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

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.

Grant Number: 5U01DK127422-05
NIH Institute/Center: NIH
Principal Investigator: Andrew Belmont