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RT-CDF: The study of gene expression and possible role of condensates in regulating gene expression havelargely ignored known nuclear structures. 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 “activeniches”; 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."