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A Streamlined Tethered Chromosome Conformation Capture Protocol

ABSTRACT: Background: Identification of locus-locus contacts at the chromatin level provides a valuable foundation for understanding of nuclear architecture and function and a valuable tool for inferring long-range linkage relationships. As one approach to this, chromatin conformation capture-based techniques allow creation of genome spatial organization maps. While such approaches have been available for some time, methodological advances will be of considerable use in minimizing both time and input material required for successful application. Results: Here we report a modified tethered conformation capture protocol that utilizes a series of rapid and efficient molecular manipulations. We applied the method to Caenorhabditis elegans, obtaining chromatin interaction maps that provide a sequence-anchored delineation of salient aspects of Caenorhabditis elegans chromosome structure, demonstrating a high level of consistency in overall chromosome organization between biological samples collected under different conditions. In addition to the application of the method to defining nuclear architecture, we found the resulting chromatin interaction maps to be of sufficient resolution and sensitivity to enable detection of large-scale structural variants such as inversions or translocations. Conclusion: Our streamlined protocol provides an accelerated, robust, and broadly applicable means of generating chromatin spatial organization maps and detecting genome rearrangements without a need for cellular or chromatin fractionation. Application of modified version of TCC protocl using different C. elegans strains (N2 and glp-1) in L1, and adult life stages.

ORGANISM(S): Caenorhabditis elegans

SUBMITTER: Idan Gabdank

PROVIDER: E-GEOD-76930 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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A Streamlined Tethered Chromosome Conformation Capture Protocol

Project description:Background: Identification of locus-locus contacts at the chromatin level provides a valuable foundation for understanding of nuclear architecture and function and a valuable tool for inferring long-range linkage relationships. As one approach to this, chromatin conformation capture-based techniques allow creation of genome spatial organization maps. While such approaches have been available for some time, methodological advances will be of considerable use in minimizing both time and input material required for successful application. Results: Here we report a modified tethered conformation capture protocol that utilizes a series of rapid and efficient molecular manipulations. We applied the method to Caenorhabditis elegans, obtaining chromatin interaction maps that provide a sequence-anchored delineation of salient aspects of Caenorhabditis elegans chromosome structure, demonstrating a high level of consistency in overall chromosome organization between biological samples collected under different conditions. In addition to the application of the method to defining nuclear architecture, we found the resulting chromatin interaction maps to be of sufficient resolution and sensitivity to enable detection of large-scale structural variants such as inversions or translocations. Conclusion: Our streamlined protocol provides an accelerated, robust, and broadly applicable means of generating chromatin spatial organization maps and detecting genome rearrangements without a need for cellular or chromatin fractionation.

2016-01-20 | GSE76930 | GEO

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Project description:Dynamic 3D chromatin conformation is a critical mechanism for gene regulation during development and disease. Despite this, profiling of 3D genome structure from complex tissues with cell-type specific resolution remains challenging. Recent efforts have demonstrated that cell-type specific epigenomic features can be resolved in complex tissues using single-cell assays. However, it remains unclear whether single-cell Chromatin Conformation Capture (3C) or Hi-C profiles can effectively identify cell types and reconstruct cell-type specific chromatin conformation maps. To address these challenges, we have developed single-nucleus methyl-3C sequencing (sn-m3C-seq) to capture chromatin organization and DNA methylation information and robustly separate heterogeneous cell types. Applying this method to >4,200 single human brain prefrontal cortex cells, we reconstruct cell-type specific chromatin conformation maps from 14 cortical cell types. These datasets reveal the genome-wide association between cell-type specific chromatin conformation and differential DNA methylation, suggesting pervasive interactions between epigenetic processes regulating gene expression.

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HiCrayon reveals distinct layers of multi-state 3D chromatin organization

Project description:The co-visualization of chromatin conformation with 1D 'omics data is key to the multi-omics driven data analysis of 3D genome organization. Chromatin contact maps are often shown as 2D heatmaps and visually compared to 1D genomic data by simple juxtaposition. While common, this strategy is imprecise, placing the onus on the reader to align features with each other. To remedy this, we developed HiCrayon, an interactive tool that facilitates the integration of 3D chromatin organization maps and 1D datasets. This visualization method integrates data from genomic assays directly into the chromatin contact map by coloring interactions according to 1D signal. HiCrayon is implemented using R shiny and python to create a graphical user interface (GUI) application, available in both web or containerized format to promote accessibility. HiCrayon is implemented in R, and includes a graphical user interface (GUI), as well as a slimmed-down web-based version that lets users quickly produce publication-ready images. We demonstrate the utility of HiCrayon in visualizing the effectiveness of compartment calling and the relationship between ChIP-seq and various features of chromatin organization. We also demonstrate the improved visualization of other 3D genomic phenomena, such as differences between loops associated with CTCF/cohesin vs. those associated with H3K27ac. We then demonstrate HiCrayon's visualization of organizational changes that occur during differentiation and use HiCrayon to detect compartment patterns that cannot be assigned to either A or B compartments, revealing a distinct 3rd chromatin compartment. Overall, we demonstrate the utility of co-visualizing 2D chromatin conformation with 1D genomic signals within the same matrix to reveal fundamental aspects of genome organization.

