Dynamic Expression of Broad-Complex Isoforms Mediates Temporal Control of an Ecdysteroid Target Gene at the Onset of Drosophila Metamorphosis

AbstractMetamorphosis in Drosophila melanogaster is orchestrated by the steroid hormone ecdysone, which triggers a cascade of primary-response transcriptional regulators and secondary effector genes during the third larval instar and prepupal periods of development. The early ecdysone-response Broad-Complex (BR-C) gene, a key regulator of this cascade, is defined by three complementing functions (rbp, br, and 2Bc) and encodes several distinct zinc-finger-containing isoforms (Z1 to Z4). Using isoform-specific polyclonal antibodies we observe in the fat body a switch in BR-C isoform expression from the Z2 to the other three isoforms during the third instar. We show that the 2Bc+ function that corresponds presumably to the Z3 isoform is required for the larval fat body-specific expression of a transgenic construct (AE) in which the lacZ gene is under the control of the ecdysone-regulated enhancer and minimal promoter of the fat body protein 1 (Fbp1) gene. Using hs(BR-C) transgenes, we demonstrate that overexpression of Z1, Z3, or Z4, but not Z2, is able to rescue AE activity with faithful tissue specificity in a BR-C null (npr1) genetic context, demonstrating a partial functional redundancy between Z1, Z3, and Z4 isoforms. We also show that continuous overexpression of Z2 during the third instar represses AE, while conversely, expression of Z3 earlier than its normal onset induces precocious expression of the construct. This finding establishes a tight correlation between the dynamic pattern of expression of the BR-C isoforms and their individual repressive or inductive roles in AE regulation. Altogether our results demonstrate that the balance between BR-C protein isoforms in the fat body mediates, in part, the precise timing of the ecdysone activation of the AE construct but does not modulate its tissue specificity

Identification of misexpressed genetic elements in hybrids between Drosophila-related species

International audienceCrosses between close species can lead to genomic disorders, often considered to be the cause of hybrid incompatibility, one of the initial steps in the speciation process. How these incompatibilities are established and what are their causes remain unclear. To understand the initiation of hybrid incompatibility, we performed reciprocal crosses between two species of Drosophila (D. mojavensis and D. arizonae) that diverged less than 1 Mya. We performed a genome-wide transcriptomic analysis on ovaries from parental lines and on hybrids from reciprocal crosses. Using an innovative procedure of co-assembling transcriptomes, we show that parental lines differ in the expression of their genes and transposable elements. Reciprocal hybrids presented specific gene categories and few transposable element families misexpressed relative to the parental lines. Because TEs are mainly silenced by piwi-interacting RNAs (piRNAs), we hypothesize that in hybrids the deregulation of specific TE families is due to the absence of such small RNAs. Small RNA sequencing confirmed our hypothesis and we therefore propose that TEs can indeed be major players of genome differentiation and be implicated in the first steps of genomic incompatibilities through small RNA regulation

Dynamic Expression Of Broad-Complex Isoforms Mediates Temporal Control Of An Ecdysteroid Target Gene At The Onset Of Drosophila Metamorphosis

Metamorphosis in Drosophila melanogaster is orchestrated by the steroid hormone ecdysone, which triggers a cascade of primary-response transcriptional regulators and secondary effector genes during the third larval instar and prepupal periods of development. The early ecdysone-response Broad-Complex (BR-C) gene, a key regulator of this cascade, is defined by three complementing functions (rbp, br, and 2Bc) and encodes several distinct zinc-finger-containing isoforms (Z1 to Z4). Using isoform-specific polyclonal antibodies we observe in the fat body a switch in BR-C isoform expression from the Z2 to the other three isoforms during the third instar. We show that the 2Bc+ function that corresponds presumably to the Z3 isoform is required for the larval fat body-specific expression of a transgenic construct (AE) in which the lacZ gene is under the control of the ecdysone-regulated enhancer and minimal promoter of the fat body protein 1 (Fbp1) gene. Using hs(BR-C) transgenes, we demonstrate that overexpression of Z1, Z3, or Z4, but not Z2, is able to rescue AE activity with faithful tissue specificity in a BR-C null (npr1) genetic context, demonstrating a partial functional redundancy between Z1, Z3, and Z4 isoforms. We also show that continuous overexpression of Z2 during the third instar represses AE, while conversely, expression of Z3 earlier than its normal onset induces precocious expression of the construct. This finding establishes a tight correlation between the dynamic pattern of expression of the BR-C isoforms and their individual repressive or inductive roles in AE regulation. Altogether our results demonstrate that the balance between BR-C protein isoforms in the fat body mediates, in part, the precise timing of the ecdysone activation of the AE construct but does not modulate its tissue specificity. (C) 2000 Academic Press

Dual Requirement for the EcR/USP Nuclear Receptor and the dGATAb Factor in an Ecdysone Response in Drosophila melanogaster

