Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Unbiased screening of regulatory insulators

ABSTRACT: Long-range transcriptional activation of gene promoters by abundant enhancers in animal genomes raises the need for mechanisms to limit inappropriate gene regulation. DNA elements known as insulators provide one such mechanism by shielding promoters from an enhancer when placed in between these two elements. Whereas promoters and enhancers have been extensively characterized in animal genomes, insulators have not because of the lack of a high-throughput screening assay. In the absence of unbiased insulator screening in a genome, basic questions remain about the identity, density and diversity of genomic insulators. Here, we establish “insulator-seq” as a plasmid-based massively parallel reporter assay in Drosophila tissue culture cells to perform the first unbiased screen for insulators in any genome. By screening developmental gene loci, we found that not all characterized insulator protein binding sites are able to block enhancer-promoter communication. By dissecting the sequence determinants of functional insulators, we found that an unexpectedly broad sequence context around the central insulator protein binding motif is required for functionality. The ability to screen millions of DNA sequences in parallel without any positional effect has enabled the first functional mapping of insulators and provided further insights into the determinants of functional insulators.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE253140 | GEO | 2024/09/09

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection [II]

Project description:CTCF is a key insulator-binding protein and mammalian genomes contain numerous CTCF-binding sites (CBSs), many of which are organized in tandem arrays. Here we provide direct evidence that CBSs, if located between enhancers and promoters in the clustered Pcdh and b-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CBS or not. Moreover, computational simulation and experimental capture revealed balanced promoter usage in vivo in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem variable CBSs. Finally, gene expression levels are negatively correlated with CBS insulators located between enhancers and promoters on a genome-wide scale. Thus, single CBS insulators ensure proper enhancer insulation and promoter activation while tandem-arrayed CBS insulators determine balanced promoter choice. This finding has interesting implications on the role of topological insulators in 3D genome folding and developmental gene regulation.

2020-03-01 | GSE136328 | GEO

Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection [I]

2020-03-01 | GSE135394 | GEO

Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection [III]

2020-03-01 | GSE138645 | GEO

Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection [V]

2020-03-01 | GSE139679 | GEO

Massively parallel characterization of insulator activity across the genome [GWLP_pools]

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.

2023-01-27 | GSE223389 | GEO

Massively parallel characterization of insulator activity across the genome [pilot_rBC]

2023-01-27 | GSE223384 | GEO

Massively parallel characterization of insulator activity across the genome [insulator_libraries]

2023-01-27 | GSE223402 | GEO

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

Genomewide analysis of Insulator protein binding sites in Drosophila embryos at E0-12

Project description:Insulators are DNA sequences that control the interactions among genomic regulatory elements and act as chromatin boundaries. A thorough understanding of their location and function is necessary to address the complexities of metazoan gene regulation. We studied by ChIP-chip the genome-wide binding sites of 6 insulator-associated proteins – dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4) and GAF – to obtain the first comprehensive map of insulator elements in Drosophila embryos. We identify over 14,000 putative insulators, including all previously known insulators. We find two major classes of insulators defined by dCTCF/CP190/BEAF-32 and Su(Hw) respectively. Distributional analyses of insulators revealed that particular sub-classes of insulator elements are excluded between cis-regulatory elements and their target promoters, divide differentially expressed, alternative, and divergent promoters, act as chromatin boundaries, are associated with chromosomal breakpoints among species, and are embedded within active chromatin domains. Together, these results provide a map demarcating the boundaries of gene regulatory units, and a framework for understanding insulator function during the development and evolution of Drosophila. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

2009-05-29 | GSE16245 | GEO

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

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data