A view through a chromatin loop: insights into the ecdysone activation of early genes in Drosophila.

The early genes are a key group of ecdysone targets that function at the top of the signaling hierarchy. In the presence of ecdysone, early genes exhibit a highly characteristic rapid and powerful induction that represents a primary response. Multiple isoforms encoded by early genes then coordinate the activation of a larger group of late genes. While the general mechanism of ecdysone-dependent transcription is well characterized, it is not known whether a distinct mechanism governs the hormonal response of early genes. We previously found that one of the Drosophila early genes, E75, harbors multiple functional ecdysone response elements (EcREs). In this study we extended the analysis to Broad and E74 and found that EcRE multiplicity is a general feature of the early genes. Since most of the EcREs within early gene loci are situated distantly from promoters, we employed the chromosome conformation capture method to determine whether higher order chromatin structure facilitates hormonal activation. For each early gene we detected chromatin loops that juxtapose their promoters and multiple distant EcREs prior to ecdysone activation. Our findings suggest that higher order chromatin structure may serve as an important mechanism underlying the distinct response of early genes to ecdysone.

Developmental roles of Drosophila tRNA processing endonuclease RNase ZL as revealed with a conditional rescue system.

Drosophila RNase Z(L) (dRNaseZ) belongs to a family of endoribonucleases with a major role in tRNA 3'-end processing. The biochemical function of RNase Z(L) is conserved from yeast to human. Here we present a study of its biological function during Drosophila development. In flies, dRNaseZ provides a non-redundant function, as the RNZ(ED24) knockout (KO) mutation causes early larval lethality. Mosaic and conditional rescue techniques were employed to determine dRNaseZ requirements at later stages. We found that dRNaseZ activity is essential for all phases of fly development that involve cell division, including growth of adult tissue progenitors during larval and metamorphic stages, and gametogenesis in adults. At the cellular level, two major phenotypes were identified-cell growth deficiency in endoreplicating tissues and cell cycle arrest in mitotic tissues. While cell growth and proliferation are both dependant on protein synthesis, the two phenotypes displayed reliance on different dRNaseZ functions. We found that dRNaseZ KO completely blocks tRNA maturation without diminishing the abundance of mature tRNA molecules. Our data indicate that growth arrest of endoreplicating cells is primarily attributed to the relocation of the pool of mature tRNAs into the nuclei causing a decrease in translation efficiency. Mitotically dividing cells appear to be less dependent on translation machinery as they maintain their normal size when deprived of dRNaseZ activity, but rather display a cell cycle arrest at the G2-M transition.

The Drosophila FTZ-F1 nuclear receptor mediates juvenile hormone activation of E75A gene expression through an intracellular pathway.

Juvenile hormone (JH) regulates a wide variety of biological activities in holometabolous insects, ranging from vitellogenesis and caste determination in adults to the timing of metamorphosis in larvae. The mechanism of JH signaling in such a diverse array of processes remains either unknown or contentious. We previously found that the nuclear receptor gene E75A is activated in S2 cells as a primary response to JH. Here, by expressing an intracellular form of JH esterase, we demonstrate that JH must enter the cell in order to activate E75A. To find intracellular receptors involved in the JH response, we performed an RNAi screen against nuclear receptor genes expressed in this cell line and identified the orphan receptor FTZ-F1. Removal of FTZ-F1 prevents JH activation of E75A, whereas overexpression enhances activation, implicating FTZ-F1 as a critical component of the JH response. FTZ-F1 is bound in vivo to multiple enhancers upstream of E75A, suggesting that it participates in direct JH-mediated gene activation. To better define the role of FTZ-F1 in JH signaling, we investigated interactions with candidate JH receptors and found that the bHLH-PAS proteins MET and GCE both interact with FTZ-F1 and can activate transcription through the FTZ-F1 response element. Removal of endogenous GCE, but not MET, prevents JH activation of E75A. We propose that FTZ-F1 functions as a competence factor by loading JH signaling components to the promoter, thus facilitating the direct regulation of E75A gene expression by JH.

RNAi knockdown of dRNaseZ, the Drosophila homolog of ELAC2, impairs growth of mitotic and endoreplicating tissues.

