Project Summary Gene regulation is a highly complex process that involves the recruitment of numerous regulatory factors as well as dynamic 3-dimensional spatial rearrangements of DNA, RNA, and protein molecules that are important for quantitatively controlling the rate of gene regulation. Yet, how these various components interact simultaneously and what their quantitative contributions are to gene regulation remains unresolved largely because of the lack of methods that can integrate combinatorial molecular binding, spatial information, and quantitative measurements of various aspects of gene regulation within the same individual cell. Here we will develop highly innovative methods that will allow us to generate dynamic molecular movies that monitor the movement of DNA, RNA. and protein molecules at high resolution in a manner that provides information about the spatial arrangement of molecules along with simultaneous information about transcription rates and mRNA splicing rates within single cells. To achieve this, we will develop pioneering new genomic methods for measuring the spatial interactions of RNA, DNA, and protein with single cell capabilities and build novel quantitative and computational modeling approaches to generate high resolution temporal “movies” from snapshots derived from tens of thousands of synchronized single cells. We will use these approaches to quantitatively understand the dynamic assembly of RNA-protein complexes, localization to DNA, and structural dynamics of genomic DNA and how these integrated components impact gene regulation across time. Specifically, we will dissect the dynamics of three RNA-mediated processes that link dynamic 3D nuclear structure and gene regulation in unique regulatory paradigms in biology and disease: (i) chromosome-wide transcriptional silencing, (ii) kinetic coupling of mRNA transcription and splicing, and (iii) RNA-induced aggregation and cellular toxicity in neurodegenerative disorders. Together, the results of this proposal will generate highly innovative new approaches for quantitatively measuring molecular and spatial dynamics of various regulators and their role in gene regulation.