Identification and characterization of a Drosophila nuclear proteasome regulator. A homolog of human 11 S REGgamma (PA28gamma ).

We report the cloning and characterization of a Drosophila proteasome 11 S REGgamma (PA28) homolog. The 28-kDa protein shows 47% identity to the human REGgamma and strongly enhances the trypsin-like activities of both Drosophila and mammalian 20 S proteasomes. Surprisingly, the Drosophila REG was found to inhibit the proteasome's chymotrypsin-like activity against the fluorogenic peptide succinyl-LLVY-7-amino-4-methylcoumarin. Immunocytological analysis reveals that the Drosophila REG is localized to the nucleus but is distributed throughout the cell when nuclear envelope breakdown occurs during mitosis. Through site-directed mutagenesis studies, we have identified a functional nuclear localization signal present in the homolog-specific insert region. The Drosophila PA28 NLS is similar to the oncogene c-Myc nuclear localization motif. Comparison between uninduced and innate immune induced Drosophila cells suggests that the REGgamma proteasome activator has a role independent of the invertebrate immune system. Our results support the idea that gamma class proteasome activators have an ancient conserved function within metazoans and were present prior to the emergence of the alpha and beta REG classes.

Serpent regulates Drosophila immunity genes in the larval fat body through an essential GATA motif.

Insects possess a powerful immune system, which in response to infection leads to a vast production of different antimicrobial peptides. The regulatory regions of many immunity genes contain a GATA motif in proximity to a kappaB motif. Upon infection, Rel proteins enter the nucleus and activate transcription of the immunity genes. High levels of Rel protein-mediated Cecropin A1 expression previously have been shown to require the GATA site along with the kappaB site. We provide evidence demonstrating that the GATA motif is needed for expression of the Cecropin A1 gene in larval fat body, but is dispensable in adult fat body. A nuclear DNA-binding activity interacts with the Cecropin A1 GATA motif with the same properties as the Drosophila GATA factor Serpent. The GATA-binding activity is recognized by Serpent-specific antibodies, demonstrating their identity. We show that Serpent is nuclear in larval fat body cells and haemocytes both before and after infection. After overexpression, Serpent increases Cecropin A1 transcription in a GATA-dependent manner. We propose that Serpent plays a key role in tissue-specific expression of immunity genes, by priming them for inducible activation by Rel proteins in response to infection.

Adjacent GATA and kappa B-like motifs regulate the expression of a Drosophila immune gene.

The GATA motif is a well known positive cis -regulatory element in vertebrates. In this work we report experimental evidence for the direct participation of a GATA motif in the expression of the Drosophila antibacterial peptide gene Cecropin A1 . Previously we have shown that a kappaB-like site is necessary for Cecropin A1 gene expression. Here we present evidence that the Drosophila Rel protein which binds to the kappaB-like site requires an intact GATA site for maximal Dif-mediated transactivation of the Cecropin A1 gene. We show that a Drosophila blood cell line contains factors binding specifically to the GATA motif of the Cecropin A1 gene. The GATA binding activity is likely to include member(s) of the GATA family of transcriptional regulators. We show that the promoters of several inducible insect immune genes possess GATA sites 0-12 base pairs away from kappaB-like sites in functionally important promoter regions. Clusters of GATA and kappaB sites are also observed in the promoters of two important mammalian immune genes, namely IL6 and IL3. The consistent proximity of GATA and kappaB sites appears to be a common theme in the immune gene expression of insects and mammals.

The dorsal-related immunity factor, Dif, is a sequence-specific trans-activator of Drosophila Cecropin gene expression.

A new member of the Rel family of transcription factors, the dorsal-related immunity factor, Dif, was recently cloned and suggested to be involved in regulating the immune response in Drosophila. Despite its classification as a Rel family member, the Dif cDNA-encoded product has not been proven previously to be a transcription factor. We now present evidence that the Dif gene product trans-activates the Drosophila Cecropin A1 gene in co-transfection assays. The transactivation requires a 40 bp upstream element including an insect kappa B-like motif. A dimer of the kappa B-like motif 5'-GGGGATTTTT inserted into a minimal promoter conferred high levels of reporter gene expression by Dif, while a multimer of several mutated versions of this motif was not activated, demonstrating the sequence specificity of Dif. Full trans-activation by Dif requires the C-terminal part of the protein. The morphogen dorsal (dl) can also activate the Cecropin A1 promoter, but to a lesser extent and in a less sequence-specific manner than Dif. Simultaneous overexpression of Dif and dl in co-transfection assays revealed that dl possesses a dominant negative effect on Dif transactivation. This study establishes that Dif is a sequence-specific transcription factor and is probably a key activator of the immune response in Drosophila.