| 1. |
- Dantoft, Widad, et al.
(författare)
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The Oct1 homolog Nubbin is a repressor of NF-kappa B-dependent immune gene expression that increases the tolerance to gut microbiota
- 2013
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Ingår i: BMC Biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Background: Innate immune responses are evolutionarily conserved processes that provide crucial protection against invading organisms. Gene activation by potent NF-kappa B transcription factors is essential both in mammals and Drosophila during infection and stress challenges. If not strictly controlled, this potent defense system can activate autoimmune and inflammatory stress reactions, with deleterious consequences for the organism. Negative regulation to prevent gene activation in healthy organisms, in the presence of the commensal gut flora, is however not well understood. Results: We show that the Drosophila homolog of mammalian Oct1/POU2F1 transcription factor, called Nubbin (Nub), is a repressor of NF-kappa B/Relish-driven antimicrobial peptide gene expression in flies. In nub(1) mutants, which lack Nub-PD protein, excessive expression of antimicrobial peptide genes occurs in the absence of infection, leading to a significant reduction of the numbers of cultivatable gut commensal bacteria. This aberrant immune gene expression was effectively blocked by expression of Nub from a transgene. We have identified an upstream regulatory region, containing a cluster of octamer sites, which is required for repression of antimicrobial peptide gene expression in healthy flies. Chromatin immunoprecipitation experiments demonstrated that Nub binds to octamer-containing promoter fragments of several immune genes. Gene expression profiling revealed that Drosophila Nub negatively regulates many genes that are involved in immune and stress responses, while it is a positive regulator of genes involved in differentiation and metabolism. Conclusions: This study demonstrates that a large number of genes that are activated by NF-kappa B/Relish in response to infection are normally repressed by the evolutionarily conserved Oct/POU transcription factor Nub. This prevents uncontrolled gene activation and supports the existence of a normal gut flora. We suggest that Nub protein plays an ancient role, shared with mammalian Oct/POU transcription factors, to moderate responses to immune challenge, thereby increasing the tolerance to biotic stress.
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| 2. |
- Junell, Anna, et al.
(författare)
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Isolation of regulators of Drosophila immune defense genes by a double interaction screen in yeast
- 2007
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Ingår i: Insect Biochemistry and Molecular Biology. - : Elsevier BV. - 0965-1748 .- 1879-0240. ; 37:3, s. 202-212
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Tidskriftsartikel (refereegranskat)abstract
- Innate immunity is a universal and ancient defense system in metazoans against microorganisms. Antimicrobial peptides, which are synthesized both in insects and humans, constitute an endogenous, gene-encoded defense arsenal. In Drosophila, antimicrobial peptides, such as the potent cecropins, are expressed both constitutively in barrier epithelia, as well as systemically in response to infection. Rel/NF-κB proteins are well-known regulators of antimicrobial peptide genes, but very few Rel/NF-κB co-factors and/or tissue-specific regulators have been identified. We performed a double interaction screen in yeast to isolate Drosophila cDNAs coding for direct regulators, as well as Dif co-regulators, of the CecropinA1 gene. Three classes of positive cDNA clones corresponding to 15 Drosophila genes were isolated and further characterized. One of the Dif-independent cDNAs encoded the Rel/NF-κB protein Relish; a well-known activator of antimicrobial peptide genes in Drosophila, demonstrating the applicability of this type of screen for isolating regulators of immune defense. Most interestingly, three transcription factors belonging to the POU domain class of homeodomain proteins, Pdm1, Pdm2 and Dfr/Vvl were isolated as Dif-interacting partners, and subsequently verified as regulators of CecA1 expression in Drosophila cells. The importance of POU proteins in development and differentiation in Drosophila and mammals is well documented, but their role in regulation of Drosophila immune defense genes is a new and essential finding.
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| 3. |
- Junell, Anna, 1972-
(författare)
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Regulation of antimicrobial peptide gene expression in Drosophila melanogaster : Involvement of POU and NF-kB/Rel factors in innate immunity
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The fruit fly, Drosophila melanogaster, has a well-developed immune response, and microbial assault induces a rapid production of potent antimicrobial peptides (AMPs). The aim of this thesis work was to gain deeper knowledge of the regulation of AMPs in Drosophila, by the isolation and characterization of transcription factors involved in AMP gene expression. A yeast screen was designed and used to isolate Drosophila cDNAs coding for novel regulators of the CecropinA1 (CecA1) gene. Three transcription factors belonging to the POU domain (Pdm) family were isolated, Pdm1, Pdm2, and Drifter (Dfr), and subsequently verified as regulators of CecA1 in Drosophila cells. POU proteins are known to regulate a range of developmental processes, but this is the first finding of POU factors controlling AMP gene expression. Dfr and Pdm1 were further analyzed with respect to their in vivo function as AMP gene regulators. Over-expression of Dfr activated several AMP genes in non-infected flies, suggesting that Dfr is involved in constitutive expression of AMP genes. Dfr was shown to bind to a CecA1 upstream enhancer, to which the homeodomain protein Caudal (Cad) previously had been shown to bind. Co-expression of Dfr and Cad promoted very high CecA1 expression, indicating that these two transcription factors act synergistically on CecA1 in tissues where both are expressed. In Pdm1 mutant flies, several AMP genes were highly expressed prior to infection, indicating that Pdm1 functions as a repressor of those genes. However, at least one gene, AttacinA, required Pdm1 for its expression suggesting that Pdm1 has dual functions, acting both as a repressor and activator. Finally, the post-translational activation of the NF-κB/Rel protein Relish in response to infection was investigated in detail. Deletion mapping revealed different functional domains of Relish, and site-directed mutagenesis was used to exactly determine the residues required for endoproteolytic cleavage by a caspase.
