Cut-and-Run in situ factor profiling maps DNA binding and 3D contact sites at high resolution
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens Saccharomyces cerevisiae
PROVIDER: GSE84474 | GEO | 2016/12/30
SECONDARY ACCESSION(S): PRJNA329356
REPOSITORIES: GEO
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Project description:Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has exceptionally low background levels. CUT&RUN outperforms the most widely- used Chromatin Immunoprecipitation (ChIP) protocols in resolution, signal-to-noise, and depth of sequencing required. In contrast to ChIP, CUT&RUN is free of solubility and DNA accessibility artifacts and has been used to profile insoluble chromatin and to detect long-range 3D contacts without cross-linking. Here we present an improved CUT&RUN protocol that does not require isolation of nuclei and provides high-quality data starting with only 100 cells for a histone modification and 1000 cells for a transcription factor. From cells to purified DNA CUT&RUN requires less than a day at the lab bench and requires no special skills.
2018-03-21 | GSE104550 | GEO
Project description:We introduce CUT&RUNTools (https://bitbucket.org/qzhudfci/cutruntools/) as a flexible, general pipeline for facilitating the identification of chromatin-associated protein binding and genomic footprinting analysis from antibody-targeted CUT&RUN primary cleavage data. CUT&RUNTools extracts endonuclease cut site information from sequences of short read fragments and produces single-locus binding estimates, aggregate motif footprints, and informative visualizations to support the high-resolution mapping capability of CUT&RUN. We illustrate the functionality of CUT&RUNTools through analysis of CUT&RUN data acquired for GATA1, a master regulator in erythroid lineage cells. Results were compared initially to published GATA1 ChIP-seq data for cells under the same conditions. We performed de novo analysis of CUT&RUN peaks to retrieve not only GATA1’s primary motif, but also the GATA1-TAL1 composite motif, and co-factor motifs GCCCCGCCTC, CMCDCCC, and RTGASTCA that correspond to SP1, KLF1, and NFE2 co-factors. Cofactor binding was verified by independent TAL1 and KLF1 CUT&RUN, and other ChIP-seq experiments. CUT&RUNTools also generated base-pair resolution motif footprint for sequence-specific binding factors, and located likely direct binding sites by quantifying log-odds of binding scores. Overall, CUT&RUNTools should enable biologists to realize advantages of cleavage data provided by CUT&RUN, and make high-quality footprinting analysis accessible to a broad audience.
2019-08-24 | GSE136251 | GEO
Project description:Endosperm is an essential seed tissue with a unique epigenetic landscape. During endosperm development, differential epigenetic regulation of the maternal and paternal genomes plays important roles in regulating gene expression, especially at imprinted genes. Profiling the endosperm epigenetic landscape on a genome-wide scale is challenging due to its small size, mode of development, and close association with maternal tissue. Here, we applied a low input chromatin profiling method, CUT&RUN (cleavage under targets and release using nuclease), to profile parental-specific chromatin modifications using low numbers of Arabidopsis endosperm nuclei. We demonstrate that CUT&RUN generates genome-wide H3K27me3 landscapes with high sensitivity, specificity and reproducibility using around 20,000 endosperm nuclei purified by flow cytometry and fluorescence-activated cell sorting. H3K27me3 peaks identified by CUT&RUN and previous ChIP (chromatin immunoprecipitation) approaches were largely overlapping, with some distinctions in heterochromatin. The versatility and simplicity of CUT&RUN makes it a viable alternative to ChIP, which requires greater amounts of starting material, and will enable the study of tissue or even cell-type specific epigenomes in Arabidopsis and other plant species.
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Project description:Characterizing the relationship between genome form and function requires methods both for zooming out, to globally survey the genomic architectural landscape, and for zooming in, to investigate regions of interest at high resolution. High throughput methods based on chromosome conformation capture (3C) have greatly advanced our understanding of the three-dimensional (3D) organization of genomes, but are limited in resolution by their reliance on restriction enzymes. Here we describe a method for comprehensively mapping global chromatin contacts that uses DNaseI for chromatin fragmentation, leading to greatly improved efficiency and resolution. Coupling this method with DNA capture technology provides a high-throughput approach for targeted mapping of fine-scale chromatin architecture. We applied targeted DNase Hi-C to characterize the 3D organization of 1,030 lincRNA promoters in two human cell lines, and identified thousands of high-confidence lincRNA promoter-associated chromatin contacts at 1 kilobase pair (kbp) resolution. Detailed analysis of these contacts reveals that expression of lincRNAs is tightly controlled by complex mechanisms involving both super-enhancers and the polycomb repressive complex. Our results provide the first glimpse of the cell-type-specific 3D organization of lincRNA genes. DNase Hi-C assay (x1 replicate) in H1 and K562 cells, respectively. Targeted DNase Hi-C assays of the 220kb promoter-enhancer library (x1 replicate) and the 5Mb lincRNA promoter library (x2 replicate), in H1 and K562 cells, respectively.
