The helix-loop-helix proteins dAP-4 and daughterless bind both in vitro and in vivo to SEBP3 sites required for transcriptional activation of the Drosophila gene Sgs-4.

The expression of Sgs genes in the salivary gland of the third instar larva of Drosophila is a spatially restricted response to signalling by the steroid hormone 20-hydroxyecdysone. For Sgs-4, we have previously demonstrated that its strictly tissue and stage-specific expression is the result of combined action of the ecdysone receptor and secretion enhancer binding proteins (SEBPs). One of these SEBPs, SEBP2, was shown to be the product of the homeotic gene fork head. Together with SEBP3, SEBP2 appears to be responsible for the spatial restriction of the hormone response of Sgs-4. Here, we show that SEBP3 is a heterogeneous binding activity that consists of different helix-loop-helix (HLH) proteins. We cloned the Drosophila homologue of human transcription factor AP-4 (dAP-4) and identified it as one of these HLH proteins. The dAP-4 protein shows great similarity to its human and Caenorhabditis counterparts within the bHLHZip domain, the second leucine zipper dimerization motif, and a third region of unknown function. The expression pattern of dAP-4 indicates that it is a ubiquitously expressed HLH protein in Drosophila. As a second component of SEBP3 we identified the Daughterless (Da) protein, which is also ubiquitously expressed and binds to SEBP3 sites independent of dAP-4. Since both dAP-4 and Da can be detected in situ at transposed Sgs-4 transcriptional control elements in polytene salivary gland chromosomes, we propose that each of the two proteins contributes to the transcriptional control of Sgs-4.

The Hippo pathway promotes cell survival in response to chemical stress.

Cellular stress defense mechanisms have evolved to maintain homeostasis in response to a broad variety of environmental challenges. Stress signaling pathways activate multiple cellular programs that range from the activation of survival pathways to the initiation of cell death when cells are damaged beyond repair. To identify novel players acting in stress response pathways, we conducted a cell culture RNA interference (RNAi) screen using caffeine as a xenobiotic stress-inducing agent, as this compound is a well-established inducer of detoxification response pathways. Specifically, we examined how caffeine affects cell survival when Drosophila kinases and phosphatases were depleted via RNAi. Using this approach, we identified and validated 10 kinases and 4 phosphatases that are essential for cell survival under caffeine-induced stress both in cell culture and living flies. Remarkably, our screen yielded an enrichment of Hippo pathway components, indicating that this pathway regulates cellular stress responses. Indeed, we show that the Hippo pathway acts as a potent repressor of stress-induced cell death. Further, we demonstrate that Hippo activation is necessary to inhibit a pro-apoptotic program triggered by the interaction of the transcriptional co-activator Yki with the transcription factor p53 in response to a range of stress stimuli. Our in vitro and in vivo loss-of-function data therefore implicate Hippo signaling in the transduction of cellular survival signals in response to chemical stress.

Downregulation of the tissue-specific transcription factor Fork head by Broad-Complex mediates a stage-specific hormone response.

Drosophila development is coordinated by pulses of the steroid hormone 20-hydroxyecdysone (20E). During metamorphosis, the 20E-inducible Broad-Complex (BR-C) gene plays a key role in the genetic hierarchies that transduce the hormone signal, being required for the destruction of larval tissues and numerous aspects of adult development. Most of the known BR-C target genes, including the salivary gland secretion protein (Sgs) genes, are terminal differentiation genes that are thought to be directly regulated by BR-C-encoded transcription factors. Here, we show that repression of Sgs expression is indirectly controlled by the BR-C through transcriptional down-regulation of fork head, a tissue-specific gene that plays a central role in salivary gland development and is required for Sgs expression. Our results demonstrate that integration of a tissue-specific regulatory gene into a 20E-controlled genetic hierarchy provides a mechanism for hormonal repression. Furthermore, they suggest that the BR-C is placed at a different position within the 20E-controlled hierarchies than previously assumed, and that at least part of its pleiotropic functions are mediated by tissue-specific regulators.

