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) P/CAF and its role in the genetic and epigenetic control of the development and (ii) the analysis of the mechanisms of post-transcriptional silencing by double-stranded RNA and micro-RNA.
1. The dP/CAF histone-acétyltransférase
How the epigenetic histone code generated by histone-acetyltransferases contributes to the specificity of genetic programs during development ? The study of the Drosophila P300/CBP associated factor (P/CAF) should help to understand the role of histone-acetyltransferases in the regulation of genetic programs during the development of complex organisms.
Our main goal is the isolation and analysis of Drosophila Pcaf mutants. We have characterized a P transposable element insertion 500 bp upstream of the Pcaf transcription start site. This P element insertion is not a Pcaf allele and causes a loss-of-function of a nearby gene. However, imprecise P excision allowed us to recover a short deficiency encompassing Pcaf. We are currently using this deficiency in a secondary EMS genetic screen. We will analyze the developmental defects as well as the perturbations of the genetic cascades associated to the generated Pcaf mutant.
Meanwhile, we have established transgenic lines for a UAS-[IR]Pcaf construct which expresses a double-stranded RNA targeted to the Pcaf mRNA. Using the appropriate GAL4 driver lines, we are able to trigger RNAi against Pcaf in a tissue- and stage-specific manner. Therefore, it is possible to initiate the study of the role of Pcaf in a given cell lineage or in a particular organ. Because it is possible to inactivate the Pcaf function in the larval salivary glands, we are also going to analyze by immunostaining how the loss-of-function of Pcaf modifies the acetylation and methylation profiles as well as the binding of other transcription or chromatin factors to the giant polytene chromosomes. This should help us to define functional Pcaf target genes.
Functional Domains of the P/CAF protein
We have established a collection of transgenic lines expressing the P/CAF protein mutated in its various remarkable domains, including the catalytic HAT domain, the putative nuclear receptor interaction domain and the bromodomain. We are going to analyze by immunostaining how these variants localize to the polytene chromosomes from the larval salivary glands. Rescue experiments using the variants will also help us to perform a structure-function analysis of the P/CAF protein.
2. Analysis of Genetic Interference by double-stranded RNA and micro-RNA
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-RNA 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 (Fig. 3).
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. Various experimental approaches will be conducted, including the use of RNAi inhibitors from plant viruses, the ectopic overexpression of micro-RNA and the overexpression of micro-RNA 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.
Figure 1 : Genetic scheme of an EMS mutagenesis designed to isolate Pcaf mutant alleles
Figure 2. A transgenic system to target RNAi in drosophila
A transgenic line expressing GAL4 under the control of a specific driver is crossed with a transgenic line expressing an inverted repeat under the control of UAS regulatory sites.
Figure 3. RNAi targeted to Pcaf using a transgenic system.
The expression pattern of a double-stranded producing transgene target to Pcaf is revealed by coexpression with a GFP reporter transgene (green), in a leg (top) and a wing (bottom) imaginal disc. Expression of the P/CAF protein revealed by a specific antibody (red) is specifically abolished in the corresponding area.
Keywords: Drosophila, Genetics, Epigenetics, Histone acetyltransferace, RNA interference, micro-RNA