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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. 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(2019)", "date_published": "2019-02-07", "uuid": "015e1588-0290-44bf-a9f0-d4cad281c221", "abstract": "The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization  genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch,  weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these \"early replication control elements\" (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.", "status": "current", "@id": "/publications/015e1588-0290-44bf-a9f0-d4cad281c221/", "title": "Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication.", "display_title": "Sima J et al. (2019) PMID:30595451", "ID": "PMID:30595451", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "publications_of_exp": [{"ID": "PMID:30595451", "@type": ["Publication", "Item"], "status": "current", "@id": "/publications/015e1588-0290-44bf-a9f0-d4cad281c221/", "abstract": "The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization  genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch,  weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these \"early replication control elements\" (ERCEs) was validated by deletions and inversions at additional domains. 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