{"lab": {"display_title": "Chuck Murry, UW", "status": "current", "correspondence": [{"contact_email": "bXVycnlAdS53YXNoaW5ndG9uLmVkdQ==", "@id": "/users/ab0b589c-01e1-4484-b58a-2a3bd9dbfe1e/", "display_title": "Chuck Murry"}], "@id": "/labs/chuck-murry-lab/", "title": "Chuck Murry, UW", "uuid": "a7e19574-4def-456e-9de9-29dd7bdc2e68", "@type": ["Lab", "Item"], "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.a7e19574-4def-456e-9de9-29dd7bdc2e68"]}}, "award": {"project": "4DN", "center_title": "NOFIC - Shendure", "description": "NOFIC: A current grand challenge in genomics involves accurately assaying, at all relevant scales, the 3D conformation of DNA in vivo and then linking conformational changes to dynamic processes such as the cell cycle, differentiation and disease. Here we propose to create the University of Washington Center for Nuclear Organization and Function, bringing together an interdisciplinary team of investigators whose diverse areas of expertise - technology development, computational modeling, and mouse and human biology - make them ideally suited to this challenge. Our overall hypothesis is that characterizing and understanding changes in genome architecture over time (the 4D nucleome) will lead to fundamental insights into human biology and disease. We will address this hypothesis by developing a combination of experimental and computational methods development, coupled with their systematic biological validation and application to development- and disease-relevant systems. On the experimental side, we will further optimize our recently developed DNase Hi- C assay, including combinatorial methods for single cells, ultimately aiming to concurrently assay nuclear architecture and gene expression within each of many single cells. On the computational side, we will extend our existing 3D modeling algorithms to account for diploidy, cell-to-cell variabilit, the hierarchical nature of genome architecture, and to explicitly model architectural changes over cell cycle and cell differentiation time scales. We will then employ several complementary computational methods to link our 4D nucleome models to existing, 1D genomics data sets. The outputs of these new experimental and computational technologies will be subjected to orthogonal validation in several well-understood model systems: human cell lines, in vivo tissues from interspecific F1 hybrid mice, mouse embryonic stem cells (ESCs) and skeletal myoblasts. We will also test specific predictions of the models in response to targeted (genome editing) or large-scale (chromosome silencing) perturbations. After initial validation and in parallel with further methods development, we will apply our new tools to the analysis of three biological systems: we will characterize the dynamics of nuclear architecture during the directed differentiation of na\u00efve human ESCs into cardiomyocytes and endothelial cells; we will test the hypothesis that cardiomyopathy-inducing mutations in the nuclear scaffolding protein, lamin A, are associated with derangements in cardiomyocyte nuclear architecture; and we will determine the changes in human cardiomyocyte nuclear architecture induced by trisomy 21. The proposed center will produce new experimental protocols for ascertaining 4D nucleome architecture, two new software toolkits for modeling the 4D nucleome and linking features of the nucleome to other types of genomic data, a variety of publicly available, large-scale 4D nucleome data sets in mouse and human systems, and fundamental insights into human biology and disease. 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A whole-genome chromatin interaction library is constructed by shearing the DNA, selecting the biotin-containing fragments with streptavidin magnetic beads and PCR amplification. After high throughput sequencing, analysis of the resulting paired-end sequences produces a matrix that quantitatively characterizes the whole-genome chromatin contacts.\n\nSee [Ramani et al 2016](https://www.ncbi.nlm.nih.gov/pubmed/27685100) for more details on in situ DNase Hi-C.\n\n<dl> <img style=\"width:600px;\" src=\"https://s3.amazonaws.com/4dn-dcic-public/static-pages/InfoBoxes/DNAse_HIC.png\" />\n  <br/><br/>\n  <em>Image source: Ramani et. al. 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DNase Hi-C was designed to overcome the limitations associated with restriction enzyme-based Hi-C approaches. Advances in molecular techniques led to the development of an improved version of this technique known as in situ DNase Hi-C that is dramatically simplified and easy to use.