Reference

Insect immunity. A transgenic analysis in Drosophila defines several functional domains in the diptericin promoter.

Meister M Braun A Kappler C Reichhart JM Hoffmann JA
The EMBO journal. 1994.

Abstract

Diptericins are antibacterial polypeptides which are strongly induced in the fat body and blood cells of dipteran insects in response to septic injury. The promoter of the single-copy, intronless diptericin gene of Drosophila contains several nucleotide sequences homologous to mammalian cis-regulatory motifs involved in the control of acute phase response genes. Extending our previous studies on the expression of the diptericin gene, we now report a quantitative analysis of the contribution of various putative regulatory elements to the bacterial inducibility of this gene, based on the generation of 60 transgenic fly lines carrying different elements fused to a reporter gene. Our data definitively identify two Kappa B-related motifs in the proximal promoter as the sites conferring inducibility and tissue-specific expression to the diptericin gene. These motifs alone, however, mediate only minimal levels of expression. Additional proximal regulatory elements are necessary to attain some 20% of the full response and we suspect a role for sequences homologous to mammalian IL6 response elements and interferon-gamma responsive sites in this up-regulation. The transgenic experiments also reveal the existence of a distal regulatory element located upstream of -0.6 kb which increases the level of expression by a factor of five.

Main claims

  1. Transgenic analysis reveals that two κB-related motifs are absolutely required for Diptericin DptA expression while two additional motifs in the promoter are required for full expression. [v1]
    Subsequent studies show that Diptericin has three kB sites that function in inducibility, and that additional motifs such as GATA sites also contribute to expression (Busse et al., 2007; Senger et al., 2006; Zhang and Palli, 2009).

    Last change on 2020-08-28 10:36 by Hannah Westlake Orcid 0009-0000-0013-8980

Major claims

  1. Two κB-related motifs in the proximal promoter confer inducibility and tissue-specific expression of the Diptericin DptA gene in larvae. [v1]
    Previously shown by (Kappler et al., 1993), subsequent studies show that at least one additional kB site is present (Busse et al., 2007) and that other elements in the promoter such as GATA sites are required for full inducibility and tissue specificity (Senger et al., 2006).

    Last change on 2020-08-28 10:37 by Hannah Westlake Orcid 0009-0000-0013-8980

    1. 2.2 kb of the upstream sequence of the Diptericin gene only conferred full inducibility by bacterial challenge to a lacZ reporter gene when it carried both 17 bp κB-related motifs; indicibility was totally lost when these two motifs were mutated. [v1]

      To be assessed

      Last change on 2020-08-28 10:47 by Hannah Westlake Orcid 0009-0000-0013-8980

    2. Histochemical analysis showed intense lacZ staining in all cells of wild type lines; however, mutated lines with 8 copies of the 17 bp κB-related sequence fused to the reporter expressed cell-autonomously in the fat body (some cells reacted strongly and some weakly), displayed an aberrant local reaction around puncture wounds, and had reduced expression in hemocytes. [v1]

      To be assessed

      Last change on 2020-08-28 10:49 by Hannah Westlake Orcid 0009-0000-0013-8980

  2. The two κB-related motifs alone mediate only minimal levels of Diptericin DptA expression in larvae. [v1]
    Consistent with the fact that full inducibility of AMPs requires multiple regulatory elements (Busse et al., 2007) and that other elements in the Diptericin promoter such as GATA sites are required for full inducibility and tissue specificity (Senger et al., 2006)

    Last change on 2022-09-06 15:19 by Hannah Westlake Orcid 0009-0000-0013-8980

    1. Mutation of one of the two 17 bp κB-related motifs (proximal or distal) resulted in inducibility at ~10% of the wild type response. [v1]

      To be assessed

      Last change on 2020-08-28 10:52 by Hannah Westlake Orcid 0009-0000-0013-8980

    2. A transgene with a minimal promoter carrying 8 copies of the 17bp κB-related repeat fused to the lacZ reporter conferred less than 10% of wild-type inducibility. [v1]

      To be assessed

      Last change on 2020-08-28 10:53 by Hannah Westlake Orcid 0009-0000-0013-8980

  3. All elements regulating Diptericin DptA expression are contained within the 2.2 kb of sequence upstream of the open reading frame [v1]
    Supported by further studies that show many regulatory elements of AMPs are present within the 200bp upstream (Busse et al., 2007), and many studies that use AMP reporters driven by this upstream sequence (e.g. (Tzou et al., 2000)). Seemingly contradicted by (Reichhart et al., 1992) (same group) who showed that inducibility of a Dpt-lacZ reporter was reduced compared to endogenous Diptericin in first and second instar larvae and adults in response to E. coli or CFA, but this difference is here explained by variability among treatments or differences between sexes.

    Last change on 2022-09-06 15:20 by Hannah Westlake Orcid 0009-0000-0013-8980

    1. • Lines containing 0.6 kb of upstream Diptericin coding sequences were able to sustain ~20% of the 2.2 kb response in larvae and adults; a distal regulatory element located between 0.6 and 2.2 kb which may be a third somewhat divergent kB site at 0.8 kB increases the level of expression 5-fold (from 20% to 100%) [v1]

      Supported by (Busse et al., 2007; Senger et al., 2006; Zhang and Palli, 2009), this kB site has a slightly different sequence than the other two and appears to require co-operation of an ecdysone-mediated factor to induce full expression of the gene.