The Drosophila RNase Z(L) (dRNaseZ) gene encodes a member of the ELAC1/ELAC2 protein family with homologs in every living organism. All RNase Z proteins tested so far were found to possess endoribonuclease activity, which is responsible for the removal of a 3' trailer from a primary tRNA transcript. Given that tRNA 3'-end processing has been delineated using in vitro, bacterial and cell culture models, its relevance to RNase Z functions in vivo has yet to be established. In this study, we employed heritable RNA interference (RNAi) in combination with the GAL4/UAS system to spatiotemporally knockdown the dRNaseZ gene and study its biological role in cells of a developing fruit fly. We found that dRNaseZ is an essential gene, as ubiquitous knockdown caused growth arrest and early larval lethality. Molecular analysis confirmed that dRNaseZ is necessary for 3'-end processing of nuclear and mitochondrial tRNAs: knockdown larvae displayed significant accumulation of both types of processing intermediates with extensions at the 3' end. This is the first in vivo demonstration of RNase Z(L) dependent tRNA processing in the context of a metazoan model organism. Using tissue-specific GAL4 drivers, we also showed that in mitotically growing imaginal discs dRNaseZ is required for cell proliferation and/or viability, but not for the maintenance of their differentiated progeny. In endoreplicating salivary glands, dRNaseZ controls organ size by supporting cell growth but not DNA replication. Although the mechanisms remain unclear, our results support the notion that RNase Z(L) is involved in biological pathways regulating cell growth and proliferation.

Hormonal regulation of the E75 gene in Drosophila: identifying functional regulatory elements through computational and biological analysis.

Drosophila development is regulated by two hormones, 20-hydroxyecdysone (ecdysone) and juvenile hormone. We previously found that expression of the E75 gene is induced by both hormones in cultured S2 cells. E75 occupies over 100 kb of genomic DNA; it has four alternative promoters producing isoforms E75A, E75B, E75C, and E75D. To identify hormone response elements in the 60-kb noncoding area upstream of the E75A transcription start site, we developed a novel approach combining in vitro, in vivo, and in silico techniques. Using chromatin immunoprecipitation coupled with quantitative real-time PCR, we identified five putative enhancers marked with H3K4 monomethylation and depletion of H3. Four of these are ecdysone-regulated enhancers, which possess hormone-responsive chromatin and contain sequences sufficient to confer ecdysone inducibility to a reporter gene. Using EvoPrinterHD- and Multiple Expectation Maximization for Motif Elicitation-based computational analysis, we first created a database of short sequences that are highly conserved among 12 Drosophila species. Within this database, we then identified a set of putative ecdysone response elements (EcREs). Seven of these elements represent in vivo binding sites for the ecdysone receptor and are necessary for hormone-mediated activation of gene expression in cultured cells. We found that each EcRE exhibits different binding and activation properties, and at least some of them function cooperatively.We propose that the presence of multiple EcREs with distinct features provides flexibility to the rapid and powerful response of E75A to ecdysone during Drosophila development.

Inhibition of micro-RNA-induced RNA silencing by 2'-o-methyl oligonucleotides in Drosophila S2 cells.

More than 90 different micro-ribonucleic acid (miRNA) encoding genes have been identified in Drosophila, yet the function of only two of these, bantam and DmiR-14, has been elucidated. In an effort to develop a general strategy for the analysis of miRNA function in Drosophila, two procedures were developed, in a Schneider line 2 cell culture system, which may be adapted to that end. First, we show that endogenous miRNAs can partially inhibit the expression of a transiently transfected reporter gene that has been modified to contain sequences complementary to that miRNA in the 3' UTR of a target messenger RNA (mRNA). Inhibition occurs by RNA interference (RNAi), which involves mRNA degradation. Second, we demonstrate that this miRNA-induced RNAi can be partially rescued with 2'-O-methyl oligonucleotides that contain sequences complementary to the cognate miRNA. We discuss how these techniques may be used, in vivo, both for localizing the tissue distribution of endogenous miRNAs during Drosophila development and identifying phenotypes associated with a loss of miRNA function.

Juvenile hormone regulation of the E75 nuclear receptor is conserved in Diptera and Lepidoptera.

