Project description:Whole-chromatin profile (FAIRE-seq) in three Drosophila species (D. melanogaster, D. pseudoobscura and D. virilis) in eye-antennal imaginal discs at the stage of third instar wandering larvae. By the use of Ornstein-Uhlenbeck methods, we assess the evolutionary forces acting on regulatory elements (cis-level) on chromatin activity across Drosophila eye-antennal imaginal discs at the stage of third instar larvae.
Project description:Growth of the drosophila eye imaginal discs is controlled by the activation of Notch in the dorsal-ventral boundary. Overexpression in the eye disc of the Notch ligand Delta together with lola and pipsqueak from the GS(2)88A8 line induces tumoral growth. We used microarray to analyze the expression profile of tumoral discs. Antennal-eye discs of Drosophila L3 larvae were selected for RNA extraction and hybridization on Affymetrix microarrayas. Two genetic conditions were analyzed: tumoral eye discs (ey-Gal4 GS(2)88A8 UAS-Dl) and control eye discs (GS(2)88A8 UAS-Dl). Three different biological replicates of each condition, each one consisting on 300 hundred pairs of eye discs, were analyzed.
Project description:Growth of the drosophila eye imaginal discs is controlled by the activation of Notch in the dorsal-ventral boundary. Overexpression in the eye disc of the Notch ligand Delta together with lola and pipsqueak from the GS(2)88A8 line induces tumoral growth. We used microarray to analyze the expression profile of tumoral discs.
Project description:PSC overexpression can cause phenotypes specifically in an rbf1 mutant background, likely due to a sensitization to PSC-induced phenotypes. The goal of this study is to understand the interaction between rbf1 hypomorphic mutation and the overexpression of Polycomb group gene Posterior sex combs. We used Drosophila larval eye imaginal discs that were mutant for rbf1 or overexpressing PSC and compared these to control larval eye discs to assess changes in gene expression. We identified a common set of genes that are deregulated when rbf1 is mutated or when PSC is overexpressed. RNA was extracted from eye imaginal discs dissected from third instar Drosophila larvae. Samples were amplified and hybridized to Affymetrix Drosophila Genome 2.0 Array. To better understand the effects of rbf1 mutation and PSC overexpression, we compared the gene expression of rbf1 mutant eye discs and eye discs overexpressing PSC to control eye discs.
Project description:Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-)activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif discovery, is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes. FAIRE-Seq in Drosophila wild type eye-antennal imaginal discs (2 wt strains); ATAC-Seq in Drosophila wild type eye-antennal imaginal discs (3 wt strains) ; FAIRE-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila eye discs with Unpaired over-expression (2 biological replicates); CTCF ChIP-seq in Drosophila eye discs; ChIP-seq input in Drosophila eye discs
Project description:Drosophila mosaic eye-antennal discs from the listed genotypes generated using the MARCM system were dissected from 3rd instar larvae at day 5 after egg deposition. 20 pairs of discs for the Abrupt and scrib- + Abrupt samples and 50 pairs from FRT control, NACT scrib- +/- BskDN and RasACT scrib- +/- BskDN were used to prepare RNA. Samples were prepared in triplicate, and the RNA isolated using TRIZOL, before being column purified (Qiagen). Probes were hybridized to GeneChip Drosophila 2.0 Genome Arrays (Affymetrix). To compare the expression profile of Abrupt when overexpressed in the eye-antennal discs with tumours formed by Abrupt overexpression in scrib- clones, and to reveal JNK responsive genes in RasV12 (RasACT) scrib- versus NotchICD (NACT) scrib- eye-antennal mosaic discs
Project description:Whole-chromatin profile (FAIRE-seq) in three Drosophila species (D. melanogaster, D. pseudoobscura and D. virilis) in eye-antennal imaginal discs at the stage of third instar wandering larvae.
Project description:In this study we use Tag-sequencing in eye-antennal and wing imaginal discs across Drosophila species to determine a set of conserved eye-specific developmental genes. Next, we perform motif discovery analysis using the tool i-cisTarget, to depict the core gene developmental network underlying compound eye photoreceptor. The Glass position weight matrix appears as the most highly overrepresented motif, thus positioning Glass as a master regulator in compound eye photoreceptor development. Differential gene expression analysis by RNA-seq in D.melanogaster wild-type eye-antennal versus glass mutant [gl 60j] shows that the majority of our predicted Glass targets show strong downregulation in the glass mutant. This SuperSeries is composed of the following subset Series: GSE39781: RNA-seq in wild-type and glass mutant eye-antennal discs in Drosophila melanogaster GSE39782: Tag-seq profiling in eye-antennal and wing imaginal discs of D. melanogaster, D. yakuba and D. virilis