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The Drosophila transcription factor Pdm1 inhibits antimicrobial peptide gene expression
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
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Identifiers
URN: urn:nbn:se:su:diva-22720OAI: oai:DiVA.org:su-22720DiVA: diva2:189315
Note
Part of urn:nbn:se:su:diva-1051Available from: 2006-05-01 Created: 2006-05-01 Last updated: 2011-03-28Bibliographically approved
In thesis
1. Signaling and transcriptional regulation of antimicrobial peptide genes in Drosophila melanogaster
Open this publication in new window or tab >>Signaling and transcriptional regulation of antimicrobial peptide genes in Drosophila melanogaster
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Insects rely solely on innate immune reactions for protection against infect-ing microbes in their environment. In Drosophila, one major defense mechanism is the production of a battery of antimicrobial peptides (AMPs). The expression of AMPs is primarily regulated at the level of transcription and constitutes both constitutive expression in a tissue-specific manner and inducible systemic expression in response to infection. The aim of my thesis has been to investigate the regulation of AMP gene expression at different levels. I have studied a novel cis-regulatory element, Region 1 (R1) found in the proximal promoter of all Cecropin genes in Drosophila melanogaster, as well as in other species of Drosophila. We found that the R1 element was important for the expression of CecropinA1 (CecA1) both in vitro and in vivo. A signaling-dependent R1-binding activity (RBA) was identified in nuclear extracts from Drosophila cells and flies. The molecular nature of the RBA, has despite considerable effort, not yet been identified. I also have studied the role of the JNK pathway in transcriptional regulation of AMP genes. The role of the JNK pathway in the regulation of AMP genes has long been elusive, however, in this study we showed that the pathway is directly involved in the expression of AMP genes. Analysis of cells mutant for JNK pathway components showed severely reduced AMP gene expression. Fur-thermore, over-expression of a JNK pathway-inhibitor also inhibited AMP gene expression. Lastly, I have studied transcription factors that have not previously been implicated in transcriptional regulation of AMP genes. In a yeast screen, three members of the POU family of transcription factors were identified as regulators of CecA1. Two of them, Drifter (Dfr) and POU do-main protein 1 (Pdm1) were further characterized. We showed that Dfr was able to promote AMP gene expression in the absence of infection, suggest-ing it to play a role in constitutive expression of AMP genes. Indeed, down-regulation of Dfr expression using RNAi severely reduced the constitutive expression of AMP genes in the male ejaculatory duct. We also identified an enhancer element important for Dfr-mediated expression of CecA1. Pdm1, on the other hand, was shown to be important for the systemic expression of AMP genes. In Pdm1 mutant flies, several AMP genes are systemically expressed even in the absence of infection, suggesting that Pdm1 works as a repressor of those genes. However, at least on AMP gene, AttacinA (AttA) requires Pdm1 for its expression, suggesting that Pdm1 works as an activator for this gene. Upon infection, Pdm1 was rapidly degraded, but, regenerated shortly after infection. We propose that the degradation of Pdm1 is important for the activation of the Pdm1-repressed genes and that regeneration sup-ports the expression of AttA.

Place, publisher, year, edition, pages
Stockholm: Institutionen för molekylärbiologi och funktionsgenomik, 2006. 70 p.
Keyword
antimicrobial peptides, drosophila, immunity, transcriptiona regulation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-1051 (URN)91-7155-263-4 (ISBN)
Public defence
2006-06-08, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 10:00
Opponent
Supervisors
Available from: 2006-05-01 Created: 2006-05-01 Last updated: 2011-03-28Bibliographically approved
2. Regulation of antimicrobial peptide gene expression in Drosophila melanogaster: Involvement of POU and NF-kB/Rel factors in innate immunity
Open this publication in new window or tab >>Regulation of antimicrobial peptide gene expression in Drosophila melanogaster: Involvement of POU and NF-kB/Rel factors in innate immunity
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:nbn:se:su:diva-6614 (URN)91-7155-370-3 (ISBN)
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

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