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.
The functional relationship between nuclear compartments and gene expression remains an enigma. Here we propose to identify components of known and still unknown nuclear ?active niches?, using a novel combination of proteomics and omics proximity-labeling. Live-cell imaging will then determine the movements of genes and mRNAs to and from these active niches and determine the functional significance of these movements on gene expression exploiting the design of novel, low-cost, high-end microscopes.