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Transcription profiling of human lymphoblastoid cell line GM06690 in conjunction with Hi-C to determine the three-dimensional architecture of the genome

Project description:We describe Hi-C, a method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. We constructed spatial proximity maps of the human genome with Hi-C at a resolution of 1Mb. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes. We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments. At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes. These expression data are used in the paper to show that there are marked differences in mRNA expression between loci in the open and closed chromatin compartments. Experiment Overall Design: Affy Expression array for GM06690 cells

2009-10-10 | E-GEOD-18350 | biostudies-arrayexpress

Comprehensive mapping of long-range interactions reveals folding principles of the human genome.

Project description:We describe Hi-C, a method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. We constructed spatial proximity maps of the human genome with Hi-C at a resolution of 1Mb. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes. We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments. At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes. The processed heatmap data, showing the number of read pairs corresponding to particular locus pairs in the genome, are linked to the Series record below. The processed eigenvector data used to generate the heatmaps are linked to the Series record below. Additional DNAseI and ChIP-Seq data tracks were used in the paper, and are available at the UCSC browser (http://genome.ucsc.edu/) DNAseI: wgEncodeUwDnaseSeqRawSignalRep2K562 wgEncodeUwDnaseSeqRawSignalRep2Gm06990 ChIP-Seq: wgEncodeBroadChipSeqSignalGm12878H3k36me3 wgEncodeBroadChipSeqSignalGm12878H3k27me3 Interaction maps in 2 different cell types.

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Single-cell multi-omic profiling of chromatin conformation and DNA methylome

Project description:Recent advances in the development of single cell epigenomic assays have facilitated the analysis of the gene regulatory landscapes in complex biological systems. Single-cell variations of methods such as DNA methylation-sequencing and ATAC-seq hold tremendous promise for delineating distinct cell types and identifying their critical cis-regulatory sequences. Emerging evidence in recent years has shown that in addition to cis-regulatory sequences, dynamic regulation of 3D chromatin conformation is a critical mechanism for the modulation of gene expressions during development and disease. While assays for the investigation of single-cell 3D chromatin structure have been developed, cell-type specific chromatin conformation in primary human tissues has not been extensively explored. It remains unclear whether single-cell Chromatin Conformation Capture (3C) or Hi-C profiles are suitable for cell type identification and allow the reconstruction of cell-type specific chromatin conformation maps. To address these challenges, we have developed a multi-omic method single-nucleus methyl-3C sequencing (sn-m3C) to profile chromatin conformation and DNA methylation from the same cell. We have shown that bulk m3C and sn-m3C accurately capture chromatin organization information and robustly separate mouse cell types. We have developed a fluorescent-activated nuclei sorting strategy based on DNA content that eliminates nuclei multiplets caused by crosslinking. The sn-m3C-seq method allows high-resolution cell-type classification using two orthogonal types of epigenomic information and the reconstruction of cell-type specific chromatin conformation maps.

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Genome-wide CPD repair maps of Caenorhabditis elegans

Project description:Genome-wide CPD repair maps of Caenorhabditis elegans

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Whole-genome analysis of transcription-coupled repair reveals novel transcription events in Caenorhabditis elegans

Project description:We have adapted the eXcision Repair-sequencing (XR-seq) method to generate single-nucleotide resolution dynamic repair maps of UV-induced cyclobutane pyrimidine dimers (CPD) photoproducts in the Caenorhabditis elegans (C. elegans) genome. We focus on the C. elegans ortholog of the human XPC-deficient strain (xpc-1) and its exclusive use of transcription-coupled repair. We provide evidence demonstrating the utility of xpc-1 XR-seq as a remarkable tool for detecting nascent transcription and identifying new transcripts. The integration of epigenetic markers, chromatin states, and non-coding RNA annotations supports the robust detection of intergenic nascent transcription by XR-seq. Overall, our results provide a comprehensive view of the transcription-coupled repair landscape in C. elegans, highlighting their potential contributions to our understanding of DNA repair mechanisms and non-coding RNA biology.

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