Reference

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.

Kappler C Meister M Lagueux M Gateff E Hoffmann JA Reichhart JM
The EMBO journal. 1993.

Abstract

The Drosophila diptericin gene codes for a 9 kDa antibacterial peptide and is rapidly and transiently expressed in larvae and adults after bacterial challenge. It is also induced in a tumorous Drosophila blood cell line by the addition of lipopolysaccharide (LPS). The promoter of this gene contains two 17 bp repeats located closely upstream of the TATA-box and harbouring a decameric kappa B-related sequence. This study reports that the replacement of the two 17 bp repeats by random sequences abolishes bacteria inducibility in transgenic fly lines. In transfected tumorous blood cells, the replacement of both or either of the 17 bp motifs reduces dramatically LPS inducibility, whereas multiple copies significantly increase the level of transcriptional activation by LPS challenge. A specific DNA-protein binding activity is evidenced in cytoplasmic and nuclear extracts of induced blood cells and fat body. It is absent in controls. It is proposed that induction of the diptericin gene mediated by the two 17 bp repeats occurs via a mechanism similar to that of mammalian NF-kappa B.

Main claims

  1. Two 17 bp repeats containing mammalian kB-like sequences confer inducibility to the Diptericin (DptA) gene and bind a polypeptide in immune-challenged mbn-2 cells and Drosophila. [v1]
    Validated by the identification of Relish: many studies have shown that Relish binds the Diptericin gene in response to immune challenge and activates transcription, and that this requires the kB sites (Busse et al., 2007; Meister et al., 1994; Zhang and Palli, 2009).

    Last change on 2022-08-15 09:36 by Anonymous

Major claims

  1. Two 17bp repeats in promoter of the Diptericin (DptA) gene are required for inducibility of a reporter in vitro and in vivo. [v1]
    (Busse et al., 2007) found three consensus kB sites in DptA all of which probably affect level of induction, although one kB site is sufficient to confer pathway specificity. Also directly supported by (Meister et al., 1994) (same group) who did similar experiments and found two kB sites were absolutely required for Dpt expression while additional motifs were necessary for full wild-type inducibility, and by (Zhang and Palli, 2009) who also found that both of these sites were required for full expression.

    Last change on 2022-08-15 09:36 by Anonymous

    1. Dpt-CAT reporter activity was enhanced (7-fold) upon addition of LPS to mbn-2 cells. [v1]

      To be assessed

      Last change on 2022-08-14 15:09 by Anonymous

    2. Replacement of either of the 17bp motifs with random sequence reduced dramatically LPS inducibility in transfected mbn-2 cells; replacement of both abolished LPS inducibility. [v1]

      To be assessed

      Last change on 2022-08-14 15:09 by Anonymous

    3. After injection of live bacteria (E. coli) in larvae and adult flies, transgenic Dpt-lacZ flies had strong reporter activity in the fat body while controls with the 17bp motifs replaced with random sequence did not (confirmed with Northern blot). [v1]

      To be assessed

      Last change on 2022-08-14 15:10 by Anonymous

  2. Reporter inducibility in response to LPS challenge varies with copy number of the 17bp repeat from the Diptericin (DptA) promoter. [v1]
    Other studies have shown that many AMPs have multiple kB sites and full inducibility requires several or all sites (e.g. (Busse et al., 2007; Charlet et al., 1996) showed both upstream and downstream kB sequences were required for full inducibility of Drosocin). Other studies have also used the technique of placing many repeat kB sites in front of a reporter construct to strengthen its expression, e.g. (Georgel et al., 1995).

    Last change on 2022-08-15 09:37 by Anonymous

    1. Mbn-2 cells transfected with 8 copies of the 17bp repeat fused in tandem upstream of a reporter cassette had a 40-fold enhanced CAT activity. [v1]

      To be assessed

      Last change on 2022-08-14 15:11 by Anonymous

    2. Replacement of either endogenous 17bp repeat reduced but did not abolish reporter activity. [v1]

      To be assessed

      Last change on 2022-08-14 15:11 by Anonymous

  3. Proteins interact with the 17bp motifs of the Diptericin (DptA) reporter in vitro and in vivo. [v1]
    Supported by (Busse et al., 2007; Copley et al., 2007), although the consensus kB binding sequence is shorter than 17bp.

    Last change on 2022-08-15 09:37 by Anonymous

    1. EMSA showed that mbn-2 cells induced with extracts of LPS, or live or heat-killed bacteria- contained a protein-DNA complex detected by an oligonucleotide probe for the 17bp repeats. [v1]

      To be assessed

      Last change on 2022-08-14 15:11 by Anonymous

    2. The 17bp oligonucleotide probe detected protein-DNA complexes similar to those detected in mbn-2 cells in fat body but not gut extracts of whole flies following bacterial challenge. [v1]

      To be assessed

      Last change on 2022-08-14 15:11 by Anonymous

    3. A stronger binding signal was detected in cytoplasmic extracts than nuclear extracts. [v1]

      To be assessed

      Last change on 2022-08-14 15:12 by Anonymous

    4. The presence of two bands in fat body extracts but not mbn-2 cells may indicate protein modification. [v1]

      To be assessed

      Last change on 2022-08-14 15:12 by Anonymous

Minor claims

  1. Protein synthesis is not required for formation of the Diptericin (DptA) protein-DNA complex, but is required to maintain it. [v1]
    Validated by the observation that Relish protein is present in untreated cells but strongly induced by infection (De Gregorio et al., 2001; Dushay et al., 1996); multiple other components of the Imd pathway are also actively synthesized and degraded during the immune response.

