PROJECT SUMMARY/ABSTRACT Regulating when and where genes are expressed is essential to the proper development, health, and viability of all living organisms. The processes that regulate gene expression are choreographed across a broad range of spatial and temporal scales spanning from molecular scales where regulatory proteins bind and unbind DNA at sub-second to second time scales, to the organization of the nucleus where proteins and DNA form dynamic sub-micrometer sized domains that fluctuate over seconds and minutes, to the coordination of these events across distinct tissue types over hours and across hundreds of micrometers to millimeters. Despite the dynamic nature of these processes, most of our knowledge about them comes from experiments on fixed samples that provide population and time-averaged data. Recently, the advent of high-resolution live imaging techniques have granted the ability to quantify the dynamics of gene regulation and have highlighted what has been missed by studies in fixed samples. Although these new imaging approaches have already provided remarkable insights, due to technical constraints they are generally applied to cells grown on glass coverslips and isolated from the tissue contexts in which they have evolved to function. The premise of this proposal is that in order to build a holistic and quantitative framework to understand gene regulation, we must develop and apply experimental approaches that access the broad range of spatial and temporal scales involved, and do so in endogenous contexts. To achieve this goal I propose to integrate cutting edge light-sheet microscopy, label-free interferometry, and molecular imaging tools that will allow quantification of single-molecule protein kinetics, transcriptional dynamics at individual gene loci, chromatin dynamics, and the compartmentalization of nuclei in actively developing animal embryos. I will apply these technologies to study the dynamics of gene regulation during early development in Drosophila Melanogaster embryos. These embryos provide an ideal context for studying fundamental aspects of gene regulation. They proceed from fertilization to differentiated tissue in around just 3 hours during which chromatin and nuclear organization is progressively established along with patterns of gene expression across the embryo. I propose experiments that leverage the new integrated technological approaches I will develop to ask: (1) How do the dynamics of transcription factor protein-protein and protein-DNA interactions affect their ability to find and bind their specific genomic targets and shape the nuclear environment? and (2) How are functional sub-nuclear compartments formed during embryonic development, and what is their role in shaping chromatin dynamics and gene expression patterns? Together this proposal will lead to new experimental capabilities that will provide fundamental insights on the dynamics of how gene expression is regulated from the molecular scale up to the organismal scale. These new types of integrated datasets will lay the foundations for developing a quantitative and predictive framework which may allow us to develop new therapeutic approaches for correcting aberrant gene expression in disease.

PROJECT NARRATIVE All the cells that compose our bodies arise from a single fertilized ovum and contain the same genomic information, thus during embryonic development the correct genes must be turned on and off in the right cells at the right times to grow and maintain an individual capable of independent life. The work proposed here will integrate cutting edge techniques to directly visualize and quantify how this regulation of gene expression is orchestrated during embryonic development. The critical new information that will be gained from the proposed experiments have the potential to lead to novel therapeutic approaches to prevent or repair defects that arise from aberrant gene expression during development, in aging, and in cancer.