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Project description:Regulatory transcription factors control many important biological processes including cellular differentiation, responses to environmental perturbations and stresses, and host-pathogen interactions. Determining the genome-wide binding of regulatory transcription factors to DNA is essential to understanding the function of transcription factors in these often complex biological processes. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a modern method for genome-wide mapping of in vivo protein-DNA binding interactions that is an attractive alternative to the traditional and widely used chromatin immunoprecipitation followed by sequencing (ChIP-seq) method. CUT&RUN is amenable to a higher throughput experimental setup and has a substantially higher dynamic range with lower per-sample sequencing costs compared to ChIP-seq. Here, we describe a comprehensive CUT&RUN protocol and accompanying data analysis workflow that is tailored for genome-wide analysis of transcription factor-DNA binding interactions in the human fungal pathogen Candida albicans. This detailed protocol describes all necessary experimental procedures, from epitope tagging of transcription factor coding genes, to library preparation for sequencing; additionally, it includes our customized computational workflow for CUT&RUN data analysis.
2022-07-15 | GSE193803 | GEO
Project description:Here we describe successful implementation of CUT&RUN for profiling protein-DNA interactions in zebrafish embryos. We apply modified a CUT&RUN method to generate high resolution maps of enrichment for H3K4me3, H3K27me3, H3K9me3, and RNA polymerase II during zebrafish gastrulation. Using this data, we identify a conserved subset of developmental genes that are enriched in both H3K4me3 and H3K27me3 during gastrulation, and we demonstrate the increased effectiveness of CUT&RUN for detecting protein enrichment at repetitive sequences with reduced mappability. Our work demonstrates the power of combining CUT&RUN with the strengths of the zebrafish system to better understand the changing embryonic chromatin landscape and its roles in shaping development.
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Project description:Characterizing the relationship between genome form and function requires methods both for zooming out, to globally survey the genomic architectural landscape, and for zooming in, to investigate regions of interest at high resolution. High throughput methods based on chromosome conformation capture (3C) have greatly advanced our understanding of the three-dimensional (3D) organization of genomes, but are limited in resolution by their reliance on restriction enzymes. Here we describe a method for comprehensively mapping global chromatin contacts that uses DNaseI for chromatin fragmentation, leading to greatly improved efficiency and resolution. Coupling this method with DNA capture technology provides a high-throughput approach for targeted mapping of fine-scale chromatin architecture. We applied targeted DNase Hi-C to characterize the 3D organization of 1,030 lincRNA promoters in two human cell lines, and identified thousands of high-confidence lincRNA promoter-associated chromatin contacts at 1 kilobase pair (kbp) resolution. Detailed analysis of these contacts reveals that expression of lincRNAs is tightly controlled by complex mechanisms involving both super-enhancers and the polycomb repressive complex. Our results provide the first glimpse of the cell-type-specific 3D organization of lincRNA genes.
2014-11-01 | GSE56869 | GEO
Project description:Our understanding of eukaryotic gene regulation is limited by the complexity of protein-DNA interactions that comprise the chromatin landscape and inefficient methods for characterizing these interactions. We recently introduced CUT&RUN, an antibody-targeted method that profiles DNA-binding proteins, histones and chromatin modifying proteins with high sensitivity and resolution. Here we describe an automated CUT&RUN platform and apply it to profile the chromatin landscapes of human cell lines. We develop a metric to quantitatively compare chromatin features between cell types and identify their distinctive gene expression programs. Finally, we use this method to identify gene expression features of two different pediatric diffuse midline gliomas using frozen tissue from patient-derived xenografts. Our easy, cost-effective workflow makes automated CUT&RUN an attractive tool for high-throughput characterization of cell types and patient samples.
2018-12-22 | GSE120011 | GEO
Project description:Methods derived from CUT&RUN and CUT&Tag enable genome-wide mapping of the localization of proteins on chromatin from as few as one cell. These and other mapping approaches focus on one protein at a time, preventing direct measurements of co-localization of different chromatin proteins in the same cells and requiring prioritization of targets where samples are limiting. Here we describe multi-CUT&Tag, an adaptation of CUT&Tag that overcomes these hurdles by using antibody-specific barcodes to simultaneously map multiple proteins in the same cells. Highly specific multi-CUT&Tag maps of histone marks and RNA Polymerase II uncovered sites of co-localization in the same cells, active and repressed genes, and candidate cis-regulatory elements. Single-cell multi-CUT&Tag profiling facilitated identification of distinct cell types from a mixed population and inference of cell type-specific gene expression. In sum, multi-CUT&Tag increases the “per cell” information content of epigenomic maps, facilitating direct analysis of the interplay of different proteins on chromatin.
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