecropin, and that this reduced but significant expression is regulated via the imd pathway. This inference in the case of Cecropin is further substantiated by the following observations for Drosomycin. In larvae, have shown that Dif and dl are functionally redundant in controlling the inducibility of Drosomycin (Manfruelli, 1999). This inducibility is not markedly affected in a Dif background but is significantly reduced in Dif1 and Dif2 hemizygous larvae where one wild-type copy of dl is left (Rutschmann, 2000).
In contrast to that of Defensin or Cecropin, the immune induction of Attacin does not depend on either Dif or dl, whereas it is strongly decreased in spz, Tl, and pll mutants. These results suggest that a branch point in the Tl pathway exists downstream of pll to regulate an undefined transcription factor, possibly Relish, that controls the inducibility of Attacin in adults (Rutschmann, 2000).
The Dif mutations generated in this study do not fully abolish Drosomycin induction by immune challenge with a mix of gram-positive and gram-negative bacteria. Indeed, some 25% of wild-type levels of induction were consistently observed in these conditions. In contrast, fungi or gram-positive bacteria failed to induce any induction of Drosomycin in Dif1 mutants. The residual Drosomycin expression is abolished in Dif-kenny double mutants challenged with the mix (kenny is a mutant that shows a phenotype similar to that of Relish). It is proposed that the low level of Drosomycin inducibility triggered by gram-negative bacteria is partially controlled by a Dif-independent pathway, namely, the imd-kenny-Relish pathway. This hypothesis is supported by the result that an infection with E. coli triggers a short-lived induction of Drosomycin, similar to that of fast reactants such as Cecropin and Attacin, that are predominantly controlled by imd-kenny-Relish. Similar observations have been reported in the case of the other genes of the Toll-signaling cassette, where some 25% of wild-type Drosomycin induction was observed after challenge with the mix, whereas no induction was detected when Tl mutants were coated with fungal spores. Furthermore, the low level of Drosomycin expression observed in Tl pathway mutants was totally abolished in Tl-imd double mutants. In conclusion, it is proposed that natural infections with B. bassiana spores trigger Drosomycin expression exclusively by the Tl-Dif-dependent pathway, whereas another pathway, most likely the Relish-kenny-imd pathway, induces, at least partially, Drosomycin expression in response to gram-negative bacteria (Rutschmann, 2000).
It has been shown that Relish is required for the induction of most antimicrobial peptides in response to a gram-negative immune challenge. Specifically as regards Drosomycin, only 20% of wild-type levels were induced in Relish null mutants 6 hr after a challenge with the gram-negative Enterobacter cloacae. The data presented above are compatible with the hypothesis that expression of the Drosomycin gene is controlled by a Relish-Dif heterodimer, which is also in keeping with tissue culture experiments performed with transfected S2 cells. However, the possibility that Relish mediates the gram-negative-induced, Dif-independent component of Drosomycin induction cannot be excluded (Rutschmann, 2000).
It has been demonstrated (Manfruelli, 1999) by a clonal analysis of the TW119 deficiency that Dif and dl are functionally redundant in the control of Drosomycin inducibility in larvae. In contrast, another study (Meng, 1999) has observed that Dif but not dl regulates the expression of Drosomycin in adults. This latter result was obtained by complementation with either a Dif or a dorsal transgene of the small J4 deficiency that removes both genes. The analysis of the Dif point mutants presented in this study resolves this paradox and shows that the regulation of Drosomycin expression in the adult fat body differs from that in the larval fat body. The reasons for this difference are at present unclear. It has to be keep in mind that larval and fat body cells are not fully equivalent and have distinct developmental origins. One explanation for the difference in regulation could be that larval and adult fat body cells produce different levels of either Dif or DL. Another explanation could relate to the presence or absence in larvae versus adults of various cofactors required for full activation of response genes by DL or Dif (Rutschmann, 2000).
Five distinct Rel family members are present in mammals where they play a major role in the host defense by controlling the expression of such diverse immune-response molecules as immunoreceptors, cytokines, adhesion molecules, and acute phase proteins. In addition, they can provide protection against TNFα-mediated apoptosis. The analysis of the respective roles of the five Rel proteins is hampered by partial redundancies and by the complexities or lethality of the mutant phenotypes. In particular, the identity of the Rel proteins that mediate the TLR2-dependent response to peptidoglycans and the TLR4-dependent response to LPS has not been established, since in both cases only the activation of a 'generic' NF-κB binding activity was investigated. Yet, distinct responses are likely to be elicited by different pathogens. In this respect, it is striking that p50 knockout mice are highly susceptible to infections by the pathogenic gram-positive Streptococcus pneumoniae and not by the gram-negative Haemophilus influenzae or E. coli K1, which raises the possibility that p50 is the Rel protein mediating the response to gram-positive bacteria (Rutschmann, 2000).
