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Regulation of antimicrobial peptide gene expression in Drosophila melanogaster: Involvement of POU and NF-kB/Rel factors in innate immunity
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Institutionen för molekylärbiologi och funktionsgenomik , 2007. , 136 p.
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-6614ISBN: 91-7155-370-3 (print)OAI: oai:DiVA.org:su-6614DiVA: diva2:196754
Public defence
2007-02-22, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 10:00
Opponent
Supervisors
Available from: 2007-02-01 Created: 2007-02-01 Last updated: 2011-03-28Bibliographically approved
List of papers
1. The POU protein Drifter activates antimicrobial peptide gene expression in Drosophila
Open this publication in new window or tab >>The POU protein Drifter activates antimicrobial peptide gene expression in Drosophila
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Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-22719 (URN)
Note
Part of urn:nbn:se:su:diva-1051Available from: 2006-05-01 Created: 2006-05-01 Last updated: 2011-03-28Bibliographically approved
2. The Drosophila transcription factor Pdm1 inhibits antimicrobial peptide gene expression
Open this publication in new window or tab >>The Drosophila transcription factor Pdm1 inhibits antimicrobial peptide gene expression
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Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-22720 (URN)
Note
Part of urn:nbn:se:su:diva-1051Available from: 2006-05-01 Created: 2006-05-01 Last updated: 2011-03-28Bibliographically approved
3. Isolation of regulators of Drosophila immune defense genes by a double interaction screen in yeast
Open this publication in new window or tab >>Isolation of regulators of Drosophila immune defense genes by a double interaction screen in yeast
2007 (English)In: Insect Biochemistry and Molecular Biology, ISSN 0965-1748, E-ISSN 1879-0240, Vol. 37, no 3, 202-212 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Innate immunity, Cecropin, Gene regulation, POU transcription factors, NF-kappaB, Antimicrobial peptides, Yeast screen
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-24100 (URN)10.1016/j.ibmb.2006.10.008 (DOI)
Available from: 2007-02-01 Created: 2007-02-01 Last updated: 2017-12-13Bibliographically approved
4. Caspase-mediated processing of the Drosophila NF-κB factor Relish
Open this publication in new window or tab >>Caspase-mediated processing of the Drosophila NF-κB factor Relish
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2003 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 100, no 10, 5991-5996 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-24103 (URN)10.1073/pnas.1035902100 (DOI)
Available from: 2007-02-01 Created: 2007-02-01 Last updated: 2017-12-13Bibliographically approved

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