\n\nThe in situ DNase Hi-C protocol involves cross-linking the cells with formaldehyde to form links between physically adjacent DNA regions. The cells are then permeabilized with their nuclei intact. Chromatin is then randomly fragmented by DNase I. The resulting chromatin fragments are end-repaired and dA-tailed, then marked with a biotinylated internal adaptor, and proximity ligation is carried out in the intact nucleus to favor ligation events between the cross-linked DNA fragments. The resulting DNA sample contains ligation products consisting of chimeric DNA fragments that were originally in close spatial proximity in the nucleus, marked with biotin at the junction. A whole-genome chromatin interaction library is constructed by shearing the DNA, selecting the biotin-containing fragments with streptavidin magnetic beads and PCR amplification. After high throughput sequencing, analysis of the resulting paired-end sequences produces a matrix that quantitatively characterizes the whole-genome chromatin contacts.\n\nSee [Ramani et al 2016](https://www.ncbi.nlm.nih.gov/pubmed/27685100) for more details on in situ DNase Hi-C.\n\n<dl> <img style=\"width:600px;\" src=\"https://s3.amazonaws.com/4dn-dcic-public/static-pages/InfoBoxes/DNAse_HIC.png\" />\n  <br/><br/>\n  <em>Image source: Ramani et. al. Nature Protocols 2016, Figure 1 </em>\n</dl>   ", "filetype": "md", "content_as_html": "<div class=\"markdown-container\"><p><strong>DNase Hi-C</strong></p>\n<p>DNase Hi-C is a method to detect and quantify pairwise interactions between chromosome regions across the entire genome. DNase Hi-C was designed to overcome the limitations associated with restriction enzyme-based Hi-C approaches. Advances in molecular techniques led to the development of an improved version of this technique known as in situ DNase Hi-C that is dramatically simplified and easy to use.</p>\n<p>The in situ DNase Hi-C protocol involves cross-linking the cells with formaldehyde to form links between physically adjacent DNA regions. The cells are then permeabilized with their nuclei intact. Chromatin is then randomly fragmented by DNase I. The resulting chromatin fragments are end-repaired and dA-tailed, then marked with a biotinylated internal adaptor, and proximity ligation is carried out in the intact nucleus to favor ligation events between the cross-linked DNA fragments. The resulting DNA sample contains ligation products consisting of chimeric DNA fragments that were originally in close spatial proximity in the nucleus, marked with biotin at the junction. A whole-genome chromatin interaction library is constructed by shearing the DNA, selecting the biotin-containing fragments with streptavidin magnetic beads and PCR amplification. After high throughput sequencing, analysis of the resulting paired-end sequences produces a matrix that quantitatively characterizes the whole-genome chromatin contacts.</p>\n<p>See <a href=\"https://www.ncbi.nlm.nih.gov/pubmed/27685100\" rel=\"noopener noreferrer\" target=\"_blank\">Ramani et al 2016</a> for more details on in situ DNase Hi-C.</p>\n<dl> <img src=\"https://s3.amazonaws.com/4dn-dcic-public/static-pages/InfoBoxes/DNAse_HIC.png\" style=\"width:600px;\"/>\n<br/><br/>\n<em>Image source: Ramani et. al. 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We identify a network of such gene loci that increase their association inter-chromosomally, and are targets of the muscle-specific splicing factor RBM20. Genome editing studies show that TTN pre-mRNA, the main RBM20-regulated transcript in the heart, nucleates RBM20 foci that drive spatial proximity between the TTN locus and other inter-chromosomal RBM20 targets such as CACNA1C and CAMK2D. 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While chromatin accessibility and gene expression show complex on/off dynamics, large-scale genome architecture changes are mostly unidirectional. Many large cardiac genes transition from a repressive to an active compartment during differentiation, coincident with upregulation. We identify a network of such gene loci that increase their association inter-chromosomally, and are targets of the muscle-specific splicing factor RBM20. Genome editing studies show that TTN pre-mRNA, the main RBM20-regulated transcript in the heart, nucleates RBM20 foci that drive spatial proximity between the TTN locus and other inter-chromosomal RBM20 targets such as CACNA1C and CAMK2D. 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