Premature expression of the late FTZ-F1 protein has an effect on early gene induction by ecdysone. The inability of E93 to be induced by ecdysone in late-third instar larval salivary glands can be overcome by ectopic expression of FTZ-F1. FTZ-F1 also represses its own transcription (Woodard, 1994).
The steroid hormone ecdysone induces a precise sequence of gene activity in Drosophila melanogaster salivary glands in late third instar larvae. The acquisition of competence for this response does not result from a single event or pathway but requires factors that accumulate throughout the instar. Individual transcripts become competent to respond at different times and their expression is differentially affected in ecd1, dor22 and BR-C mutants. ecd1 mutants are deficient in ecdysone. dor encodes a protein with a zinc-finger like motif. The induction of early-late transcripts, originally assumed to necessarily follow early transcripts, is partially independent of early transcript activation. Attempts to inhibit the synthesis of regulatory proteins reveal transcript-specific superinduction effects. Furthermore these inhibitors led to the induction of betaFTZ-F1 and E93 transcripts at levels normally found in prepupal glands. These studies reveal the complexity of the processes underlying the establishment of a hormonal response (Richards, 1999).
The beta FTZ-F1 orphan nuclear receptor functions as a competence factor for stage-specific responses to the steroid hormone ecdysone during Drosophila metamorphosis. beta FTZ-F1 mutants pupariate normally in response to the late larval pulse of ecdysone but display defects in stage-specific responses, adult head eversion, leg elongation and salivary gland death, in response to the subsequent ecdysone pulse in prepupae. The ecdysone-triggered genetic hierarchy that directs these developmental responses is severely attenuated in beta FTZ-F1 mutants, although ecdysone receptor expression is unaffected. Both E74A and E75A, whose levels of expression are normally increased several orders of magnitude by ecdysone, are significantly affected in betaFTZ-F1 mutants. The severity of these effects correlates with the intensity of polytene chromosome staining by FTZ-F1 antibodies. The Br-C locus is only weakly stained, while E74 is strongly stained, and E75 is the most intensely stained site in the genome. It thus appears that betaFTZ-F1 exerts specificity to the degree to which it can enhance the ecdysone-induction of different promoters. The E93 early gene is also submaximally induced in betaFTZ-F1 mutants, consistent with the proposal that this stage-specific response is dependent on betaFTZ-F1 function. In contrast, the levels of Ecdysone receptor and Ultraspiracle mRNA are not significanty affected by betaFTZ-F1. EDG84A, a gene that encodes a pupal cuticle protein that is specifically expressed in the imaginal discs of mid-prepupae, contains a betaFTZ-F1 binding site upstream from the start site, and EDG84A transcription is delayed and reduced in betaFTZ-F1 mutants. Thus this study defines beta FTZ-F1 as an essential competence factor for stage-specific responses to a steroid signal and implicates interplay among nuclear receptors as a mechanism for achieving hormonal competence (Broadus, 1999).
The nuclear localization of E93 in larval salivary glands provided an opportunity to determine if E93 binds to the salivary gland polytene chromosomes and, if so, to identify the sites bound by the protein. Salivary glands were dissected 12-14 hr after puparium formation, fixed, squashed, and photographed to acquire accurate cytology of the banding and puffing patterns for mapping. The chromosomes were then stained with affinity-purified E93 antibodies, and these patterns were compared with the original set of photographs to allow accurate mapping of the bound sites. E93 clearly binds to the polytene chromosomes in a reproducible and site-specific manner and is consistently detected at 65 chromosome sites, many of which contain ecdysone-regulated genes or programmed cell death genes. Among these sites are the 74EF and 75B early puffs, which contain the E74 and E75 ecdysone-inducible genes, as well as the 93F puff, which contains E93. In addition, 1B, 21C, 59F, and 99B are bound by E93 and contain the programmed cell death genes dredd, crq, dcp-1, and drICE, respectively. The 2B5 early puff, containing the BR-C ecdysone-inducible gene, and 75CD, containing βFTZ-F1 and the programmed cell death genes rpr, hid, and grim, were not bound by E93. These data indicate that E93 may directly regulate the genes in bound chromosome loci and may either encode a site-specific DNA binding protein or a chromatin-associated protein that functions as a transcriptional regulator (Lee, 2000).
