Gene expression after single and dual insulator protein knock-downs in Drosophila Kc cells.
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE36944 | GEO | 2012/06/18
SECONDARY ACCESSION(S): PRJNA157275
REPOSITORIES: GEO
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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. 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.
2012-06-17 | E-GEOD-36944 | biostudies-arrayexpress
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
Project description:Chromatin insulators are DNA-protein complexes that establish higher order independent DNA domains to influence transcriptional regulation. Insulators are defined by two different functions: they can block communication between an enhancer and a promoter and also act as a barrier between heterochromatin and euchromatin. In Drosophila, the gypsy-insulator complex contains three core components: Su(Hw), CP190 and Mod(mdg4)67.2. Here we identify a novel role for Chromatin-linked adaptor for MSL proteins (CLAMP) in promoting gypsy chromatin insulator function. When Clamp is depleted by RNAi, gypsy-dependent enhancer blocking activity decreases and barrier activity is reduced in all tissues. Furthermore, Clamp RNAi knockdowns and mutation result in disorganized insulator complex localization in the nucleus. Co-immunoprecipitation experiments showed that CLAMP physically associates with core gypsy-insulator proteins. Co-localization of CLAMP with gypsy components on polytene chromosomes and ChIP-seq analysis demonstrates co-localization of CLAMP with a subset of insulator sites across the genome. Thus, our findings suggest a ubiquitous, genome-wide role for CLAMP in promoting gypsy-dependent chromatin insulator activity.
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Project description:Chromatin insulators are DNA-protein complexes that establish higher order independent DNA domains to influence transcriptional regulation. Insulators are defined by two different functions: they can block communication between an enhancer and a promoter and also act as a barrier between heterochromatin and euchromatin. In Drosophila, the gypsy-insulator complex contains three core components: Su(Hw), CP190 and Mod(mdg4)67.2. Here we identify a novel role for Chromatin-linked adaptor for MSL proteins (CLAMP) in promoting gypsy chromatin insulator function. When Clamp is depleted by RNAi, gypsy-dependent enhancer blocking activity decreases and barrier activity is reduced in all tissues. Furthermore, Clamp RNAi knockdowns and mutation result in disorganized insulator complex localization in the nucleus. Co-immunoprecipitation experiments showed that CLAMP physically associates with core gypsy-insulator proteins. Co-localization of CLAMP with gypsy components on polytene chromosomes and ChIP-seq analysis demonstrates co-localization of CLAMP with a subset of insulator sites across the genome. Thus, our findings suggest a ubiquitous, genome-wide role for CLAMP in promoting gypsy-dependent chromatin insulator activity.
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Project description:Chromatin insulators and Polycomb group (PcG) complexes control nuclear organization to effect changes in gene expression. In Drosophila, RNA silencing pathways influence long range interactions mediated by PcG proteins and nuclear localization of the gypsy insulator; however, the underlying mechanisms are unknown. Here, we identify a singular requirement for Argonaute2 (AGO2) for the activity of the CCCTC-binding factor (CTCF)/Centrosomal protein 190 (CP190) dependent Fab-8 insulator. AGO2 and CP190 interact physically, and genome wide localization of AGO2 by chromatin immunoprecipitation and sequencing (ChIP-seq) reveals extensive colocalization of AGO2 with insulators and Polycomb Response Elements (PREs) but minimal overlap with regions of endogenous small interfering RNA (endo-siRNA) production. Finally, depletion of either CTCF or CP190 results in loss of AGO2 association with insulators, PREs, and other cis-regulatory regions. Our findings suggest that Dicer-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome. ChIP-seq of AGO2 in two Drosophila cell types (S2 and S3)
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Project description:Insulators are cis-regulatory sequences (CRSs) that can block enhancers from activating target promoters or act as barriers to block the spread of heterochromatin. Their name derives from their ability to ‘insulate’ transgenes from genomic position effects, an important function in gene therapy and biotechnology applications that require high levels of sustained transgene expression. In theory, flanking transgenes with insulators protects them from position effects, but in practice, efforts to insulate transgenes meet with mixed success because the contextual requirements for insulator function in the genome are not well understood. A key question is whether insulators are modular elements that can function anywhere in the genome or whether they are adapted to function only in certain genomic locations. To distinguish between these two possibilities we developed MPIRE (Massively Parallel Integrated Regulatory Elements) and used it to measure the effects of three insulators (A2, cHS4, ALOXE3) and their mutants at thousands of locations across the genome. Our results show that each insulator functions in only a small number of genomic locations, and that insulator function depends on the sequence motifs that comprise each insulator. All three insulators can block enhancers in the genome, but specificity arises because each insulator blocks enhancers that are bound by different sets of transcription factors. In contrast, only ALOXE3 can act as a heterochromatin barrier. We conclude that insulator function is highly context dependent and that MPIRE is a robust and systematic method for revealing the context dependencies of insulators and other cis-regulatory elements across the genome.