Despite longstanding efforts, the juvenile hormone (JH) signaling pathway remains unknown. In Drosophila melanogaster (Diptera), JH activates expression of the E75A nuclear receptor. The E75 gene encodes a family of related proteins. A homologue of Drosophila E75 was previously identified and two isoforms, mE75A and mE75B, were reported in Manduca sexta (Lepidoptera). Here, we describe the identification of two additional isoforms, mE75C and mE75D, and the hormonal regulation of mE75 gene expression in Manduca CH1 cultured cells. mE75A and mE75B isoforms are specifically induced by ecdysone in CH1 cells. One isoform, mE75C, shows constitutive expression. The mE75D isoform exhibits dual hormonal regulation; it can be activated by either ecdysone or the JH analog, methoprene. E75-encoded proteins represent the first example of transcription factors directly induced by JH. E75 activation by JH, in both Diptera and Lepidoptera, suggests a conserved function in the JH signaling pathway.

Hormonal regulation and functional role of Drosophila E75A orphan nuclear receptor in the juvenile hormone signaling pathway.

Ecdysone and juvenile hormone (JH) are important regulators of insect growth and development. While ecdysone initiates a transition from one developmental stage to another, JH determines the nature of the transition. How these two hormones interact at the molecular level is not known. Here we report the JH inducibility of the E75A nuclear receptor encoded by the E75 early ecdysone-inducible gene. In Drosophila S2 cells, E75A transcription is specifically activated by JH at concentrations well within the physiological range found in larvae and adults. The induction is rapid and does not require a concurrent protein synthesis, and thus represents a primary hormone response. Consistent with JH regulation, E75A mRNA levels are reduced in ovaries of apterous(4) mutant adults defective in JH secretion. Expression is rescued by topical methoprene application. We further provide evidence that ectopic E75A is sufficient to perform several functions in the JH signaling pathway. First, it can down-regulate its own transcription. Second, E75A can potentiate the JH inducibility of a secondary response gene, JhI-21. Finally, in the presence of JH, E75A can repress ecdysone activation of early genes including Broad-Complex. Based on these data, we propose a model for the role of E75A in the ecdysone-JH regulatory interplay.

Drosophila RNase Z processes mitochondrial and nuclear pre-tRNA 3' ends in vivo.

Although correct tRNA 3' ends are crucial for protein biosynthesis, generation of mature tRNA 3' ends in eukaryotes is poorly understood and has so far only been investigated in vitro. We report here for the first time that eukaryotic tRNA 3' end maturation is catalysed by the endonuclease RNase Z in vivo. Silencing of the JhI-1 gene (RNase Z homolog) in vivo with RNAi in Drosophila S2 cultured cells causes accumulation of nuclear and mitochondrial pre-tRNAs, suggesting that JhI-1 encodes both forms of the tRNA 3' endonuclease RNase Z, and establishing its biological role in endonucleolytic tRNA 3' end processing. In addition our data show that in vivo 5' processing of nuclear and mitochondrial pre-tRNAs occurs before 3' processing.

The expression of the let-7 small regulatory RNA is controlled by ecdysone during metamorphosis in Drosophila melanogaster.

In Caenorhabditis elegans, the heterochronic pathway controls the timing of developmental events during the larval stages. A component of this pathway, the let-7 small regulatory RNA, is expressed at the late stages of development and promotes the transition from larval to adult (L/A) stages. The stage-specificity of let-7 expression, which is crucial for the proper timing of the worm L/A transition, is conserved in Drosophila melanogaster and other invertebrates. In Drosophila, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) control the timing of the transition from larval to pupal to adult stages. To test whether let-7 expression is regulated by ecdysone in Drosophila, we used Northern blot analysis to examine the effect of altered ecdysone levels on let-7 expression in mutant animals, organ cultures, and S2 cultured cells. Experiments were conducted to test the role of Broad-Complex (BR-C), an essential component in the ecdysone pathway, in let-7 expression. We show that ecdysone and BR-C are required for let-7 expression, indicating that the ecdysone pathway regulates the temporal expression of let-7 in Drosophila. These results demonstrate an interaction between steroid hormone signaling and the heterochronic pathway in insects.