In Drosophila, mutants for all three individual Rel proteins are now available. Remarkably, the viability of these mutants is not impaired under normal conditions. Dif plays a critical role in mediating the antifungal response that is activated through the Tl pathway in the adult. Strikingly, Dif does not seem to play a role in the humoral immune response against gram-negative bacteria, as can be judged from the lack of effect of Dif mutations on the inducibility of Drosocin, Diptericin, and Attacin, all of which are essentially active on such bacteria. Studies underway should reveal whether the three Rel proteins are indeed complementary in mediating a humoral immune response against diverse pathogens or whether there is some degree of functional redundancy like that observed between Dif and dl in larvae (Rutschmann, 2000).
Because excessive or inadequate responses can be detrimental, immune responses to infection require appropriate regulation. Networks of signaling pathways establish versatility of immune responses. Drosophila melanogaster is a powerful model organism for dissecting conserved innate immune responses to infection. For example, the Toll pathway, which promotes activation of NF-kappaB transcription factors Dorsal/Dorsal-related immune factor (Dif), was first identified in Drosophila. Together with the IMD pathway, acting upstream of NF-kappaB transcription factor calcineurin A1, acts on Relish during infection. However, it is not known whether there is a role for calcineurin in Dorsal/Dif immune signaling. This article demonstrates involvement of specific calcineurin isoforms, protein phosphatase at 14D (Pp2B-14D)/calcineurin A at 14F (CanA-14F), in Toll-mediated immune signaling. These isoforms do not affect IMD signaling. In cell culture, pharmacological inhibition of calcineurin or RNA interference against homologous calcineurin isoforms Pp2B-14D/CanA-14F, but not against isoform calcineurin A1, decreased Toll-dependent Dorsal/Dif activity. A Pp2B-14D gain-of-function transgene promoted Dorsal nuclear translocation and Dorsal/Dif activity. In vivo, Pp2B-14D/CanA-14F RNA interference attenuated the Dorsal/Dif-dependent response to infection without affecting the Relish-dependent response. Altogether, these data identify a novel input, calcineurin, in Toll immune signaling and demonstrate involvement of specific calcineurin isoforms in Drosophila NF-kappaB signaling (Li, 2014).
Dif maps to the 36C region on chromosome 2, which corresponds to the map position of dorsal. The two genes are separated by at least 15 kb; it is probable that they map to between 15 and 88 kb of one another. A 1.8 kb RNA species related in sequence to Dif is specified by a convergently transcribed gene that is tightly linked to Dif; it is related to Dif solely on the basis of a 250 base pair region in the Dif untranslated trailer sequence (Ip, 1993).
Bases in 5' UTR - 562
Bases in 3' UTR - 277
The Rel domain contains 295 amino acid residues, spanning residues 78 to 372. This Rel sequence is most closely related to the Dorsal sequence; they share 142 identical residues. Among the vertebrate Rel proteins, Dif is most similar to the turkey c-Rel (40% identity in the Rel domain). The Dorsal Rel domain is about equally related to Dif, mouse p65 and Xenopus Rel. The Rel domains of Dif, Dorsal, turkey c-Rel and mouse p65 share 86 invariant residues. Sequences that reside in the Dif Rel domain do not share significant identities with the unique regions of Dorsal or other Rel-containing proteins. The C-terminal region of Dif (amino acid residues 582 to 631) is rich in glutamine, proline, and hydrophobic residues, suggesting that it might function as a transcriptional activation domain (Ip, 1993).
Evolutionary homologs for Dif are the same as for Dorsal, and information on these homologs will be found at the Dorsal site.
