Project description:The subject of the current study is the finding of possible molecular partners of drosophila EcR receptor. The whole-genome experiments revealed that the sites of EcR receptor are partially overlapped with ERR binding sites. As ERR receptor specifically binds regulatory regions of glycolytic genes and genes of glycogen metabolism, the presence of EcR on ERR targets signifies involvement of the ecdysone signaling in regulation of carbohydrate metabolism.
Project description:In order to identify interaction partner of the Drosophila melanogaster TFIIA protein, we have immunoprecipitated an endogenously 3xFLAG-AID tagged TFIIA-L from Drosophila Schneider S2 cells
Project description:The centromere-specific Histone H3-variant CENH3 (also known as CENP-A) is considered to be an epigenetic mark for establishment and propagation of centromere identity. Pulse-induction of CENH3 (Drosophila CID) in Schneider S2 cells incorporates into noncentromeric regions and generates CID islands that resist clearing from chromosome arms for multiple cell generations. We demonstrate that CID islands represent functional ectopic kinetochores, which are non-randomly distributed on the chromosome and display a preferential localization near telomeres and pericentric heterochromatin in transcriptionally silent, intergenic chromatin domains. Although overexpression of heterochromatin protein 1 (HP1) or increasing Histone acetylation interferes with CID islands formation on a global scale, induction of a locally defined region of synthetic heterochromatin by targeting HP1-LacI fusions to stably integrated Lac Operator arrays produces a proximal hotspot for CID islands formation. These data suggest that the characteristics of regions bordering heterochromatin promote de novo kinetochore assembly and thereby contribute to centromere identity.
Project description:Neurogenesis in all animals is dependent on the function of different Notch receptors and their ligands. Drosophila is an excellent system to understand the principles and mechanisms governing this conserved process. Since the different Notch receptors and their ligands are expressed and function in the same cells, or overlapping stages of neurogenesis, and since there is extensive feedback regulation between them, it is very difficult to identify the target genes of each receptor or ligand in vivo. We have developed an excellent model system based on Drosophila cultured Schneider (S2) cells that faithfully represents in vivo activities. We propose microarray analysis with this system to identify the suite of putative target genes that are independently regulated by each protein in our study. We also expect to gather information about the signaling pathways used by the different receptors and their ligands, and develop hypotheses for testing during neurogenesis in vivo. These efforts will contribute significantly towards achieving the Specific Objectives of the grant parent (R01 NS043122,Different Notch Receptors in Drosophila Development).,To determine the sets of genes regulated by NFull, NDCterm, and Delta through microarray analyses of RNA extracted from S2 cells expressing these proteins that were treated or not treated with S2 cells expressing their ligand or receptor. ,Neurogenesis in Drosophila initiates within clusters of cells that have acquired the potential to become neuronal cells. The action of the full-length Notch receptor, NFull, and the ligand Delta make the majority of these cells to differentiate the epidermis. The action of a truncated Notch receptor, NDCterm, and Delta make the remaining cells to differentiate the nervous system. We have recently shown that Delta also has activities independent of Notch receptors that promote neurogenesis. We will test the hypothesis that NFull would promote expression of epidermal differentiation genes while NDCterm and Delta that would promote expression of neuronal differentiation genes. We will also test the hypothesis that genes regulated by NDCterm and Delta would be the same or would complement each otherâs activities.,We request the Microarray Consortium to process our samples and help us analyze the data. We will select a set of genes for validation by northern blotting or QPCR on similar samples of RNA from S2 cells and in vivo materials. We have available in the lab fly stocks carrying mutants (nulls) alleles or inducible transgenes for all the proteins used in this microarray experiment. Funds from the parent grant will be used for these in vitro and in vivo validation experiments. This project proposal is a part of the Supplementary Proposal approved by NIH/NINDS. Project proposals covering other parts, the effects on secreted ligands of Scabrous and Wingless, will be submitted when we have ready the RNAs from these experiments.
Project description:HITS-CLIP was carried out on wild-type Schneider (S2) cells using specific MLE antibodies to identify binding sites for MLE in the Drosophila transcriptome
Project description:Cis-natural antisense transcripts (cis-NATs) have been speculated to be substrates for endogenous RNA interference (RNAi), but little experimental evidence for such a pathway in animals has been reported. Analysis of massive Drosophila melanogaster small RNA data sets now reveals that endogenous small interfering RNAs (siRNAs) are produced via bidirectional transcription. >100 cis-NATs with overlapping 3' exons generate 21-nt, Dicer-2 (Dcr-2)Âdependent, 3'-end modified siRNAs. To determine whether any co-expressed cisNATs are denied entry into the RNAi pathway, we analyzed the gene expression profile of S2 cells. The analysis suggested that the processing of cis-NATs by RNAi are actively restricted, and the selected loci are enriched for nucleic acidÂbased functions and include Argonaute-2 (AGO2) itself. Experiment Overall Design: Drosophila Schneider cells (S2) were treated with dsRNA against GFP for 8 days. The treatment was done in duplicate. Total RNA was extracted from the dsRNA treated cells using trizol. Experiment Overall Design: Gene expression analysis was conducted using Drosophila Genome 2.0 Genechip® arrays (Affymetrix, Santa Clara, CA). Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3¹ Amplification One-Cycle Target labeling kit according to manufacturer¹s protocol. For each array, 10ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000 and data was obtained using the Genechip® Operating Software (Version 1.2.0.037).
Project description:Transcriptional profiling of Schneider 2 cells comparing control cells with stable inducible dGcn2-V5 expressing cells after 12h of induction with 0.7mM copper sulphate.
Project description:Transcriptional profiling of Schneider 2 cells comparing control cells with stable inducible dGcn2-V5 expressing cells after 6h of induction with 0.7mM copper sulphate.
Project description:Transcriptional profiling of Schneider 2 cells comparing control stable inducible HA-expressing cells with stable inducible HA-crc expressing cells after 6h of induction with 0.7mM copper sulphate.