Identification of the active nuclear niche(s) using novel proteomic, genomic, transgenic, and live-cell microscopy technologies

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The study of gene expression and possible role of condensates in regulating gene expression have largely ignored known nuclear structures. This proposal is significant because we propose a novel model for the role of nuclear organization in regulating gene expression: 1) Nuclear speckles and still unknown nuclear compartments/bodies help organize other phase-separated condensates to modulate gene expression; 2) Nuclear speckles together with surrounding nuclear compartments/bodies and associated phase-separated condensates together represent active nuclear niches which may have different functional properties; 3) Small distances matter: gene movements of only a few hundred nm between repressive and these different active nuclear niches may differentially regulate gene expression; 4) Action-at-a distance: component flux into and out of these nuclear compartments will have global effects on gene expression; 5) These same nuclear compartments/bodies may similarly modulate RNA processing and organize nuclear export. Here we propose to: 1) Identify multiple components of known and still unknown nuclear “active niches”; 2) Map genome-wide the positions and predicted movements of genes relative to these active niches during physiological transitions; 3) Visualize nuclear body/compartment dynamics and fluxes of proteins between nuclear bodies in steady-state and through physiological transitions; 4) Visualize movements of reporter transgenes, endogenous genes, and rewired chromosome loci relative to these nuclear bodies/compartments and temporally correlate changes in gene expression with their dynamic movements and compartment associations; 5) Visualize movements of pre-mRNAs and nuclear mRNAs during RNA processing and export; 6) Measure fluxes of nuclear body components to and from adjacent transcribing chromatin. Additionally, we propose developing relatively low-cost, novel microscope platforms and software specifically 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) using iterative rounds of TSA-MS-Ratio, validation by light microscopy, and TSA-Seq; 2. Measure dynamics of bodies, components of nuclear bodies using live-cell imaging; 3. Measure temporal correlation between changes in gene expression and gene movement relative to nuclear bodies and visualize the export path of expressed transcripts; 4. Design and deliver two novel microscopes designed to facilitate Aims 1-3 at a modest cost. Successful completion of these Aims should significantly change our current understanding of the role of nuclear organization in regulating gene expression with impact across a wide range of research fields.