Gene name - Dorsal-related immunity factor
Cytological map position - 36C2
Function - Transcription factor
Keywords - immune response
Symbol - Dif
Genetic map position - 2-
Classification - rel homolog
Cellular location - cytoplasmic and nuclear
|Recent literature||Zhou, B., Lindsay, S. A. and Wasserman, S. A. (2015). Alternative NF-κB isoforms in the Drosophila neuromuscular junction and brain. PLoS One 10: e0132793. PubMed ID: 26167685
The Drosophila NF-κB protein Dorsal is expressed at the larval neuromuscular junction, where its expression appears unrelated to known Dorsal functions in embryonic patterning and innate immunity. Using confocal microscopy with domain-specific antisera, this study demonstrates that larval muscle expresses only the B isoform of Dorsal, which arises by intron retention. Dorsal B interacts with and stabilizes Cactus at the neuromuscular junction, but exhibits Cactus independent localization and an absence of detectable nuclear translocation. It was further found that the Dorsal-related immune factor Dif encodes a B isoform, reflecting a conservation of B domains across a range of insect NF-kappaB proteins. Carrying out mutagenesis of the Dif locus via a site-specific recombineering approach, it was demonstrated that Dif B is the major, if not sole, Dif isoform in the mushroom bodies of the larval brain. The Dorsal and Dif B isoforms thus share a specific association with nervous system tissues as well as an alternative protein structure.
Insects resist bacterial infections through the induction of both cellular and humoral immune responses. The cellular response involves the mobilization of hemocytes, whereas the humoral response 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.
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: 15 March 2015
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.