CAPTURE-3C-seq analysis of cis-element-mediated long-range DNA interactions
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PROVIDER: PRJNA387464 | 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. 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:Capture-C using probes at the Cdkn1b promoter and the Lockd promoter Many long non-coding (lnc) RNAs are reported to regulate gene expression and protein functions. However, the proportion of lncRNAs with biological activities among the thousands expressed in mammalian cells is controversial. We studied Lockd (LncRNA downstream of Cdkn1b), a 434 bp polyadenylated lncRNA originating 4 kb 3â to the Cdkn1b gene. Heterozygous and homozygous deletion of the 25 kb Lockd locus reduced Cdkn1b transcription by approximately 35 and 70% respectively in a mouse erythroid cell line. In contrast, homozygous insertion of a polyadenylation cassette 80 bp downstream of the Lockd transcription start site reduced the entire lncRNA transcript level by > 90%, but had no effect on Cdkn1b transcription. The promoter of the Lockd gene contains a DNase hypersensitive site, binds numerous transcription factors (TFs), and physically associates with the Cdkn1b promoter in chromosomal conformation capture (NG Capture-C) studies. Thus, the Lockd gene positively regulates Cdkn1b transcription through an enhancer-like cis element, while the lncRNA itself is dispensable. These findings demonstrate that the biological activities of a lncRNA cannot be inferred from phenotypes that arise after deleting the corresponding gene. Rather, the model of an inert transcript arising from a functional genomic cis element should be considered while investigating the biology of any lncRNA.
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