In situ CAPTURE of chromatin interactions by biotinylated dCas9
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ABSTRACT:
PROVIDER: PRJNA348725 | ENA |
REPOSITORIES: ENA
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Project description:Cis-regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs in chromatin remains unknown. Here we describe a CRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated endonuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show high-resolution and selective isolation of chromatin interactions at a single copy genomic locus. Purification of human telomeres using CAPTURE identifies known and new telomeric factors. In situ capture of individual constituents of the enhancer cluster controlling human β-globin genes establishes evidence for composition-based hierarchical organization of enhancer structure. Furthermore, unbiased analysis of chromatin interactions at disease-associated cis-elements and developmentally controlled super-enhancers reveals spatial features causally regulate gene transcription. Thus, comprehensive analysis of locus-specific regulatory composition provides mechanistic insight into genome structure and function in development and disease.
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Project description:The spatiotemporal control of 3D chromatin structure is fundamental for gene regulation, yet it remains challenging to obtain high-resolution chromatin interacting profiles at cis-regulatory elements (CREs) by chromatin conformation capture (3C)-based methods. Here, we describe the redesigned dCas9-based CAPTURE method for multiplexed, high-throughput and high-resolution analysis of locus-specific chromatin interactions. Using C-terminally biotinylated dCas9, endogenous biotin ligase and pooled sgRNAs, the new system enables quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling systematic comparisons across CREs within and between gene clusters. Multiplexed analyses of erythroid super-enhancers (SEs) reveal SE hierarchical structure and distinct modes of SE-gene interactions. Multiplexed capture of temporal dynamics of promoter-centric interactions establishes the instructive function of enhancer-promoter looping in transcriptional regulation during lineage differentiation. These applications illustrate the ability of multiplexed CAPTURE for decoding the organizational principles of genome structure and function.
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Project description:The spatiotemporal control of 3D chromatin structure is fundamental for gene regulation, yet it remains challenging to obtain high-resolution chromatin interacting profiles at cis-regulatory elements (CREs) by chromatin conformation capture (3C)-based methods. Here, we describe the redesigned dCas9-based CAPTURE method for multiplexed, high-throughput and high-resolution analysis of locus-specific chromatin interactions. Using C-terminally biotinylated dCas9, endogenous biotin ligase and pooled sgRNAs, the new system enables quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling systematic comparisons across CREs within and between gene clusters. We reveal the hierarchical structure of super-enhancers (SEs) and distinct modes of SE-gene interactions. Multiplexed capture of temporal dynamics of promoter-centric interactions establishes the instructive function of enhancer-promoter looping in transcriptional regulation during lineage differentiation. These applications illustrate the ability of multiplexed CAPTURE for decoding the organizational principles of genome structure and function.
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Project description:Cis-regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs in chromatin remains unknown. Here we describe a CRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated endonuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show high-resolution and selective isolation of chromatin interactions at a single copy genomic locus. Purification of human telomeres using CAPTURE identifies known and new telomeric factors. In situ capture of individual constituents of the enhancer cluster controlling human β-globin genes establishes evidence for composition-based hierarchical organization of enhancer structure. Furthermore, unbiased analysis of chromatin interactions at disease-associated cis-elements and developmentally controlled super-enhancers reveals spatial features causally regulate gene transcription. Thus, comprehensive analysis of locus-specific regulatory composition provides mechanistic insight into genome structure and function in development and disease.
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Project description:The spatiotemporal control of 3D chromatin structure is fundamental for gene regulation, yet it remains challenging to obtain high-resolution chromatin interacting profiles at cis-regulatory elements (CREs) by chromatin conformation capture (3C)-based methods. Here, we describe the redesigned dCas9-based CAPTURE method for multiplexed, high-throughput and high-resolution analysis of locus-specific chromatin interactions. Using C-terminally biotinylated dCas9, endogenous biotin ligase and pooled sgRNAs, the new system enables quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling systematic comparisons across CREs within and between gene clusters. We reveal the hierarchical structure of super-enhancers (SEs) and distinct modes of SE-gene interactions. Multiplexed capture of temporal dynamics of promoter-centric interactions establishes the instructive function of enhancer-promoter looping in transcriptional regulation during lineage differentiation. These applications illustrate the ability of multiplexed CAPTURE for decoding the organizational principles of genome structure and function.
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Project description:Cis-regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs in chromatin remains unknown. Here we describe a CRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated endonuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show high-resolution and selective isolation of chromatin interactions at a single copy genomic locus. Purification of human telomeres using CAPTURE identifies known and new telomeric factors. In situ capture of individual constituents of the enhancer cluster controlling human β-globin genes establishes evidence for composition-based hierarchical organization of enhancer structure. Furthermore, unbiased analysis of chromatin interactions at disease-associated cis-elements and developmentally controlled super-enhancers reveals spatial features causally regulate gene transcription. Thus, comprehensive analysis of locus-specific regulatory composition provides mechanistic insight into genome structure and function in development and disease.
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