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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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These lamina-associated domains (LADs) are involved in gene regulation and may provide a backbone for the folding of interphase chromosomes. Little is known about the dynamics of LADs during interphase, in particular at the onset of G1 phase and during DNA replication. We developed an antibody-based variant of the DamID technology (named pA-DamID) that allows us to map and visualize genome-NL interactions with high temporal resolution. Application of pA-DamID combined with synchronization and cell sorting experiments reveals that LAD-NL contacts are generally rapidly established early in G1 phase. However, LADs on the distal ~25 Mb of most chromosomes tend to contact the NL first and then gradually detach, while centromere-proximal LADs accumulate gradually at the NL. Furthermore, our data indicate that S-phase chromatin shows transiently increased lamin interactions. These findings highlight a dynamic choreography of LAD-NL contacts during interphase progression and illustrate the usefulness of pA-DamID to study the dynamics of genome compartmentalization.", "ID": "PMID:32893442", "@id": "/publications/f1218a92-1f37-4519-85d6-ccedd5f7ad39/", "title": "Cell cycle dynamics of lamina-associated DNA.", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, {"journal": "Nucleic acids research", "date_published": "2022-09-23", "@type": ["Publication", "Item"], "status": "current", "authors": ["Friskes A", "Koob L", "Krenning L", "Severson TM", "Koeleman ES", "Vergara X", "Schubert M", "van den Berg J", "Evers B", "Manjon AG", "Joosten S", "Kim Y", "Zwart W", "Medema RH"], "display_title": "Friskes A et al. (2022) PMID:36107780", "uuid": "3a03fb4c-e4f5-45a4-83b4-0bfdc5fcd5e0", "abstract": "Cells respond to double-strand breaks (DSBs) by activating DNA damage response  pathways, including cell cycle arrest. 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We show that the  toxicity of a DSB is highly similar across the different ChromHMM states. Rather,  we find that the major determinants of toxicity of a sgRNA are cutting efficiency  and off-target effects. 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