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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.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Edward Ramos

PROVIDER: E-GEOD-36944 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Gene expression after single and dual insulator protein knock-downs in Drosophila Kc cells.

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.

2012-06-18 | GSE36944 | GEO

Gene expression analysis of Kc cells from Drosophila melanogaster during ecdysone treatment and CP190 knockdown

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.

2011-10-13 | E-GEOD-30686 | biostudies-arrayexpress

Kc cells from Drosophila melanogaster during ecdysone treatment and CP190 knockdown

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

2011-10-13 | E-GEOD-30741 | biostudies-arrayexpress

NURF301 contributes to gypsy chromatin insulator-mediated nuclear organization

Project description:Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and their normal localization is correlated with proper insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) are also required for recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy binding site DNA, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.

2022-10-14 | PXD032165 | Pride

BEAF-32 insulator regulates gene expression of genes involved in cell polarity and neoplastic growth.

Project description:This SuperSeries is composed of the following subset Series: GSE36735: Distribution of Drosophila insulator protein BEAF-32 in Wing imaginal tissue (Wildtype) [ChIP-seq] GSE36736: Genome wide transcriptional profiling of BEAF-32 in wing imaginal tissues of wildtype and mutants [expresion array] Refer to individual Series

2012-07-05 | E-GEOD-36737 | biostudies-arrayexpress

Genome-wide localization of exosome components to active promoters and chromatin insulators in Drosophila

Project description:Chromatin insulators are functionally conserved DNA-protein complexes that are situated throughout the genome and organize independent transcriptional domains. Previous work implicated RNA as an important cofactor in chromatin insulator activity, although the mechanisms by which RNA affects insulator activity are not yet understood. Here we identify the exosome, the highly conserved major cellular 3’ to 5’ RNA degradation machinery, as a physical interactor of CP190-dependent chromatin insulator complexes in Drosophila. High resolution genome-wide profiling of exosome by ChIP-seq in two different embryonic cell lines reveals extensive and specific overlap with the CP190, BEAF-32, and CTCF insulator proteins. Colocalization occurs mainly at promoters but also well-characterized boundary elements, such as scs, scs’, Mcp, and Fab-8. Surprisingly, exosome associates primarily with promoters but not gene bodies, arguing against simple cotranscriptional recruitment to RNA substrates. We find that exosome is recruited to chromatin in a transcription dependent manner, preferentially to highly transcribed genes. Similar to insulator proteins, exosome is also significantly enriched at divergently transcribed promoters. Directed ChIP of exosome in cell lines depleted of insulator proteins shows that CTCF is specifically required for exosome association at Mcp and Fab-8 but not other sites, suggesting that alternate mechanisms must also contribute to exosome chromatin recruitment. Taken together, our results reveal a novel relationship between exosome and chromatin insulators throughout the genome. ChIP-seq of exosome components. RNA-seq after control and exosome subunit knockdown in Drosophila cell lines.

2013-01-29 | E-GEOD-41950 | biostudies-arrayexpress

Transcription profiling of Drosophila Myb-MuvB/dREAM

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).

2008-06-16 | E-GEOD-8751 | biostudies-arrayexpress

Mapping the inter- and intra-chromosomal interactions of specific insulator binding sites with circular chromosome conformation capture (4C) assay (Gondor et al., 2008 Nature Protocol) [NimbleGen Array data]

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.

2013-09-15 | E-GEOD-50768 | biostudies-arrayexpress

Effects of FSH knockdown on gene expression

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).

2013-02-06 | E-GEOD-43180 | biostudies-arrayexpress

Genome wide transcriptional profiling of BEAF-32 in wing imaginal tissues of wildtype and mutants [expresion array]

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.

2012-07-05 | E-GEOD-36736 | biostudies-arrayexpress

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