PROJECT SUMMARY/ABSTRACT Gastrulation is a pivotal process for the formation of human body plan and its disruptions can lead to miscarriage or birth defects such as caudal dysgenesis. During this process, pluripotent cells differentiate into the primary germ layers or the endoderm, mesoderm, and ectoderm lineages that later create all of the body’s cell types. Such differentiation events involve extensive three-dimensional (3D) reorganizations of the genome that are specific to each lineage. However, the functional effects and the underlying mechanisms of these reorganization as well as consequences of their disruption are poorly understood. Our objective is to determine the dynamics of lineage-specific genome 3D reorganization during gastrulation and how they respond to disruptions in the epigenetic landscape. Our hypothesis is that genome 3D changes in each lineage help establish the cell fates it generates later in development by poising it to assume those cells’ transcriptional programs. We further hypothesize that such important 3D reorganizations should be more faithfully inherited through mitosis and and that chromatin epigenetic marks, such as histone modifications and DNA methylation, are important for their establishment. Members of this team have developed tools that create new opportunities for understanding the role of genome 3D organization in embryonic development. Among them are proximity ligation assays that allow for global determination of genome 3D organization in cell populations and single cells. We have also developed a cutting-edge and powerful in vivo combinatorial barcoding system in mice that enables us to decipher the lineal relationship between cells. Here we propose using these tools to identify lineage-specific higher-order features in gastrulating mouse embryos, assess their association with gene expression later in development, evaluate how faithfully they re-establish after mitotic divisions, and examine how they are affected by disease-related epigenetic perturbations. Our long-term goal is to understand the role of genome structure dynamics in fate determination during embryogenesis when cells undergo a succession of lineage commitments in highly programmed fashion. Our proposed research will broadly impact the field by characterizing lineage-specific genome 3D organization during embryogenesis, as well as its functional dynamics and connection with chromatin epigenetic marks.