Gene expression after single and dual insulator protein knock-downs in Drosophila Kc cells.
ABSTRACT: Transcriptional changes assayed with two bilogical replicates in wild type and RNAi mediated insulator knock-downs RNAi mediated insulator knock-downs cause changes in the H3K27me3 levels and spread of Topoisomerase II In order to understand the role of insulators in gene expression and regulation we used Drosophila Kc cells to knock-down single and multiple insulators in combination to assay for transcriptional changes. Two biologial replicates were prepared in independent experiments. Each cDNA samples were labeled with Cy3 dye and hybridized and scaned as per manufactures instructions at Florida State University Nimblegen facility.
Project description:This study examines the changes in genes expression that occur in Drosophila melanogaster during the ecdysone response as well as during RNAi knockdown of the insulator protein, CP190. Analysis was performed in Kc cells after 0, 3, and 48 hours of ecdysone treatment in the presence of either control or CP190 knockdown. Six conditions were analyzed, and each condition was performed for 2 biological replicates making a total of 12 chips. Each chip measures the expression level of 16,637 genes from D.melanogaster with eight, 60-mer probes per gene.
Project description:Background: The question of how cells re-establish gene expression states after cell division is still poorly understood. Genetic and molecular analyses have indicated that Trithorax group (TrxG) proteins are critical for the long-term maintenance of active gene expression states in many organisms. A generally accepted model suggests that TrxG proteins contribute to maintenance of transcription by protecting genes from inappropriate Polycomb group (PcG)-mediated silencing, instead of directly promoting transcription. Results: Here we report a physical and functional interaction in Drosophila between two members of the TrxG, the histone methyltransferase ASH1 and the bromodomain and extraterminal family protein FSH. We investigated this interface at the genome level, uncovering a widespread colocalization of both proteins at promoters and PcG-bound intergenic elements. Our integrative analysis of chromatin maps and gene expression profiles revealed that the observed ASH1-FSH binding pattern at promoters is a hallmark of active genes. Inhibition of FSH-binding to chromatin resulted in global down-regulation of transcription. In addition, we found that genes displaying marks of robust PcG-mediated repression also have ASH1 and FSH bound to their promoters. Conclusions: Our data strongly favor a global coactivator function of ASH1 and FSH during transcription, as opposed to the notion that TrxG proteins impede inappropriate PcG-mediated silencing, but are dispensable elsewhere. Instead, our results suggest that PcG repression needs to overcome the transcription-promoting function of ASH1 and FSH in order to silence genes. Refer to individual Series
Project description:see Super Series Summary Gene expression profiles of Drosophila S2-DRSC FSH knockdown cells were generated by Illumna RNA sequencing and compared to profiles derived from control cells (eGFP knockdown).
Project description:Transcriptional changes assayed with two bilogical replicates in wild type and RNAi mediated insulator knock-downs RNAi mediated insulator knock-downs cause changes in the H3K27me3 levels and spread of Topoisomerase II In order to understand the role of insulators in gene expression and regulation we used Drosophila Kc cells to knock-down single and multiple insulators in combination to assay for transcriptional changes. Overall design: Two biologial replicates were prepared in independent experiments. Each cDNA samples were labeled with Cy3 dye and hybridized and scaned as per manufactures instructions at Florida State University Nimblegen facility.
Project description:This SuperSeries is composed of the following subset Series: GSE30686: Gene expression analysis of Kc cells from Drosophila melanogaster during ecdysone treatment and CP190 knockdown GSE30740: Distribution of Drosophila insulator proteins after ecdysone treatment in Kc cells Refer to individual Series
Project description:This SuperSeries is composed of the following subset Series: GSE15660: Gene Expression analysis of Kc and Mbn2 cell lines from Drosophila melanogaster. GSE15661: Genome-wide binding profiles of Drosophila melanogaster insulator proteins in Kc and Mbn2 cells (Set1) GSE15662: Genome-wide binding profiles of Drosophila melanogaster insulator proteins in Kc and Mbn2 cells (Set2) GSE15663: Genome-wide binding profiles of Drosophila melanogaster insulator proteins in Kc and Mbn2 cells (Set3) Refer to individual Series
Project description:Drosophila Insulator proteins mediate long-range chromosomal interactions. ChIP-seq revealed that binding of insulator proteins to some specific DNA sites was regulated by poly(ADP-ribosyl)ation in S2 cells. Three insulator sites regulated by poly(ADP-ribosyl)ation were used as baits to map their distant interacting sites using 4C assay in control S2 cells. Mapping the chromosomal interactions of three specific insulator binding sites with 4C assay in control S2 cells.
Project description:The mutants in BEAF-32 gene in Drosophila caused the Neoplastic growth. In order to understand the rold of BEAF-32 insulator function in regualtion of gene expression we used the imagial tissue as a model to assay the transcriptional changes. The Imagianl tissues were disected in the icecold PBS and Total RNA was prepared using RNaeasy ( qiagen ) kit. This is followed by cDNA synthesis using Oligo dT. Transcriptional changes assayed with two biological replicates and two experimental replicates Two biologial replicates and two experimental replicates were prepared in an independent experiments. Each cDNA samples were labeled with Cy3 due and hybridised and scaned as per manufactures instructions at Florida state university Nimblegen fecility.
Project description:Myb-MuvB (MMB)/dREAM is a nine subunit complex first described in Drosophila as a repressor of transcription, dependent upon E2F2 and the RBFs. Myb, an integral member of MMB, curiously plays no role in the silencing of the test genes previously analyzed. Moreover, Myb plays an activating role in DNA replication in Drosophila egg chamber follicle cells. The essential functions for Myb are executed as part of MMB. This duality of function lead to the hypothesis that MMB, which contains both known activator and repressor proteins, might function as part of a switching mechanism that is dependent upon DNA sites and developmental context. Here, we used proliferating Drosophila Kc tissue culture cells to explore both the network of genes regulated by MMB (employing RNAi and micro-array expression analysis) and the genomic locations of MMB following chromatin immunoprecipitation (ChIP) and tiling array analysis. MMB occupies thousands of chromosomal sites where a substantial number are proximal to repressed genes that are normally expressed in a wide range of developmental pathways. At many of these sites, E2F2 was critical for repression whereas at other non-overlapping sites, Myb was critical for repression. These data highlight that the MMB factors are utilized in a combinatorial way for targeting gene regulation. We also found sites where MMB was a positive regulator of transcript levels that included genes required for mitotic functions (G2/M), which may explain some of the chromosome instability phenotypes attributed to loss of Myb function in myb mutants. Experiment Overall Design: RNAi to deplete Lin-52, Mip40, Myb, Mip120, Mip130, E2F2, both RBFs (RBF1 and RBF2) and L(3)MBT were performed in triplicate. RNAi with a nonspecific RNA derived from a pBSK+ plasmid (named SK+) was used as control. Total RNA was extracted from RNAi-transfected cells after 4 days using RNeasy Mini Kit (QIAGEN).