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| 4. |
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| 5. |
- Junell, Anna, et al.
(författare)
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The POU Transcription Factor Drifter/Ventral veinless Regulates Expression of Drosophila Immune Defence Genes
- 2010
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Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 30:14, s. 3672-3684
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Innate immunity operates as a first line of defense in multicellular organisms against infections caused by different classes of microorganisms. Antimicrobial peptides (AMPs) are synthesized constitutively in barrier epithelia to protect against microbial attack and are also upregulated in response to infection. Here, we implicate Drifter/Ventral veinless (Dfr/Vvl), a class III POU domain transcription factor, in tissue-specific regulation of the innate immune defense of Drosophila. We show that Dfr/Vvl is highly expressed in a range of immunocompetent tissues, including the male ejaculatory duct, where its presence overlaps with and drives the expression of cecropin, a potent broad-spectrum AMP. Dfr/Vvl overexpression activates transcription of several AMP genes in uninfected flies in a Toll pathway- and Imd pathway-independent manner. Dfr/Vvl activates a CecA1 reporter gene both in vitro and in vivo by binding to an upstream enhancer specific for the male ejaculatory duct. Further, Dfr/Vvl and the homeodomain protein Caudal (Cad) activate transcription synergistically via this enhancer. We propose that the POU protein Dfr/Vvl acts together with other regulators in a combinatorial manner to control constitutive AMP gene expression in a gene-, tissue-, and sex-specific manner, thus promoting a first-line defense against infection in tissues that are readily exposed to pathogens.
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| 6. |
- Stöven, Svenja, et al.
(författare)
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Caspase-mediated processing of the Drosophila NF-kappaB factor Relish.
- 2003
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Ingår i: Proc Natl Acad Sci U S A. - 0027-8424. ; 100:10, s. 5991-6
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Tidskriftsartikel (refereegranskat)abstract
- The NF-kappaB-like transcription factor Relish plays a central role in the innate immune response of Drosophila. Unlike other NF-kappaB proteins, Relish is activated by endoproteolytic cleavage to generate a DNA-binding Rel homology domain and a stable IkappaB-like fragment. This signal-induced endoproteolysis requires the activity of several gene products, including the IkappaB kinase complex and the caspase Dredd. Here we used mutational analysis and protein microsequencing to demonstrate that a caspase target site, located in the linker region between the Rel and the IkappaB-like domain, is the site of signal-dependent cleavage. We also show physical interaction between Relish and Dredd, suggesting that Dredd indeed is the Relish endoprotease. In addition to the caspase target site, the C-terminal 107 aa of Relish are required for endoproteolysis and signal-dependent phosphorylation by the Drosophila IkappaB kinase beta. Finally, an N-terminal serine-rich region in Relish and the PEST domain were found to negatively regulate Relish activation.
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| 7. |
- Stöven, Svenja, et al.
(författare)
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Caspase-mediated processing of the Drosophila NF-κB factor Relish
- 2003
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 100:10, s. 5991-5996
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Tidskriftsartikel (refereegranskat)abstract
- The NF-κB-like transcription factor Relish plays a central role in the innate immune response of Drosophila. Unlike other NF-κB proteins, Relish is activated by endoproteolytic cleavage to generate a DNA-binding Rel homology domain and a stable IκB-like fragment. This signal-induced endoproteolysis requires the activity of several gene products, including the IκB kinase complex and the caspase Dredd. Here we used mutational analysis and protein microsequencing to demonstrate that a caspase target site, located in the linker region between the Rel and the IκB-like domain, is the site of signal-dependent cleavage. We also show physical interaction between Relish and Dredd, suggesting that Dredd indeed is the Relish endoprotease. In addition to the caspase target site, the C-terminal 107 aa of Relish are required for endoproteolysis and signal-dependent phosphorylation by the Drosophila IκB kinase β. Finally, an N-terminal serine-rich region in Relish and the PEST domain were found to negatively regulate Relish activation.
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| 8. |
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| 9. |
- Wiklund, Magda-Lena, et al.
(författare)
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The N-terminal half of the Drosophila Rel/NF-kappa B factor Relish, REL-68, constitutively activates transcription of specific Relish target genes
- 2009
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Ingår i: Developmental and Comparative Immunology. - New York : Elsevier BV. - 0145-305X .- 1879-0089. ; 33:5, s. 690-696
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Tidskriftsartikel (refereegranskat)abstract
- The Rel/NF-kappa B transcription factor Relish is a major regulator of the antimicrobial response in Drosophila. Upon immune challenge, Relish is cleaved to generate two fragments, the DNA-binding transcription factor REL-68 and the I kappa B-like REL-49. Using transgenic fly strains we show here that overexpression of REL-68 separately from REL-49 is sufficient to activate strong constitutive transcription of the Diptericin gene, but little constitutive or inducible transcription of Attacin and Cecropin, two other Relish target genes. Their transcription may therefore require additional modifications of Relish. However, phosphorylation of the conserved serine residue S431 is not involved in such modifications. This is unlike p65 and Dorsal, which are modulated by phosphorylation at their homologous site. In contrast to other I kappa B proteins, overexpression of REL-49 had no inhibitory effect on Relish-dependent transcription. Instead, we propose that the C-terminal I kappa B-like domain executes a scaffolding and recruiting function for full activation of Relish.
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