{"ID": "PMID:29599440", "aka": "http://biorxiv.org/content/early/2017/03/01/104653", "lab": {"title": "David Gilbert, SDBRI", "display_title": "David Gilbert, SDBRI", "@id": "/labs/david-gilbert-lab/", "@type": ["Lab", "Item"], "correspondence": [{"contact_email": "Z2lsYmVydEBzZGJyaS5vcmc=", "@id": "/users/1dbb8e49-38e4-4154-a97c-af8176a58c44/", "display_title": "David Gilbert"}], "status": "current", "uuid": "6423b207-8176-4f06-a127-951b98d6a53a", "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.6423b207-8176-4f06-a127-951b98d6a53a"]}}, "url": "https://www.ncbi.nlm.nih.gov/pubmed/29599440", "award": {"display_title": "COMBINED CYTOLOGICAL, GENOMIC, AND FUNCTIONAL MAPPING OF NUCLEAR GENOME ORGANIZATION", "name": "1U54DK107965-01", "status": "current", "description": "NOFIC: Decades of microscopy have revealed that the nucleus is not a homogeneous organelle, but rather consists of distinct compartments such as nucleoli, nuclear speckles, the nuclear lamina, among other structures. Increasing evidence indicates that specific genomic regions each associate with these compartments. This genome compartmentalization has been linked to various functions, but these links are still poorly understood. Interestingly, Lamina Associated Domains (LADs) share specific heterochromatin marks, defining chromatin domains with distinct genetic and epigenetic properties. Genomic regions associating with other nuclear compartments may similarly define distinct classes of chromatin domains. One major bottleneck towards a deeper understanding of nuclear organization has been the inability to convert microscopy views of nuclear compartments into genome-wide maps that show which loci are associated with which compartment, and how the chromosomal fiber traverses between compartments. In addition, there is an urgent need for more efficient methods to dissect the mechanisms by which large genomic regions are targeted to specific nuclear compartments. Finally, there is an urgent need for high-throughput approaches that query the functional relevance of genome compartmentalization. For this Center grant, we propose to meet these needs through the following Aims: 1. Develop a strategy that connects microscopy views to genome-wide maps that, together with modeling, reveal the localization and dynamics of genomic regions relative to all major nuclear compartments. 2. Develop methods for efficient manipulation of the genome in order to elucidate mechanisms that target loci to specific compartments. 3. Develop methods to measure, model, and validate the functional relevance of nuclear compartments. The combined results of these approaches will reveal causal relationships now hidden among entangled genomic, epigenetic, and nuclear organization features. Deliverables of this proposal include a wide range of structural and functional maps of nuclear organization, reagents for visualizing endogenous chromosome loci, a powerful pipeline for synthesis of ~100kb DNA fragments, and cell lines facilitating repeated, high-fidelity insertio of these large fragments back into selected sites in the genome. These resources will provide a powerful complement to other 4D Nucleome Consortium efforts. A key strength of this Center proposal is the experience and complementary research capabilities of its five Investigators. Together they will pool their expertise for a concerted investigation into the biological functions of nuclear compartmentalization.", "@id": "/awards/1U54DK107965-01/", "center_title": "NOFIC - Belmont", "uuid": "91b694c3-f4d7-4ddd-8278-16f94e15c1c5", "@type": ["Award", "Item"], "project": "4DN", "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "title": "Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq.", "status": "current", "aliases": ["4dn-dcic-lab:PMID_29599440_repliseq"], "authors": ["Marchal C", "Sasaki T", "Vera D", "Wilson K", "Sima J", "Rivera-Mulia JC", "Trevilla-Garcia C", "Nogues C", "Nafie E", "Gilbert DM"], "journal": "Nature protocols", "abstract": "This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate  their DNA content during S phase, following a defined program called replication  timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and subnuclear position. Moreover, RT is regulated during development and is altered in diseases. Here, we describe E/L Repli-seq, an extension of our Repli-chip protocol. E/L Repli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse-labeled with BrdU, and early and late S-phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a  single bedGraph file containing the ratio of nascent DNA from early versus late S-phase fractions. The results are comparable to those of Repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic  range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single-nucleotide polymorphisms (SNPs) to analyze RT allelic asynchrony, and for direct comparison to Repli-chip data. This protocol can be performed in up to 3 d before sequencing, and requires basic cellular and  molecular biology skills, as well as a basic understanding of Unix and R.", "date_created": "2018-05-15T19:51:34.394152+00:00", "published_by": "4DN", "submitted_by": {"error": "no view permissions"}, "last_modified": {"modified_by": {"error": "no view permissions"}, "date_modified": "2018-08-08T18:38:37.897475+00:00"}, "date_published": "2018-03-29", "public_release": "2018-05-15", "schema_version": "2", "@id": "/publications/e3427217-1675-433a-b844-f40d8ec7d2b5/", "@type": ["Publication", "Item"], "uuid": "e3427217-1675-433a-b844-f40d8ec7d2b5", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}, "display_title": "Marchal C et al. (2018) PMID:29599440", "external_references": [], "short_attribution": "Marchal C et al. (2018)", "@context": "/terms/", "aggregated-items": {}, "validation-errors": []}