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TCPA: Three-dimensional (3D) genome organization is important for proper gene regulation, and impacts development and disease. Topological Associating Domains (TADs) comprise the basic unit of organization within the genome, with sharp boundaries characterized by highly transcribed housekeeping genes, short interspersed nuclear elements (SINE) or sites for the DNA-binding protein CCCTC-binding factor (CTCF) which interacts with Cohesin. This proposal addresses several major unanswered questions and explores new opportunities in the field, including: i) When does 3D genome architecture (TADs and their boundaries) begin to form during early vertebrate embryogenesis? ii) Is genome-wide transcriptional activation in the developing embryo required for the establishment of TADs? iii) How dynamic are individual TADs at the single cell level and what is their effect on gene expression and nuclear architecture? Importantly, our combination of genomics with super-resolution imaging bridges and connects two major disciplines in the 4D Nucleome community. Our central goal is to determine when and how higher-order 3D chromatin architecture is first established in early embryos, how is it remodeled during developmental reprograming and what is their impact on transcription. This proposal is significant for several reasons. First, from a standpoint of nuclear architecture, it addresses how TADs are formed over time and what is their effect on genome activation and nuclear architecture. Second, it addresses how positional information in the nucleus plays a role in the fundamental step of transcription in vivo. Third, within the context of the 4D Nucleome initiative and a technological standpoint, the zebrafish embryo is a powerful system for probing different technologies that that can be further applied across the 4D Nucleome initiatives directly relevant to ES cells, mouse embryos and differentiated tissues to understand how nuclear architecture is established in vivo. From a developmental biology and fertility standpoint, we address fundamental mechanisms of initiation of gene expression after fertilization - a universal transition across animals. In our proposal, we provide preliminary evidence supporting the following hypothesis: TADs and their boundary elements are fully established only after the onset of genome-wide transcriptional activation, to refine embryonic transcription. The experiments described below will define the true relationship of TADs to transcription, illuminating the fundamental mechanisms responsible for assembling the 4D nuclear architecture during embryogenesis when cells become totipotent. Together, our laboratories will combine advanced genomics techniques (Hi-C, PLAC-seq, others), super-resolution/EM microscopy methods and innovative genetic and molecular approaches in zebrafish embryos, toward addressing these questions.