The observations that E93 is essential for salivary gland cell death and that E93 protein binds to specific sites in the salivary gland polytene chromosomes suggest that E93 may regulate the transcription of target genes that function in steroid-triggered programmed cell death. If this hypothesis is true, then E93 mutations should impact the transcription of genes that reside in salivary gland chromosome loci bound by E93. Salivary glands were dissected from staged late third instar larvae, prepupae, and pupae of control and mutant animals. Total RNA extracted from these tissues was analyzed by Northern blot hybridization. E93 mutations have little or no effect on the timing and levels of BR-C, E74, and E75A transcription in the salivary glands of late third instar larvae and early prepupae. However, the level of expression of each of these regulatory genes is significantly reduced or absent in salivary glands 10-24 hr following puparium formation. Although the smaller E74B transcript is induced, the larger E74A RNA is not detected following the prepupal pulse of ecdysone. The levels of EcR expression in late third instar larval and prepupal salivary glands are not altered by E93 mutations, although its timing is delayed by 4-6 hr at the prepupal to pupal transition. Like EcR, βFTZ-F1 transcription is delayed but the level of this mRNA is not altered in E93 mutant salivary glands. A similar delay is observed in the parental flies that were used for mutagenesis, indicating that this effect is due to the genetic background. The induction of EcR and E74B in E93 mutant prepupae, as well as the successful completion of adult head eversion, indicates that the prepupal pulse of ecdysone occurs in these mutant animals, signaling the prepupal-pupal transition (Lee, 2000).
E93 mutant salivary glands also exhibited little or no transcription of genes that play a key role in programmed cell death. rpr and hid are induced in control animals in a stage-specific manner, immediately preceding the onset of salivary gland cell death. Interestingly, the relative of the vertebrate CD36 gene named croquemort (crq), ark, and the caspase dronc are also induced at this time, indicating that other components of the apoptotic signaling pathway are utilized during programmed cell death in salivary glands. Transcription of the caspases dredd, dcp-1, and drICE is not detected in salivary glands at these developmental stages. The cell death genes rpr, hid, crq, ark, and dronc are transcribed at reduced levels in E93 mutant salivary glands 12–24 hr following puparium formation. These observations indicate that E93 functions as a key regulator by specifying the steroid activation of cell death genes (Lee, 2000).
Programmed cell death (PCD), important in normal animal physiology and disease, can be divided into at least two morphological subtypes, including type I, or apoptosis, and type II, or autophagic cell death. While many molecules involved in apoptosis have been discovered and studied intensively during the past decade, autophagic cell death is not well characterized molecularly. This study reports the first comprehensive identification of molecules associated with autophagic cell death during normal metazoan development in vivo. During Drosophila metamorphosis, the larval salivary glands undergo autophagic cell death regulated by a hormonally induced transcriptional cascade. To identify and analyze the genes expressed, wild-type patterns of gene expression were examined in three predeath stages of Drosophila salivary glands using serial analysis of gene expression (SAGE). 1244 transcripts, including genes involved in autophagy, defense response, cytoskeleton remodeling, noncaspase proteolysis, and apoptosis, were seen to be expressed differentially prior to salivary gland death. Mutant expression analysis indicated that several of these genes were regulated by E93, a gene required for salivary gland cell death. These analyses strongly support both the emerging notion that there is overlap with respect to the molecules involved in autophagic cell death and apoptosis, and that there are important differences (Gorski, 2003).
Dronc is an apical Drosophila caspase essential for programmed cell death during fly development. During metamorphosis, dronc gene expression is regulated by the steroid hormone ecdysone, which also regulates the levels of a number of other critical cell death proteins. As dronc protein levels are important in determining caspase activation and initiation of cell death, the regulation of the dronc promoter was analyzed using transgenic flies expressing a LacZ reporter gene under the control of the dronc promoter. These results indicate that dronc expression is highly dynamic during Drosophila development, and is controlled both spatially and temporally. While a 2.3 kb dronc promoter region contains most of the information required for correct gene expression, a 1.1 kb promoter region is expressed in some tissues and not others. During larval-pupal metamorphosis, two ecdysone-induced transcription factors, Broad-Complex and E93, are required for correct dronc expression. These data suggest that the dronc promoter is regulated in a highly complex manner, and provides an ideal system to explore the temporal and spatial regulation of gene expression driven by nuclear hormone receptors (Daish, 2003).
