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This proposal is significant because we propose a novel model forthe role of nuclear organization in regulating gene expression: 1) Nuclear speckles and still unknown nuclearcompartments/bodies help organize other phase-separated condensates to modulate gene expression; 2)Nuclear speckles together with surrounding nuclear compartments/bodies and associated phase-separatedcondensates together represent active nuclear niches which may have different functional properties; 3) Smalldistances matter: gene movements of only a few hundred nm between repressive and these different activenuclear niches may differentially regulate gene expression; 4) Action-at-a distance: component flux into andout of these nuclear compartments will have global effects on gene expression; 5) These same nuclearcompartments/bodies may similarly modulate RNA processing and organize nuclear export. Here we propose to: 1) Identify multiple components of known and still unknown nuclear \u201cactiveniches\u201d; 2) Map genome-wide the positions and predicted movements of genes relative to these active nichesduring physiological transitions; 3) Visualize nuclear body/compartment dynamics and fluxes of proteinsbetween nuclear bodies in steady-state and through physiological transitions; 4) Visualize movements ofreporter transgenes, endogenous genes, and rewired chromosome loci relative to these nuclearbodies/compartments and temporally correlate changes in gene expression with their dynamic movements andcompartment associations; 5) Visualize movements of pre-mRNAs and nuclear mRNAs during RNAprocessing and export; 6) Measure fluxes of nuclear body components to and from adjacent transcribingchromatin. Additionally, we propose developing relatively low-cost, novel microscope platforms and softwarespecifically designed to facilitate these live-cell imaging goals in our laboratories as well as others. Our Aims will be to: 1. Map proteins, genes, RNAs relative to active nuclear compartment(s) usingiterative rounds of TSA-MS-Ratio, validation by light microscopy, and TSA-Seq; 2. Measure dynamics ofbodies, components of nuclear bodies using live-cell imaging; 3. Measure temporal correlation betweenchanges in gene expression and gene movement relative to nuclear bodies and visualize the export path ofexpressed transcripts; 4. Design and deliver two novel microscopes designed to facilitate Aims 1-3 at amodest cost. Successful completion of these Aims should significantly change our current understanding of therole of nuclear organization in regulating gene expression with impact across a wide range of research fields.\"", "@type": ["Award", "Item"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}, "pi": {"error": "no view permissions"}}, "md5sum": "e4457eda624b5d6fc2a8f7b73d7d7c2e", "status": "released", "aliases": ["belmont-lab:U2OS_THRAP3_2xDTT_Input_bio1_GTCCGCAC_L001_R1_001.fastq"], "filename": "U2OS_THRAP3_2xDTT_Input_bio1_GTCCGCAC_L001_R1_001.fastq.gz", "accession": "4DNFICFVN1W5", "file_size": 1785270931, "file_type": "reads", "instrument": "NovaSeq6000", "file_format": {"@type": ["FileFormat", "Item"], "@id": "/file-formats/fastq/", "status": "released", "uuid": "c13d06cf-218e-4f61-aaf0-91f226248b2c", "file_format": "fastq", "display_title": "fastq", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "read_length": 100, "date_created": "2024-12-19T15:40:03.988840+00:00", "submitted_by": {"error": "no view permissions"}, "last_modified": {"modified_by": {"error": "no view permissions"}, "date_modified": "2025-09-05T17:06:03.271488+00:00"}, "content_md5sum": "ad9dcf18739f581610da0218a19bca18", "public_release": "2025-08-27", "quality_metric": {"Sequence length": "100", "status": "released", "@type": ["QualityMetricFastqc", "QualityMetric", "Item"], "url": "https://s3.amazonaws.com/elasticbeanstalk-fourfront-webprod-wfoutput/4DNFICFVN1W5/fastqc_report.html", "Total Sequences": 37954345, "@id": "/quality-metrics-fastqc/7f7408b9-2463-4150-8a42-853f3c176822/", "uuid": "7f7408b9-2463-4150-8a42-853f3c176822", "display_title": "QualityMetricFastqc from 2024-12-20", "overall_quality_status": "PASS", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "schema_version": "2", "file_first_line": "@A00419:487:HL25CDRXY:1:2101:1127:1000 1:N:0:GTCCGCAC", "project_release": "2025-08-27", "flowcell_details": [{"barcode": "GTCCGCAC", "machine": "NovaSeq6000"}], "file_classification": "raw file", "@id": "/files-fastq/4DNFICFVN1W5/", "@type": ["FileFastq", "File", "Item"], "uuid": "8f1432ee-0f31-4c77-98ad-d0784290128f", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}, "display_title": "4DNFICFVN1W5.fastq.gz", "external_references": [], "experiments": [{"status": "released", "display_title": "TSA-seq against THRAP3 protein on U2OS (Tier 2) - 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