PROJECT SUMMARY Dynamic remodeling of the three-dimensional organization of the genome regulates gene expression to coordinate the healthy development of organisms and can impact disease progression when dysregulated. Recent advances have enabled the visualization of accessible chromatin in situ in fixed samples; however, we still do not fully understand how changes in chromatin organization occur in vivo during vertebrate development, especially at the single-cell level. Therefore, the goal of this project is to study the dynamic remodeling of chromatin organization during zygotic genome activation, when the transcriptionally silent embryo first initiates transcription of its own genes and genome architecture is dramatically remodeled. First (Aim 1), I will investigate the dynamic spatial organization of chromatin accessibility during genome activation in zebrafish. To do so, I will use Live-ATAC, a method I recently developed. Live-ATAC enables real time detection of chromatin accessibility in vivo at the single-cell level in living embryos. Using this method, I will determine precisely when and where chromatin accessibility is gained as the embryo undergoes genome activation and transcription is initiated. Second (Aim 2), I will use Live-ATAC in mutant embryos to dissect the role of pioneer transcription factors in dynamically remodeling chromatin accessibility during genome activation. Specifically, I will use embryos lacking functional Nanog, Oct4, and Sox19b which are maternally supplied transcription factors have been implicated in pluripotency and genome activation. Simultaneously, I will measure transcription of the first zygotically transcribed genes to determine the causal relationships between transcription factors, chromatin accessibility, and transcription in real time in vivo. Altogether, this work will provide an unprecedented view of how chromatin organization is dynamically remodeled during cellular reprogramming and begin elucidating the underlying mechanisms. This will inform our understanding of how three-dimensional genome organization may affect human embryogenesis and disease given the likely conserved regulatory principles.