    Last change on 2022-08-15 09:37 by Anonymous

    1. Cycloheximide did not inhibit formation of the protein-DNA complex in response to LPS, but complex formation was reduced after 2h in cycloheximide-treated cells while it persisted strongly in untreated cells. [v1]

      To be assessed

      Last change on 2022-08-14 15:12 by Anonymous

  2. The protein binding to the 17bp Diptericin (DptA) promoter sequence has a mass of 75-80 kDa. [v1]
    Gels used to reach this conclusion were run in reducing conditions, so the Rel proteins binding to DNA would probably have been present as monomers rather than dimers. This protein likely represented the active cleaved form of Relish (Rel-68, 68kDa), but the molecular weight is higher than expected. Mature DIF is 74kDa and Dorsal is either 75 or 111 kDa depending on isoform, which suggests Dif or Dl is a better match for this protein; however Diptericin kB sites specifically bind Relish and not DIF or Dorsal (Busse et al., 2007; Copley et al., 2007). This suggests this result is inaccurate, or that NF-kB protein in this study bore modifications such as phosphoryl or SUMO groups. See also (Gross et al., 1999) who discuss differences between predicted and measured molecular weights of Dorsal and provide an explanation for this.

    Last change on 2022-08-15 09:38 by Anonymous

    1. Hypothesized to be related to or identical to dorsal based on hybridization with a probe. [v1]

      To be assessed

      Last change on 2022-08-14 15:14 by Anonymous

  3. Diptericin (DptA) expression is controlled via a mechanism similar to that of the mammalian NF-kB system. [v1]
    Extensively supported (see e.g. (Busse et al., 2007; Copley et al., 2007))

    Last change on 2022-08-15 09:38 by Anonymous

    1. The two Diptericin 17bp repeats harbor a decameric kB-related sequence homologous to mammals. [v1]

      To be assessed

      Last change on 2022-08-14 15:15 by Anonymous

    2. The mammalian NF-kB subunit p50 forms a DNA-protein complex with the 17bp Drosophila diptericin promoter motif; competed by Drosophila wild-type sequence. [v1]

      To be assessed

      Last change on 2022-08-14 15:15 by Anonymous

    3. The 17bp probe-protein complex formed in bacteria-challenged Drosophila is competed by an excess of mammalian kB consensus probe. [v1]

      To be assessed

      Last change on 2022-08-14 15:15 by Anonymous

Methods

  1. Mbn-2 cells and flies transfected with wild-type and mutated versions of the Diptericin promoter fused to a reporter; EMSA using radioactive 17bp oligonucleotide probe; competitive binding; crude LPS and E. coli bacterial challenge. [v1]

    Last change on 2022-08-14 15:15 by Anonymous

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]

    Charlet M, Lagueux M, Reichhart JM, Hoffmann D, Braun A, Meister M. 1996. Cloning of the gene encoding the antibacterial peptide drosocin involved in Drosophila immunity. Expression studies during the immune response. Eur J Biochem 241:699–706. [DOI link]

    Copley RR, Totrov M, Linnell J, Field S, Ragoussis J, Udalova IA. 2007. Functional conservation of Rel binding sites in drosophilid genomes. Genome Res 17:1327–1335. [DOI link]

    De Gregorio E, Spellman PT, Rubin GM, Lemaitre B. 2001. Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays. Proc Natl Acad Sci USA 98:12590–12595. [DOI link]

    Dushay MS, Asling B, Hultmark D. 1996. Origins of immunity: Relish, a compound Rel-like gene in the antibacterial defense of Drosophila. Proc Natl Acad Sci USA 93:10343–10347. [DOI link]

    Georgel P, Kappler C, Langley E, Gross I, Nicolas E, Reichhart JM, Hoffmann JA. 1995. Drosophila immunity. A sequence homologous to mammalian interferon consensus response element enhances the activity of the diptericin promoter. Nucleic Acids Res 23:1140–1145. [DOI link]

    Gross I, Georgel P, Oertel-Buchheit P, Schnarr M, Reichhart JM. 1999. Dorsal-B, a splice variant of the Drosophila factor Dorsal, is a novel Rel/NF-kappaB transcriptional activator. Gene 228:233–242. [DOI link]

    Meister M, Braun A, Kappler C, Reichhart JM, Hoffmann JA. 1994. Insect immunity. A transgenic analysis in Drosophila defines several functional domains in the diptericin promoter. EMBO J 13:5958–5966. [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]

    [v1]

    Last change on 2022-08-14 15:18 by Anonymous