      Last change on 2022-09-06 15:20 by Hannah Westlake Orcid 0009-0000-0013-8980

    2. • Lines containing additional elements within or downstream the coding sequence responded similarly to 2.2 kb lines (upstream sequence + hsp70 termination element or 2.2 kb + 1.1kb downstream sequence) [v1]

      To be assessed

      Last change on 2022-09-06 15:20 by Hannah Westlake Orcid 0009-0000-0013-8980

    3. • Lines containing upstream sequences of longer lengths (3.7 kb or 11 kb) responded similarly to 2.2 kb [v1]

      To be assessed

      Last change on 2022-09-06 15:21 by Hannah Westlake Orcid 0009-0000-0013-8980

Minor claims

  1. Dpt-lacZ is expressed upon infection in a subset of hemocytes. [v1]
    (Foley and O’Farrell, 2003) showed that Dpt-lacZ was expressed in a subset of larval hemocytes upon natural Ecc infection; (Irving et al., 2005) found that E. coli+M. luteus infection poorly induced Diptericin in larval hemocytes (Affymetrix)

    Last change on 2020-08-28 10:40 by Hannah Westlake Orcid 0009-0000-0013-8980

  2. Dpt-lacZ is expressed in the integument of larvae at the injury site. [v1]
    (Tingvall et al., 2001) found Imd-dependent expression of CecA1 in the epidermis underlying wound sites in larvae; if CecA1 is expressed in response to wounding it is logical that other Imd-dependent genes like Dpt would also be.

    Last change on 2020-08-28 10:40 by Hannah Westlake Orcid 0009-0000-0013-8980

  3. Diptericin is more strongly induced in larvae than in adults, and in females than in males. [v1]
    (Tapadia and Verma, 2012) also found induction of Diptericin in response to infection was higher in larvae than adults, although this may be due primarily to differences in basal levels between stages. No direct verification is available that shows Diptericin is more highly expressed in females than males, but there is sexual dimorphism in various aspects of the immune response, including differences in AMP expression (Belmonte et al 2020). Some results also suggest that basal levels of AMPs are higher in males, resulting in a lesser apparent induction (Junell et al., 2010).

    Last change on 2020-08-28 10:42 by Hannah Westlake Orcid 0009-0000-0013-8980

Methods

  1. Generation of transgenic fly lines with wild-type and mutated versions of the Diptericin promoter fused to a lacZ reporter, B-galactosidase assays. [v1]

    Last change on 2020-08-28 10:42 by Hannah Westlake Orcid 0009-0000-0013-8980

Additional context

Not annotated yet

Additional files

Not annotated yet

References

  1. Busse MS, Arnold CP, Towb P, Katrivesis J, Wasserman SA. 2007. A kappaB sequence code for pathway-specific innate immune responses. EMBO J 26:3826–3835. [DOI link]

    Foley E, O’Farrell PH. 2003. Nitric oxide contributes to induction of innate immune responses to gram-negative bacteria in Drosophila. Genes Dev 17:115–125. [DOI link]

    Irving P, Ubeda J-M, Doucet D, Troxler L, Lagueux M, Zachary D, Hoffmann JA, Hetru C, Meister M. 2005. New insights into Drosophila larval haemocyte functions through genome-wide analysis. Cell Microbiol 7:335–350. [DOI link]

    Junell A, Uvell H, Davis MM, Edlundh-Rose E, Antonsson A, Pick L, Engström Y. 2010. The POU transcription factor Drifter/Ventral veinless regulates expression of Drosophila immune defense genes. Mol Cell Biol 30:3672–3684. [DOI link]

    Kappler C, Meister M, Lagueux M, Gateff E, Hoffmann JA, Reichhart JM. 1993. Insect immunity. Two 17 bp repeats nesting a kappa B-related sequence confer inducibility to the diptericin gene and bind a polypeptide in bacteria-challenged Drosophila. EMBO J 12:1561–1568.

    Reichhart JM, Meister M, Dimarcq JL, Zachary D, Hoffmann D, Ruiz C, Richards G, Hoffmann JA. 1992. Insect immunity: developmental and inducible activity of the Drosophila diptericin promoter. EMBO J 11:1469–1477.

    Senger K, Harris K, Levine M. 2006. GATA factors participate in tissue-specific immune responses in Drosophila larvae. Proc Natl Acad Sci USA 103:15957–15962. [DOI link]

    Tapadia MG, Verma P. 2012. Immune Response and Anti-Microbial Peptides Expression in Malpighian Tubules of Drosophila melanogaster Is under Developmental Regulation. PLOS ONE 7:e40714. [DOI link]

    Tingvall TO, Roos E, Engström Y. 2001. The GATA factor Serpent is required for the onset of the humoral immune response in Drosophila embryos. Proc Natl Acad Sci USA 98:3884–3888. [DOI link]

    Tzou P, Ohresser S, Ferrandon D, Capovilla M, Reichhart JM, Lemaitre B, Hoffmann JA, Imler JL. 2000. Tissue-specific inducible expression of antimicrobial peptide genes in Drosophila surface epithelia. Immunity 13:737–748. [DOI link]

    Zhang Z, Palli SR. 2009. Identification of a cis-regulatory element required for 20-hydroxyecdysone enhancement of antimicrobial peptide gene expression in Drosophila melanogaster. Insect Mol Biol 18:595–605. [DOI link]

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    Last change on 2022-09-06 15:23 by Hannah Westlake Orcid 0009-0000-0013-8980