{"lab": {"@id": "/labs/david-gilbert-lab/", "correspondence": [{"contact_email": "Z2lsYmVydEBzZGJyaS5vcmc=", "@id": "/users/1dbb8e49-38e4-4154-a97c-af8176a58c44/", "display_title": "David Gilbert"}], "@type": ["Lab", "Item"], "title": "David Gilbert, SDBRI", "display_title": "David Gilbert, SDBRI", "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"]}}, "award": {"@type": ["Award", "Item"], "project": "4DN", "@id": "/awards/1U54DK107965-01/", "center_title": "NOFIC - Belmont", "name": "1U54DK107965-01", "uuid": "91b694c3-f4d7-4ddd-8278-16f94e15c1c5", "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.", "display_title": "COMBINED CYTOLOGICAL, GENOMIC, AND FUNCTIONAL MAPPING OF NUCLEAR GENOME ORGANIZATION", "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "status": "released", "aliases": ["gilbert-lab:auxin_treatment_for_multires_repliseq"], "chemical": "Auxin", "description": "HCT116 RAD21-mAID cells were synchronized and treated as in (Oldach and Nieduszynski 2019 PMID: 30836708). 2.5 mM Thymidine was added to the media for 24 hours followed by wash out and a 3-hour release. 100 ng/mL nocodazole was then added to the media for 8 hours and mitotic cells were collected by shake off. Percentage of mitotic cells was estimated by metaphase spread. Collected cells were released into fresh media and 500mM 3-Indoleacetic acid (Auxin, I2886 Sigma) was added 30 minutes after release to degrade RAD21. Cells were released into the cell cycle for 4, 6, 8, and 10 hours. 400uM BrdU was added to the media 30 minutes before each time point after which the cells were collected and fixed in ethanol. Equal numbers of cells from each time point were pooled together for high resolution repli-seq sorting. High resolution repli-seq was performed as in (Zhao et al 2020 PMID: 32209126).", "date_created": "2021-02-22T19:36:10.881716+00:00", "submitted_by": {"error": "no view permissions"}, "concentration": 500, "last_modified": {"modified_by": {"error": "no view permissions"}, "date_modified": "2021-03-17T14:46:56.129659+00:00"}, "public_release": "2021-03-17", "schema_version": "1", "treatment_type": "Chemical", "project_release": "2021-03-17", "concentration_units": "mM", "@id": "/treatments-agent/34a262d2-cc44-4d79-bb67-ccb1986f2a07/", "@type": ["TreatmentAgent", "Treatment", "Item"], "uuid": "34a262d2-cc44-4d79-bb67-ccb1986f2a07", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}, "display_title": "Auxin treatment (500 mM)", "external_references": [], "@context": "/terms/", "aggregated-items": {}, "validation-errors": []}