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Project description:Insulators are cis-regulatory sequences (CRSs) that can block enhancers from activating target promoters or act as barriers to block the spread of heterochromatin. Their name derives from their ability to ‘insulate’ transgenes from genomic position effects, an important function in gene therapy and biotechnology applications that require high levels of sustained transgene expression. In theory, flanking transgenes with insulators protects them from position effects, but in practice, efforts to insulate transgenes meet with mixed success because the contextual requirements for insulator function in the genome are not well understood. A key question is whether insulators are modular elements that can function anywhere in the genome or whether they are adapted to function only in certain genomic locations. To distinguish between these two possibilities we developed MPIRE (Massively Parallel Integrated Regulatory Elements) and used it to measure the effects of three insulators (A2, cHS4, ALOXE3) and their mutants at thousands of locations across the genome. Our results show that each insulator functions in only a small number of genomic locations, and that insulator function depends on the sequence motifs that comprise each insulator. All three insulators can block enhancers in the genome, but specificity arises because each insulator blocks enhancers that are bound by different sets of transcription factors. In contrast, only ALOXE3 can act as a heterochromatin barrier. We conclude that insulator function is highly context dependent and that MPIRE is a robust and systematic method for revealing the context dependencies of insulators and other cis-regulatory elements across the genome.
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Project description:Insulators are cis-regulatory sequences (CRSs) that can block enhancers from activating target promoters or act as barriers to block the spread of heterochromatin. Their name derives from their ability to ‘insulate’ transgenes from genomic position effects, an important function in gene therapy and biotechnology applications that require high levels of sustained transgene expression. In theory, flanking transgenes with insulators protects them from position effects, but in practice, efforts to insulate transgenes meet with mixed success because the contextual requirements for insulator function in the genome are not well understood. A key question is whether insulators are modular elements that can function anywhere in the genome or whether they are adapted to function only in certain genomic locations. To distinguish between these two possibilities we developed MPIRE (Massively Parallel Integrated Regulatory Elements) and used it to measure the effects of three insulators (A2, cHS4, ALOXE3) and their mutants at thousands of locations across the genome. Our results show that each insulator functions in only a small number of genomic locations, and that insulator function depends on the sequence motifs that comprise each insulator. All three insulators can block enhancers in the genome, but specificity arises because each insulator blocks enhancers that are bound by different sets of transcription factors. In contrast, only ALOXE3 can act as a heterochromatin barrier. We conclude that insulator function is highly context dependent and that MPIRE is a robust and systematic method for revealing the context dependencies of insulators and other cis-regulatory elements across the genome.
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Project description:Chromatin insulators and Polycomb group (PcG) complexes control nuclear organization to effect changes in gene expression. In Drosophila, RNA silencing pathways influence long range interactions mediated by PcG proteins and nuclear localization of the gypsy insulator; however, the underlying mechanisms are unknown. Here, we identify a singular requirement for Argonaute2 (AGO2) for the activity of the CCCTC-binding factor (CTCF)/Centrosomal protein 190 (CP190) dependent Fab-8 insulator. AGO2 and CP190 interact physically, and genome wide localization of AGO2 by chromatin immunoprecipitation and sequencing (ChIP-seq) reveals extensive colocalization of AGO2 with insulators and Polycomb Response Elements (PREs) but minimal overlap with regions of endogenous small interfering RNA (endo-siRNA) production. Finally, depletion of either CTCF or CP190 results in loss of AGO2 association with insulators, PREs, and other cis-regulatory regions. Our findings suggest that Dicer-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.
2011-08-15 | GSE22623 | GEO
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.
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