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Our ultimate goal is to deliver complex chromatin interaction network maps in the context of 3D genome structures from which the dynamics of individual genomic elements can be monitored and referenced. Here, we propose to develop a Nucleome Positioning System (NPS)-comprised of 1) a robust genome- wide mapping technology platform, 2) advanced computational modeling algorithms and 3) state-of-the-art nuclear imaging methods-that will allow users community-wide to uncover the regulatory functions of 3D genome organization in human cells. NPS will be based upon the established ChIA-PET method (1,2), enhanced by process optimizations-i.e., microfluidic-based miniaturization and Tn5-transposase-based library preparation-to facilitate the study of chromatin interactions mediated by protein factors across a broader range of human cell types (Aim 1, see also Mapping Technology Development Component). We will also optimize RICh-PET for the comprehensive mapping of chromatin interactions mediated by non-coding RNAs (Aim 1). The high-quality mapping data generated through these optimization efforts will be analyzed by a new computational platform (Three-Dimensional Nucleome Modeling Engine, or 3D-NOME) that makes use of hierarchical multi-scaling to model 3D genome structures (Aim 2, Data Analysis and Modeling component). We will also complement the 3D modeling with transcriptome, epigenome and SNP data associated with genetic diseases (GWAS) to provide functional annotation to structural units (Aim 2). We will continue by developing strategies to validate the nucleome geometry predicted by 3D-NOME both structurally, using new nuclear imaging technologies, and functionally, using cutting-edge genome- and epigenome-editing approaches, in both human cell lines and mouse models (Aim 3, Biological Validation Component). Finally, we will implement NPS to generate pilot 3D genome maps from a wide range of human cell lines and primary immune cells sorted from whole blood, to elucidate the spatiotemporal dynamics of human genome organization over major developmental and hematopoietic cell lineages, as well as among differentiating lymphocytes involved in the immune response (Aim 4, Data Generation Component). Together, these efforts will yield a powerful set of sophisticated, high-quality tools and mapping data for the larger research community, and will help establish the standards for future 3D/4D nucleome studies. 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However, its precise role in  regulating gene transcription remains largely unknown. We investigated the  relationship between cohesin and RNA Polymerase II (RNAPII) using single-molecule  mapping and live-cell imaging methods in human cells. Cohesin-mediated  transcriptional loops were highly correlated with those of RNAPII and followed  the direction of gene transcription. Depleting RAD21, a subunit of cohesin,  resulted in the loss of long-range (>100 kb) loops between distal  (super-)enhancers and promoters of cell-type-specific genes. By contrast, the  short-range (<50 kb) loops were insensitive to RAD21 depletion and connected  genes that are mostly housekeeping. This result explains why only a small  fraction of genes are affected by the loss of long-range chromatin interactions  due to cohesin depletion. Remarkably, RAD21 depletion appeared to up-regulate  genes located in early initiation zones (EIZ) of DNA replication, and the EIZ  signals were amplified drastically without RAD21. Our results revealed new  mechanistic insights of cohesin's multifaceted roles in establishing  transcriptional loops, preserving long-range chromatin interactions for  cell-specific genes, and maintaining timely order of DNA replication.", "@type": ["Publication", "Item"], "title": "Multifaceted roles of cohesin in regulating transcriptional loops.", "date_published": "2024-03-27", "uuid": "a84d089a-b2a0-4da5-a454-07f73987df08", "journal": "bioRxiv : the preprint server for biology", "short_attribution": "Kim M et al. (2024)", "display_title": "Kim M et al. (2024) doi:10.1101/2024.03.25.586715", "authors": ["Kim M", "Wang P", "Clow PA", "Chien IE", "Wang X", "Peng J", "Chai H", "Liu X", "Lee B", "Ngan CY", "Yue F", "Milenkovic O", "Chuang JH", "Wei CL", "Casellas R", "Cheng AW", "Ruan Y"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "publications_of_exp": [{"abstract": "Cohesin is required for chromatin loop formation. However, its precise role in  regulating gene transcription remains largely unknown. We investigated the  relationship between cohesin and RNA Polymerase II (RNAPII) using single-molecule  mapping and live-cell imaging methods in human cells. Cohesin-mediated  transcriptional loops were highly correlated with those of RNAPII and followed  the direction of gene transcription. Depleting RAD21, a subunit of cohesin,  resulted in the loss of long-range (>100 kb) loops between distal  (super-)enhancers and promoters of cell-type-specific genes. By contrast, the  short-range (<50 kb) loops were insensitive to RAD21 depletion and connected  genes that are mostly housekeeping. This result explains why only a small  fraction of genes are affected by the loss of long-range chromatin interactions  due to cohesin depletion. Remarkably, RAD21 depletion appeared to up-regulate  genes located in early initiation zones (EIZ) of DNA replication, and the EIZ  signals were amplified drastically without RAD21. Our results revealed new  mechanistic insights of cohesin's multifaceted roles in establishing  transcriptional loops, preserving long-range chromatin interactions for  cell-specific genes, and maintaining timely order of DNA replication.", "display_title": "Kim M et al. (2024) doi:10.1101/2024.03.25.586715", "uuid": "a84d089a-b2a0-4da5-a454-07f73987df08", "ID": "doi:10.1101/2024.03.25.586715", "date_published": "2024-03-27", "journal": "bioRxiv : the preprint server for biology", "title": "Multifaceted roles of cohesin in regulating transcriptional loops.", "@type": ["Publication", "Item"], "@id": "/publications/a84d089a-b2a0-4da5-a454-07f73987df08/", "short_attribution": "Kim M et al. (2024)", "authors": ["Kim M", "Wang P", "Clow PA", "Chien IE", "Wang X", "Peng J", "Chai H", "Liu X", "Lee B", "Ngan CY", "Yue F", "Milenkovic O", "Chuang JH", "Wei CL", "Casellas R", "Cheng AW", "Ruan Y"], "status": "current", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}], "experiment_categorizer": {"field": "Enzyme", "value": "AluI", "combined": "Enzyme: AluI"}, "experiment_summary": "in situ Hi-C on HCT116 (Tier 2) with AluI", "@context": "/terms/", "aggregated-items": {"badges": [{"parent": "/biosamples/4DNBSIDXV1FM/", "embedded_path": "biosample.badges", "item": {"messages": ["Biosample missing Cell Culture Details"], "badge": {"commendation": null, "warning": "Biosample Metadata Incomplete", "uuid": "2b2cc7ff-b7a8-4138-9a6c-22884fc71690", "@id": "/badges/biosample-metadata-incomplete/", "badge_icon": "/static/img/badges/biosample-icon.svg", "description": "Biosample is missing metadata information required as part of the standards implemented by the 4DN Samples working group."}}}]}, "validation-errors": []}