Edward B. Dubrovsky

Temporal regulation of microRNA expression in Drosophila melanogaster mediated by hormonal signals and broad-Complex gene activity.

lin-4 and let-7 are founding members of an extensive family of genes that produce small transcripts, termed microRNAs (miRNAs). In Caenorhabditis elegans, lin-4 and let-7 control the timing of postembryonic events by translational repression of target genes, permitting progression from early to late developmental programs. To identify Drosophila melanogaster miRNAs that could play similar roles in the control of developmental timing, we characterized the developmental expression profile of 24 miRNAs in Drosophila, and found 7 miRNAs that are either upregulated or downregulated in conjunction with metamorphosis. The upregulation of three of these miRNAs (mir-100, mir-125, and let-7), and the downregulation of a fourth (mir-34) requires the hormone ecdysone (Ecd) and the activity of the Ecd-inducible gene Broad-Complex. Interestingly, mir-125 is a putative homologue of lin-4. mir-100, -125, and let-7 are clustered within an 800-bp region on chromosome 2L, suggesting that these three miRNAs may be coordinately regulated via common cis-acting elements during metamorphosis. In S2 cells, Ecd and the juvenile hormone analog methoprene exert opposite effects on the expression of these four miRNAs, indicating the participation of both these hormones in the temporal regulation of mir-34, -100, -125, and let-7 expression in vivo.

Hormonal cross talk in insect development

Two hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH), coordinately orchestrate insect growth and development. 20E initiates all major developmental transitions from egg, to larva, to pupa, to adult, but it is an interaction with the JH signal that transduces 20E pulses into stage-specific responses. Years of research have given us an understanding of 20E signaling pathway. By contrast, the molecular mechanism of JH action remains an enigma. Recent studies provide insight into the molecular background to JH-20E regulatory interplay. Two transcription factors--BR-C and E75A--contribute to the cross-talk between the two hormones. It appears that BR-C is a key target of JH status quo action, and E75A is a part of the mechanism whereby JH prevents BR-C activation.

Juvenile hormone signaling during oogenesis in Drosophila melanogaster

Juvenile hormone (JH) participates both in the control of insect development and the establishment of reproductive maturity. In cultured Drosophila cells and in ovarian nurse cells, JH and its synthetic analog, methoprene, induce the expression of two related genes. These genes encode highly similar amino acid transport proteins that are homologous to transporters found in a variety of eukaryotes. JhI-21 is a novel Drosophila gene, and minidiscs (mnd) is a gene that was identified earlier. Two JH-inducible genes are regulated by different molecular mechanisms; JhI-21 behaves as a secondary JH-responsive gene, while mnd behaves as a primary responsive gene. Both JhI-21 and mnd transcripts show developmental profiles, which are consistent with JH regulation. Following eclosion, transcripts from JhI-21 and mnd are synthesized in ovarian nurse cells and subsequently sequestered in the mature egg. Their ectopic accumulation in ovaries can be induced by topical methoprene application. In apterous (ap4) mutant adults defective in JH secretion, mnd and JhI-21 RNA levels are severely reduced, but normal abundance is rescued to a high degree by topical methoprene treatment. Based on the evidence, we propose that during sexual maturation of Drosophila, JH provides a signal to the ovary that leads to the production of several maternally inherited mRNAs.

The Drosophila FTZ-F1 Nuclear Receptor Mediates Juvenile Hormone Activation of E75A Gene Expression through an Intracellular Pathway

Background: JH is a critical insect hormone, but its mechanism of action is contentious. Results: JH activates E75A through an intracellular pathway utilizing FTZ-F1 and GCE, which form a transcriptionally active heterodimer. Conclusion: FTZ-F1 functions as a competence factor that facilitates JH activation of gene expression. Significance: As a competence factor for multiple insect hormones, FTZ-F1 could be a mediator of hormonal cross-talk. 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.

