Unit: Drosophila genetics and epigenetics
Director: Christophe Antoniewski
We are interested in the transcriptional and post-transcriptional mechanisms of gene regulation during the development of metazoans. The fruit fly Drosophila melanogaster is a good experimental model to address these questions using a combination of genetic and molecular approaches. Two research projects are conducted in the laboratory including (i) the study of the function of the histone-acetyltransferase (HAT) dGcn5 and its role in the genetic and epigenetic control of the development and (ii) the analysis of mechanisms of post-transcriptional silencing by double-stranded RNA and micro-RNA.
1. The dGcn5 histone-acétyltransférase
Dimitri Szymczak, Clément Carré
Gcn5 is the catalytic unit of various Gcn5-containing N-acetyltransferase (GNAT) complexes whose composition and activity appear to be conserved from yeast to human. Acetylation of histones by these complexes has been involved in the modulation of chromatin structure and the recruitment of transcription factors to gene promoters. In the lab, we are more particularly interested in understanding how the epigenetic histone code generated by histone-acetyltransferases contributes to genetic programs during development.
During the year we have conducted an extensive EMS-induced mutagenesis. We have isolated and characterized four point mutations of the Drosophila Gcn5 gene. The loss of function of Gcn5 blocks the onset of both oogenesis and metamorphosis and hypomorphic Gcn5 alleles impair the ecdysone-controlled formation of adult appendages and cuticle. Strikingly, Gcn5 is not required for larval development. In contrast there are strong cell proliferation defects in Gcn5-depleted imaginal tissues.
In vivo global histone H3 acetylation of K9 and K14 lysine residues was lost in Gcn5 mutants, while other histone modifications were not affected.
Complementation by variant proteins revealed that the Pcaf homology domain, the domain of interaction with the Ada proteins and the catalytic domain are all required to fulfill the function of Gcn5. Strikingly the Gcn5 bromodomain was dispensable. Our results point out to Gcn5 as a major histone H3 acetylase in Drosophila involved in the control of specific morphogenetic cascades during development.
2. Analysis of Genetic Interference by double-stranded RNA and micro-RNA
Hélène Thomassin, Delphine Fagegaltier et Bassam Berry.
RNA interference (RNAi) designates the process by which double-stranded RNA induce the specific degradation of their complementary mRNA. This process is involved in various regulatory pathways in eukaryotes, including defense against plant viruses, heterochromatinization of pericentromeric regions, various genetic cosuppression phenomena, repression of transposable elements and chromosome imprinting.
Micro-RNAs are short hairpin, partially double stranded RNA encoded by eukaryote genomes. Several hundreds of micro-RNA have been recently described whose maturation and mode of action involves pathways overlapping with RNAi pathway. However, instead of serving as guide for the degradation of their target mRNA, micro-RNA inhibit their translation. Although only recently characterized, it is clear that micro-RNA are involved in the regulation of essential processes such as development, cell proliferation and apoptosis. We have developed a method to trigger RNAi in vivo using double-stranded RNA producing transgenes (Fig. 2). This method turned out to be a powerful approach to inactivate drosophila genes in a tissue- or stage-specific manner.
We wish now to study the contribution of double-stranded RNA to the endogenous regulatory mechanisms.
A first project consists in analyzing the role of micro-RNA during Drosophila development.
In collaboration with Pasteur-Génopole®, we are building and testing oligonucleotide microarrays to perform genome wide analyses of micro-RNA expression profiles during development or in various experimental conditions. We are also analyzing the effect of the expression of micro-RNA inhibitors derived from plant or insect virus on the development of transgenic flies. Last, we are overexpressing natural or reprogrammed micro-RNA in cells or in transgenic animal analyzing their effect on various biological targets.
A second project consists in testing, using our transgenic systems, whether the targeting of double-stranded RNA to non-coding, regulatory sequences can induce Transcriptional Gene Silencing (TGS) and/or heterochromatinization of the targeted sequences.
Legend to Photos :
Left hand side. Spreading of polytene chromosomes from salivary glands of wild type or Gcn5 mutant third instar larvae, as indicated. Immunostaining using a specific antibody (red) revealed that acetylation of H3-K14 residue is lost in Gcn5 mutants.
Right hand side. A RNAi transgenic system (see below) was used to silence Gcn5 in the posterior compartment of imaginal discs. Acetylation of H3-K14 residue was lost in that compartment.
Figure 2. A transgenic system to target RNAi in Drosophila
Left hand side. A transgenic line expressing GAL4 under the control of a specific driver is crossed with a transgenic line transgenic for an inverted repeat under the control of UAS regulatory sites.
Right hand side. Gcn5 RNAi in transgenic larvae
The expression pattern of a RNAi transgene targeted to Gcn5 was revealed by coexpression with a UAS-GFP reporter transgene (green), in a leg (top) and a wing (bottom) imaginal disc. Expression of the Gcn5 protein revealed by a specific antibody (red) is abolished in the corresponding area.
Keywords: Drosophila, Genetics, Epigenetics, Histone acetyltransferase, RNA interference, micro-RNA