The EcR/USP nuclear receptor controls Drosophila metamorphosis by activating complex cascades of gene transcription in response to pulses of the steroid hormone ecdysone at the end of larval development. Ecdysone release provides a ubiquitous signal for the activation of the receptor, but a number of its target genes are induced in a tissue- and stage-specific manner. Little is known about the molecular mechanisms involved in this developmental modulation of the EcR/USP-mediated pathway. Fbp1 is a good model of primary ecdysone response gene expressed in the fat body for addressing this question. We show here that the dGATAb factor binds to three target sites flanking an EcR/USP binding site in a 70-bp enhancer that controls the tissue and stage specificity of Fbp1 transcription. We demonstrate that one of these sites and proper expression of dGATAb are required for specific activation of the enhancer in the fat body. In addition, we provide further evidence that EcR/USP plays an essential role as a hormonal timer. Our study provides a striking example of the integration of molecular pathways at the level of a tissue-specific hormone response unit

Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila

RNA interference (RNAi) designates the multistep process by which double-stranded RNA induces the silencing of homologous endogenous genes. Some aspects of RNAi appear to be conserved throughout evolution, including the processing of trigger dsRNAs into small 21–23-bp siRNAs and their use to guide the degradation of complementary mRNAs. Two remarkable features of RNAi were uncovered in plants and Caenorhabditid elegans. First, RNA-dependent RNA polymerase activities allow the synthesis of siRNA complementary to sequences upstream of or downstream from the initial trigger region in the target mRNA, leading to a transitive RNAi with sequences that had not been initially targeted. Secondly, systemic RNAi may cause the targeting of gene silencing in one tissue to spread to other tissues. Using transgenes expressing dsRNA, we investigated whether transitive and systemic RNAi occur in Drosophila. DsRNA-producing transgenes targeted RNAi to specific regions of alternative mRNA species of one gene without transitive effect directed to sequences downstream from or upstream of the initial trigger region. Moreover, specific expression of a dsRNA, using either cell-specific GAL4 drivers or random clonal activation of a GAL4 driver, mediated a cell-autonomous RNAi. Together, our results provide evidence that transitive and systemic aspects of RNAi are not conserved in Drosophila and demonstrate that dsRNA-producing transgenes allow powerful reverse genetic approaches to be conducted in this model organism, by knocking down gene functions at the resolution of a single-cell type and of a single isoform

TrEMOLO: accurate transposable element allele frequency estimation using long-read sequencing data combining assembly and mapping-based approaches

Abstract Transposable Element MOnitoring with LOng-reads (TrEMOLO) is a new software that combines assembly- and mapping-based approaches to robustly detect genetic elements called transposable elements (TEs). Using high- or low-quality genome assemblies, TrEMOLO can detect most TE insertions and deletions and estimate their allele frequency in populations. Benchmarking with simulated data revealed that TrEMOLO outperforms other state-of-the-art computational tools. TE detection and frequency estimation by TrEMOLO were validated using simulated and experimental datasets. Therefore, TrEMOLO is a comprehensive and suitable tool to accurately study TE dynamics. TrEMOLO is available under GNU GPL3.0 at https://github.com/DrosophilaGenomeEvolution/TrEMOLO .Transposable Element MOnitoring with LOng-reads (TrEMOLO) est un nouveau logiciel qui combine des approches basées sur l'assemblage et le mapping pour détecter de manière robuste les éléments génétiques appelés éléments transposables (TE). En utilisant des assemblages génomiques de haute ou basse qualité, TrEMOLO peut détecter la plupart des insertions et des délétions de TE, et estimer leur fréquence allélique dans les populations. Des tests avec des données simulées ont révélé que TrEMOLO surpasse d'autres outils computationnels de pointe. La détection des TE et l'estimation de leur fréquence par TrEMOLO ont été validées à l'aide de jeux de données simulées et expérimentales. Par conséquent, TrEMOLO est un outil complet et adapté pour étudier avec précision la dynamique des TE. TrEMOLO est disponible sous la licence GNU GPL3.0 à l'adresse https://github.com/DrosophilaGenomeEvolution/TrEMOLO

Transposable element misregulation is linked to the divergence between parental piRNA pathways in Drosophila hybrids

Interspecific hybridization is a genomic stress condition that leads to the activation of transposable elements (TEs) in both animals and plants. In hybrids between Drosophila buzzatii and Drosophila koepferae, mobilization of at least 28 TEs has been described. However, the molecular mechanisms underlying this TE release remain poorly understood. To give insight on the causes of this TE activation, we performed a TE transcriptomic analysis in ovaries (notorious for playing a major role in TE silencing) of parental species and their F1 and backcrossed (BC) hybrids. We find that 15.2% and 10.6% of the expressed TEs are deregulated in F1 and BC1 ovaries, respectively, with a bias toward overexpression in both cases. Although differences between parental piRNA (Piwi-interacting RNA) populations explain only partially these results, we demonstrate that piRNA pathway proteins have divergent sequences and are differentially expressed between parental species. Thus, a functional divergence of the piRNA pathway between parental species, together with some differences between their piRNA pools, might be at the origin of hybrid instabilities and ultimately cause TE misregulation in ovaries. These analyses were complemented with the study of F1 testes, where TEs tend to be less expressed than in D. buzzatii. This can be explained by an increase in piRNA production, which probably acts as a defence mechanism against TE instability in the male germline. Hence, we describe a differential impact of interspecific hybridization in testes and ovaries, which reveals that TE expression and regulation are sex-biased

Genome-wide identification of in vivo Drosophila Engrailed-binding DNA fragments and related target genes

fragments and related target gene