CUT&RUN reveals unique positioning of pre-initiated RNA polymerase II in the steady state of transcription
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE155666 | GEO | 2020/10/20
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.
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Project description:Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, Cut-and-Run is performed in situ, allowing for both high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, Cut-and-Run yielded precise transcription factor profiles while avoiding cross-linking and solubilization issues. Cut-and-Run is simple to perform and at low temperatures is inherently robust, with extremely low backgrounds that make it especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, Cut-and-Run mapped high-resolution 3D directional interactions. We conclude that Cut- and-Run is a suitable complement or replacement for ChIP-seq that can also provide 3D mapping information.
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Project description:Over the past two decades, the dominant technology for chromatin profiling has been chromatin immunoprecipitation (ChIP), in which cellular components are solubilized to fragment chromatin, and an antibody is added to precipitate a DNA/protein complex of interest. We previously described a novel alternative to ChIP, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which the antibody is added to permeabilized cells followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein to the antibody (PMID 29651053). Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. Here we introduce four extensions to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that allows simplified purification using a commercial kit; 2) a modified digestion protocol that prevents release of the enzyme and so minimizes artifactual cleavage of accessible DNA; 3) a calibration strategy based on carryover of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy that is customized for the low-background profiles obtained using CUT&RUN. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.
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Project description:Mapping DNase I hypersensitive sites (DHSs) within nuclear chromatin is a traditional and powerful method of identifying genetic regulatory elements. DHSs have been mapped by capturing the ends of long DNase I-cut fragments (>100,000 bp), or 100-1200 bp DNase I-double cleavage fragments (also called double-hit fragments). But next generation sequencing requires a DNA library containing DNA fragments of 100-500bp. Therefore, we have modified the double-hit method and use short DNA fragments to generate DNA libraries for next generation sequencing. We call this method Short DHS Assay (Short DNAse I Hypersensitive Site assay). The short segments are 100-300bp and can be directly cloned and used for high-throughput sequencing. We identified 83,897 DHSs in 2,343,479 tags across the human genome. Our results indicate that the DHSs identified by the Short DHS assay are consistent with those identified by longer fragments in previous studies.
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Project description:Samples from 20 children with B-cell precursor ALL, comprising ten with high hyperdiploidy and ten with the ETV6/RUNX1 fusion, were included in the study. Four µg of methylated and input fraction DNA were analyzed with a classic MeDIP assay and subsequent microarray hybridization using the NimbleGen CpG Island-Plus-Promoter Array (Roche NimbleGen, Madison, WI).<br><br>This array platform covers all 28,226 UCSC-annotated CpG islands and promoter regions for all RefSeq genes, including 385,000 probes of 50-75mer length per array, with an upstream and downstream promoter tiling of 800 bp and 200 bp, respectively. Signal intensity data were extracted from the scanned images. Each feature on the array has a corresponding log2 ratio, which is the ratio of the input signals for the methylated DNA and the input fraction DNA. The log2 ratio was scaled in order to center the ratio data around zero by subtracting the bi-weight mean for the log2 ratio values for all features on the array from each log2 ratio value. A modified ACME algorithm, where a fixed-length window (750 bp) is placed around each consecutive probe, and the one-sided Kolmogorov-Smirnov test were then applied on the scaled log2 ratio data to determine whether the probes were drawn from a significantly more positive distribution (peak) of intensity log2 ratios than the other probes in the array. The resulting score for each probe is the -log10 p-value from the windowed Kolmogorov-Smirnov test around that probe. Peak data were generated by searching for probes above a p-value minimum cut-off (-log10) of 2.<br><br>Peaks within 500 bp of each other were merged. Each annotated gene was subsequently searched for peaks appearing in a specified promoter region around the transcription start site. The region searched was design-specific, spanning from 5 kb upstream to 1 kb downstream of the transcription start site. Genes that had at least two probes with peaks above the p-value minimum cut-off were considered to have significant CpG island methylation scores for hypermethylation.
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Project description:Eukaryotic chromosomes are composed of chromatin, in which regularly spaced nucleosomes containing ~147 bp of DNA are separated by linker DNA. Most eukaryotic cells have a characteristic average nucleosome spacing of ~190 bp, corresponding to a ~45 bp linker. However, cortical neurons have a shorter average spacing of ~165 bp. The significance of this atypical global chromatin organization is unclear. We have compared the chromatin structures of purified mouse dorsal root ganglia (DRG) neurons, cortical oligodendrocyte precursor cells (OPCs) and cortical astrocytes. DRG neurons have short average spacing (~165 bp), whereas OPCs (~182 bp) and astrocytes (~183 bp) have longer spacing. We measured nucleosome positions by MNase-seq and gene expression by RNA-seq. Most genes in all three cell types have a promoter chromatin organization typical of active genes: a nucleosome-depleted region at the promoter flanked by regularly spaced nucleosomes phased relative to the transcription start site. In DRG neurons, the spacing of phased nucleosomes downstream of promoters (~178 bp) is longer than expected from the genomic average, whereas phased nucleosome spacing in OPCs and astrocytes is similar to the global average (~183 bp). Thus, the atypical nucleosome spacing of neuronal chromatin does not extend to promoter-proximal regions.
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Project description:Eukaryotic chromosomes are composed of chromatin, in which regularly spaced nucleosomes containing ~147 bp of DNA are separated by linker DNA. Most eukaryotic cells have a characteristic average nucleosome spacing of ~190 bp, corresponding to a ~45 bp linker. However, cortical neurons have a shorter average spacing of ~165 bp. The significance of this atypical global chromatin organization is unclear. We have compared the chromatin structures of purified mouse dorsal root ganglia (DRG) neurons, cortical oligodendrocyte precursor cells (OPCs) and cortical astrocytes. DRG neurons have short average spacing (~165 bp), whereas OPCs (~182 bp) and astrocytes (~183 bp) have longer spacing. We measured nucleosome positions by MNase-seq and gene expression by RNA-seq. Most genes in all three cell types have a promoter chromatin organization typical of active genes: a nucleosome-depleted region at the promoter flanked by regularly spaced nucleosomes phased relative to the transcription start site. In DRG neurons, the spacing of phased nucleosomes downstream of promoters (~178 bp) is longer than expected from the genomic average, whereas phased nucleosome spacing in OPCs and astrocytes is similar to the global average (~183 bp). Thus, the atypical nucleosome spacing of neuronal chromatin does not extend to promoter-proximal regions.
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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.
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Project description:Proof-of-concept for Mnase-SSP: a variant of Mnase-seq. Mnase-SSP dramatically increases the representation of short fragments of nucleolytically-digested DNA, enabling simultaneous analysis of transcription factor binding and nucleosome occupancy using the same assay. We used MNase-SSP to demarcate chromatin architecture at murine promoters and at transcription factor binding sites in murine embryonic stem cells. Are results reveal heterogeneity in the binding mode of C2H2 zinc fingers like Ctcf and Rest, demonstrating that Mnase-SSP, and SSP in general, as a flexible platform for profiling nucleolytically digested DNA for MNase-seq, MNase-ChIP, or CUT&RUN with reduced bias.
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