Single Cell Tracking of 3D Epigenetic Landscape Evolution During Embryonic Development An important question to cell biology is how cells break the symmetry during mitotic divisions. During mammalian pre-implantation embryonic development (PED), how the first cell fate decision is made remains unclear and is crucial for the understanding of how specific gene regulations can guide the life of a cell. Epigenetic modifications including chromatin remodeling are early events during PED. Histone methylation at different residues can recruit differential sets of chromatin remodeling complexes to regulate chromatin structures and silence/activate gene expressions accordingly. These histone methylations and their combinations at different genomic loci can serve as codes to determine the overall gene expression profile and phenotypic outcomes. However, it is still not understood how histone methylations and hence chromatin structures at specific loci are dynamically regulated during PED in which cells undergo a heterogeneous modulation at single cell levels. In this proposal, we will harness the power of directed evolution and high-throughput screening method to systematically develop specific/sensitive FRET (fluorescence resonance energy transfer) biosensors for the monitoring of crucial histone methylations in single cells. We will further develop and apply the mapping RNA-chromatin interactions in single cells (sciMARGI) to identify crucial RNA-genome interaction sites during PED. We will then employ the endonuclease-deficient Cas9 (dCas9), small guide RNAs (sgRNAs) and split FPs to identify and track the positons of specific loci crucial for embryonic cell differentiation. Ultimately, we will apply our controllable epigenetic modulators to guide the histone modulations at specific loci and elucidate their role in determining cell fates during PED. Given the importance of epigenetic modifications at different loci, the success of the project should have transformative impact in understanding the role of locus-specific epigenetics in determining the cell fate during PED. Accordingly, three aims are proposed: Aim 1. Spatiotemporal imaging of crucial histone methylations in single live cells and during PED; Aim 2. Visualize the locus-specific histone modifications during PED; Aim 3. Reprogram the locus-specific histone modifications during PED. While the focus of this proposal is to develop tools targeting histone methylations and chromatin structures at specific loci and differentiation outcomes, the strategies and approaches can be extended to monitor, in principle, any other epigenetic modification in single cells, including but not limited to histone acetylation and phosphorylation. The results from this project can also lead directly to the dynamic nuclear atlas illustrating how specific histone codes are encrypted in an integrative manner for the regulation of life.