Locus-Scale Massively Parallel Reporter Assays
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE341622 | GEO | 2026/07/28
REPOSITORIES: GEO
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Project description:We have combined the 4C chromosome conformation capture protocol with high throughput, genome-wide sequence analysis to characterise in full a cis-acting regulatory network at a single locus. In contrast to methods which identify large interacting regions (10-1000kb), the 4C approach provides a comprehensive, high resolution analysis of a specific locus with the aim of defining, in detail, all cis- regulatory elements controlling a single gene or gene cluster. Using the human M-NM-1-globin locus as a model we detected all known local and long-range interactions with this gene cluster. In addition we identified two interactions with genes located 300 kb (NME4) and 625 kb (FAM173a) from the M-NM-1-globin cluster. Using paired-end sequencing it was possible for the first time to identify interactions between specific subsets of cis-regulatory elements in populations of cells and within individual cells. Two biological replicates of 4C amplification from the promoters of the human HBA gene promoters. Sequenced as paired end fragments usinig Illumina GAII
2013-06-06 | E-GEOD-42382 | biostudies-arrayexpress
Project description:We have combined the 4C chromosome conformation capture protocol with high throughput, genome-wide sequence analysis to characterise in full a cis-acting regulatory network at a single locus. In contrast to methods which identify large interacting regions (10-1000kb), the 4C approach provides a comprehensive, high resolution analysis of a specific locus with the aim of defining, in detail, all cis- regulatory elements controlling a single gene or gene cluster. Using the human M-NM-1-globin locus as a model we detected all known local and long-range interactions with this gene cluster. In addition we identified two interactions with genes located 300 kb (NME4) and 625 kb (FAM173a) from the M-NM-1-globin cluster. Using paired-end sequencing it was possible for the first time to identify interactions between specific subsets of cis-regulatory elements in populations of cells and within individual cells. Two biological replicates of 4C amplification from the promoters of the human HBA and HBB genes and B-Cell controls. Quality of B-cell material was confirmed using amplification from a CTCF bound region associated with Human Axin Gene.
2013-06-06 | E-GEOD-42383 | biostudies-arrayexpress
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:We have combined the 4C chromosome conformation capture protocol with high throughput, genome-wide sequence analysis to characterise in full a cis-acting regulatory network at a single locus. In contrast to methods which identify large interacting regions (10-1000kb), the 4C approach provides a comprehensive, high resolution analysis of a specific locus with the aim of defining, in detail, all cis- regulatory elements controlling a single gene or gene cluster. Using the human α-globin locus as a model we detected all known local and long-range interactions with this gene cluster. In addition we identified two interactions with genes located 300 kb (NME4) and 625 kb (FAM173a) from the α-globin cluster. Using paired-end sequencing it was possible for the first time to identify interactions between specific subsets of cis-regulatory elements in populations of cells and within individual cells.
2013-06-06 | GSE42383 | GEO
Project description:We have combined the 4C chromosome conformation capture protocol with high throughput, genome-wide sequence analysis to characterise in full a cis-acting regulatory network at a single locus. In contrast to methods which identify large interacting regions (10-1000kb), the 4C approach provides a comprehensive, high resolution analysis of a specific locus with the aim of defining, in detail, all cis- regulatory elements controlling a single gene or gene cluster. Using the human α-globin locus as a model we detected all known local and long-range interactions with this gene cluster. In addition we identified two interactions with genes located 300 kb (NME4) and 625 kb (FAM173a) from the α-globin cluster. Using paired-end sequencing it was possible for the first time to identify interactions between specific subsets of cis-regulatory elements in populations of cells and within individual cells.
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Project description:In cellular genomes, CTCF insulators impact transcription over small distances in a one-dimensional manner and over much longer distances in a three-dimensional manner by maintaining chromatin loops. We have previously shown that the latent HSV-1 genome contains CTCF insulators that function to regulate lytic transcription of adjacent genes in a one-dimensional manner. Here, we test the hypothesis that HSV-1 CTCF insulators nucleate chromatin loops to regulate the expression of distance separated gene regions through three-dimensional organization of viral genomes. We used 4C-seq methods to identify multiple long-range cis interactions in HSV-1 genomes that generate viral chromatin domains, including those nucleated by the viral CTCF insulator CTRL2. Deletion of the CTRL2 insulator disrupted these viral chromatin domains. Loop-nucleating interactions were quantitated with a novel approach (UMI-4C-seq) that utilizes unique molecular identifiers to label and count chromatin interactions associated with specific viewpoint primers. Cis-interaction peaks across four different viewpoints were quantified. Viral genomes lacking CTRL2 displayed more cis-interaction peaks and wider ranges of interaction lengths compared to wt virus, suggesting altered chromatin organization. Furthermore, differential looping analysis showed viral genomes lacking CTRL2 displayed a more transcriptionally permissive chromatin environment. Thus, the CTRL2 insulator functions as a critical regulator of long-range chromatin interactions and its deletion reshapes the viral chromatin landscape, leading to a more accessible and dynamic regulatory environment that may influence HSV-1 transcriptional programs and latency-associated chromatin states.
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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: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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