The recent sequencing of several complete genomes has made it possible to track the evolution of large gene families by their genomic structure. Following the large-scale association of exons encoding domains with well defined functions in invertebrates could be useful in predicting the function in mammals of complex multidomain proteins produced by accretion of domains. With this objective, the genomic structure of the 14 genes in invertebrates and vertebrates that contain rel domains has been examined. The sequence encoding the rel domain is defined by intronic boundaries and has been recombined with at least three structurally and functionally distinct genomic sequences to generate coding sequences for: (1) the rel/Dorsal/NFkappaB proteins that are retained in the cytoplasm by IkB-like proteins; (2) the NFATc proteins that sense calcium signals and undergo cytoplasmic-to-nuclear translocation in response to dephosphorylation by calcineurin; and (3) the TonEBP tonicity-responsive proteins. Remarkably, a single exon in each NFATc family member encodes the entire Ca2+/calcineurin sensing region, including nuclear import/export, calcineurin-binding, and substrate regions. The Rel/Dorsal proteins and the TonEBP proteins are present in Drosophila but not Caenorhabditis elegans. However, the calcium-responsive NFATc proteins are present only in vertebrates, suggesting that the NFATc family is dedicated to functions specific to vertebrates such as a recombinational immune response, cardiovascular development, and vertebrate-specific aspects of the development and function of the nervous system (Graef, 2001).
The positions of introns in genes coding for rel domain proteins are highly conserved, with introns positioned to either side of the sequence encoding the rel domain. The exceptions to this are informative: the sequences encoding the rel domain in Relish, Dif, Dorsal, and Rel B lack an intron 5' to the coding region. If the ancestral gene contained an intron demarcating the N-terminal coding region in these genes, this intron must have been lost before the formation of Rel B, Dorsal, Dif, and Relish, because the other vertebrate genes all have retained this intron. Alternatively, if the ancestral gene lacked an intron demarcating the N-terminal coding region of the rel domain, it must have been inserted after the Relb, dif, dorsal, and relish genes had originated from the ancestral gene. By either scenario, Rel B is the closest vertebrate relative of Dorsal, Dif, and Relish. Introns could not have been randomly lost or inserted, because a number of studies have shown that their positions are highly conserved within gene families. The sequence encoding the C terminus of the rel domain is also bounded by introns for each of the proteins except Relish. Indeed, the conserved proline codon at the C terminus of all rel domains occurs within five amino acid codons of the C-terminal intronic insertion (Graef, 2001).
The most distinctive structural feature of the rel domain is the division of the dimerization and specificity domains. Remarkably, in all vertebrate rel domain-containing genes, an intron precisely separates the sequences encoding the dimerization and the DNA specificity domains within the rel domain. Again, the exceptions are informative, in that no insect gene other than Drosophila TonEBP has this intron insertion site between the recognition and dimerization domains. One possible explanation is that the ancestral gene contained an intron at this position that was lost. However, several lines of evidence bode against intron loss, particularly because there is no evidence of processing and reinsertion of the insect rel domains. A more likely scenario is that the ancestral gene gained an intron separating the sequences encoding the dimerization and specificity domains, which then allowed the rel domain to successfully recombine and disseminate in vertebrates (Graef, 2001).
In the p100, p105, and Relish proteins, a cytoplasmic retention domain is a distinct region in each protein and is characterized by the presence of ankyrin repeats. This region is processed and eventually degraded to allow translocation to the nucleus. This cis-acting cytoplasmic retention function in Relish is encoded by a single exon, which in vertebrate p105 is divided into 13 different exons and a large but as yet undetermined number of exons in the p100 gene. Cytoplasmic retention can also be provided by the cactus or IkB proteins, which have sequence similarity to p105, p100, and Relish outside the rel domain (Graef, 2001).
A rel domain related to the one found in the NFATc proteins was recently reported in the mammalian TonEBP or NFAT5. This protein is encoded by a single mammalian gene and is transcriptionally regulated by osmotic stress. A gene related to mammalian TonEBP was found in Drosophila (Misexpression Suppressor of Ras 1). This gene also has a large exon 5' to the coding sequence for the rel domain, but the protein contains neither the ankyrin repeats of the p105/Relish proteins nor the translocation domain of the NFATc family. The Drosophila protein shares some features of the human TonEBP protein outside the rel domain, including the glutamine-rich regions. The mammalian gene has been partially sequenced and found to encode a rel domain with its sequence divided by introns at sites that correspond to those present in the NFATc genes. However, outside of the rel domain, the genomic structure of TonEBP is unrelated to NFATc family members. Most definitively, TonEBP lacks the translocation exon, indicating that it is not functionally related to the NFATc proteins (Graef, 2001).
date revised: 12 September 2022
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