Comparison of the GAGA factor genes of Drosophila melanogaster and Drosophila virilis reveals high conservation of GAGA factor structure beyond the BTB/POZ and DNA-binding domains.

As a member of the trithorax-group, the Trithorax-like (Trl) gene of Drosophila melanogaster contributes to the expression of homeotic genes and many other genes. Trl encodes different isoforms of the GAGA factor which is thought to act as an "antirepressor" of transcription by remodelling chromatin structure and thereby rendering control regions accessible for transcriptional activators. A more global role of the GAGA factor in chromatin structure and function is suggested by various phenotypes of Trl mutations, such as modification of position effect variegation. To better define the molecular basis of these pleiotropic effects, we cloned cDNAs encoding the GAGA isoforms of D. melanogaster and a distantly related species, D. virilis. We also characterized the genomic organization of both the D. melanogaster and D. virilis genes, and analysed the expression patterns of isoform-specific mRNAs. The D. virilis GAGA isoforms show high similarity to their D. melanogaster counterparts, particularly within the BTB/POZ protein-interaction and the zinc finger DNA-binding domains. Interestingly, conservation clearly extends beyond the previously defined limits of these domains. Moreover, the comparison reveals a completely conserved block of amino acid residues located between the BTB/POZ and DNA-binding domains, and a high conservation of the C-terminus specific for one of the GAGA isoforms. Thus, sequences of as yet unknown functions are defined as rewarding targets for further mutational analyses. The high conservation of the GAGA proteins of the two species is in accord with the nearly identical genomic organization and expression patterns of the corresponding genes.

Mediation of interleukin-1beta-induced transforming growth factor beta1 expression by activator protein 4 transcription factor in primary cultures of bovine articular chondrocytes: possible cooperation with activator protein 1.

OBJECTIVE: Interleukin-1 (IL-1) and transforming growth factor beta1 (TGFbeta1) play major roles in osteoarticular diseases, exerting opposite effects on both the catabolism and anabolism of cartilage matrix. Previous findings suggest that IL-1 and TGFbeta1 could function in a feedback interaction. However, the effect exerted by IL-1 on expression of TGFbeta by articular chondrocytes is, so far, poorly understood. The present study was carried out to determine the influence of IL-1beta on the expression of TGFbeta1 by bovine articular chondrocytes (BACs) in primary culture. METHODS: BAC primary cultures were treated with IL-1beta, and TGFbeta1 messenger RNA (mRNA) steady-state levels and protein expression were measured by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Transient transfection of TGFbeta1 gene promoter constructs was performed to delineate the DNA sequences that mediate the IL-1beta effect. Electrophoretic mobility shift assays (EMSAs) and supershift analysis were used to characterize the transcription factors binding to these sequences. RESULTS: Cultured BACs responded to IL-1beta exposure by exhibiting an increase of TGFbeta1 expression at both the mRNA and protein levels. The effect was found to be mediated by a major 80-bp sequence located between -732 and -652 upstream of the transcription initiation site. EMSA and supershift analysis revealed that the transcription factors activator protein 4 (AP-4) and AP-1 specifically bound to the -720/-696 part of this sequence under IL-1beta treatment. Overexpression of AP-4 in the BAC cultures resulted in stimulation of the transcriptional activity of the -732/+11 TGFbeta1 promoter construct through the same IL-1beta-responsive element. CONCLUSION: IL-1beta induces an increase of TGFbeta1 in articular chondrocytes through activation of AP-4 and AP-1 binding to the TGFbeta1 gene promoter. These findings may help us understand the role of IL-1beta in the disease process. Notwithstanding its deleterious effect on cartilage, IL-1 could initiate the repair response displayed by injured cartilage in the early stages of osteoarthritis through its ability to enhance TGFbeta1 expression by local chondrocytes.