Experiments outlined in this paper demonstrate that 2.3 kb of the dronc promoter is largely sufficient for temporal expression (compared to endogenous dronc) throughout development. Previous experiments have shown that dronc is predominantly expressed in the larval and prepupal salivary glands and midgut, and larval brain lobes. 2.3 kb of the dronc promoter contains all necessary elements for correct spatial regulation of dronc expression in these tissues (Daish, 2003).
In order to identify transcription factors responsible for both temporal and spatial regulation of dronc and ecdysone-mediated PCD, it is of vital importance to elucidate the regions of the promoter essential for dronc expression in different tissues. In addition, it would be of interest to determine if there is a single promoter region controlling the spatial expression profile of dronc, or if different promoter regions are required in different tissues. LacZ transgenic reporter experiments reveal that the 2.3 kb promoter is the minimal requirement for correct expression in brain lobes and salivary glands. Furthermore, the region between 1.1 and 2.3 kb contains transcription factor-binding sites essential for expression in these tissues. This region also seems to harbor a repressor element important to keep dronc levels low during periods when ecdysone titers are low. Surprisingly, regulation of dronc transcription is markedly different in the midgut. The region between 1.1 and 2.3 kb is not important for transcription in this tissue, because 1.1 kb of the promoter is sufficient for expression. These results clearly demonstrate that distinct regions of the promoter are required for expression in different tissues, and implies that different transcription factors regulate dronc expression in a tissue-dependent manner (Daish, 2003).
The two ecdysone-induced transcription factors BR-C and E93 are essential for dronc expression in salivary glands. In the midgut, however, only E93 seems to be important. The results of dronc promoter-LacZ transgenic expression in flies deficient in BR-C and E93 are consistent with recent findings. LacZ expression driven by the 2.8 kb promoter is severely impaired in salivary glands of BR-C (rbp5 and npr) or E93 mutants, whereas expression is impaired only in the midgut of E93 mutant background animals. This further supports the idea that the mechanisms governing dronc regulation are tissue specific. The key questions arising from these experiments are: why does the BR-C Z1 isoform (rbp5 mutant) regulate dronc in the salivary glands and not in the midgut? What factors are binding to the 1.1-2.3 kb region of the promoter in salivary glands, and why are they not as important in the midgut? Previous results show that either BR-C Z1- or BR-C Z1-regulated proteins bind to the dronc proximal promoter and control its expression. Transactivation of the 2.8 kb promoter by BR-C Z1, however, was only seen in specific cell types. Given that BR-C Z1 is also expressed in the midgut, this implies that it may be acting through cofactors which are not expressed in the midgut, yet are specifically recruited to the dronc promoter. Alternatively, BR-C Z1 induces the expression of another factor which binds to the promoter, and this factor is absent in the midgut (Daish, 2003).
Since the proximal promoter alone (0.54 kb) is not sufficient for expression in the salivary gland, it is believed that BR-C Z1 (or a Z1-regulated protein) is cooperating with other transcription factors binding upstream (1.1-2.3 kb), that are essential for salivary gland expression. It has been shown that E93 acts through the first 600 bp of the dronc promoter by transactivation studies; however, no direct binding of E93 to the dronc (or any other) promoter has been shown so far. Additionally, a preliminary analysis indicates the presence of an EcR/Usp-binding site between 1.1 and 2.3 kb of the dronc promoter, and in vitro experiments show that this element may be important in regulating dronc expression. Since the proximal promoter (0.54 kb) alone is not sufficient for expression, cooperation of BR-C and E93 with EcR/Usp and other unknown factors may be important for temporal and spatial regulation of dronc expression during development. Identification of these factors will be important for fully understanding dronc transcription during development (Daish, 2003).
Overall, this study has established the minimal dronc promoter requirement for spatial and temporal expression to be within the 2.3 kb region upstream of the dronc gene. This region is important for both BR-C- and E93-mediated transcription in salivary glands and E93 transcription in the midgut. Importantly, the 1.1-2.3 kb promoter region harbors elements important for salivary gland expression and a putative repressor element. The 0.54-1.1 kb promoter region is important for expression in the midgut. These regions will form the basis of future experiments designed to identify factors necessary for the regulation of dronc expression during PCD (Daish, 2003).
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