The Drosophila Juvenile Hormone Receptor Candidates Methoprene-tolerant (MET) and Germ Cell-expressed (GCE) Utilize a Conserved LIXXL Motif to Bind the FTZ-F1 Nuclear Receptor

Background: JH signaling involves interactions between FTZ-F1 and candidate JH receptors MET and GCE. Results: Mutation of FTZ-F1 AF2 or LIXXL MET/GCE sequence disrupts interaction between the proteins. Conclusion: NR box-AF2 binding underlies FTZ-F1·MET and FTZ-F1·GCE heterodimer formation. Significance: Dissecting the interaction between FTZ-F1 and MET, GCE is critical to understanding the molecular basis of JH signaling. Juvenile hormone (JH) has been implicated in many developmental processes in holometabolous insects, but its mechanism of signaling remains controversial. We previously found that in Drosophila Schneider 2 cells, the nuclear receptor FTZ-F1 is required for activation of the E75A gene by JH. Here, we utilized insect two-hybrid assays to show that FTZ-F1 interacts with two JH receptor candidates, the bHLH-PAS paralogs MET and GCE, in a JH-dependent manner. These interactions are severely reduced when helix 12 of the FTZ-F1 activation function 2 (AF2) is removed, implicating AF2 as an interacting site. Through homology modeling, we found that MET and GCE possess a C-terminal α-helix featuring a conserved motif LIXXL that represents a novel nuclear receptor (NR) box. Docking simulations supported by two-hybrid experiments revealed that FTZ-F1·MET and FTZ-F1·GCE heterodimer formation involves a typical NR box-AF2 interaction but does not require the canonical charge clamp residues of FTZ-F1 and relies primarily on hydrophobic contacts, including a unique interaction with helix 4. Moreover, we identified paralog-specific features, including a secondary interaction site found only in MET. Our findings suggest that a novel NR box enables MET and GCE to interact JH-dependently with the AF2 of FTZ-F1.

Selective binding of Drosophila BR-C isoforms to a distal regulatory element in the hsp23 promoter

The Broad-Complex (BR-C) gene plays a key role in the ecdysone regulatory hierarchy. Together with other early ecdysone-inducible genes BR-C transmits the hormonal signal to a set of secondary response genes in a tissue-specific manner. Among its targets is the hsp23 gene. Previously we showed that expression of the hsp23 gene in late third instar is BR-C-dependent, and accompanied by the appearance of a BR-C-dependent DNase I hypersensitive site at position -1400 (DHS-1400). BR-C encodes a family of transcription factors, and we show here that at least three BR-C protein isoforms--Z1, Z2, and Z3--bind to the sequences around DHS-1400 in vitro. A DNase I footprinting assay reveals five protected regions, designated site 1 to site 5, each of which specifically associates with one or several BR-C protein isoforms. We also show that a 100 bp region overlapping site 5, which binds all three isoforms in vitro, is required for hsp23 activity in vivo. The deletion of binding site 5 in a reporter gene construct reproduced the effect of the npr class mutations, that is, hsp23 is no longer expressed in any tissue tested except brain. Thus, BR-C regulates hsp23 expression via direct interaction of the predominant isoform with the distal regulatory element.

Trans-regulation of ecdysterone-induced protein synthesis in Drosophila melanogaster salivary glands.

A set of coordinately expressed genes is actively transcribed after a dramatic increase in the ecdysterone titer in late third-instar development of Drosophila melanogaster, as shown by the appearance of a number of puffs in salivary gland chromosomes and by the synthesis of a number of new proteins. Previous work has suggested that a product of the ecs gene, which is located within the 2B3-5 puff, is necessary for providing alterations in transcriptional activity at the sites of ecdysterone-dependent puffs. The experiments reported here were designed to determine whether the ecs genes regulatory effect on puffing is confirmed by its regulatory effect on the synthesis of ecdysterone-inducible proteins (EIPs). The first series of experiments showed that in salivary glands in vivo ecdysterone induces 24 EIPs and in vitro induces 26 EIPs. The sets of polypeptides revealed are in good conformity. The second set of experiments demonstrated that mutations in the ecs locus disturb EIP synthesis: ecsl(1)t10 and ecsl(1)t143 mutations affect EIP synthesis to a lesser extent, while ecsl(1)t435 and ecsl(1)t324, as well as the 2B3-5 puff deficiency, prevent EIP synthesis completely. The experiments on dosage dependency revealed two EIPs whose rate of synthesis correlates with the dosage of the 2B3-5 X-chromosomal region. These EIPs are shown to be in fact small heat-shock proteins 23 and 28K, which are known to be encoded within the 67B puff and are under dual control--transient and developmental. The final set of experiments followed the 2B3-5 dosage dependency in vitro and showed that 15 EIPs display either an affected rate of synthesis or, mainly, a quicker induction time. Data obtained show that the ecs locus is trans-regulatory and that its product is necessary for spreading the effect of ecdysterone to other loci.

INHIBITION OF MICRO-RNA–INDUCED RNA SILENCING BY 2′-O-METHYL OLIGONUCLEOTIDES IN DROSOPHILA S2 CELLS

SummaryMore 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.