PROJECT SUMMARY Increasing evidence indicates that three-dimensional (3D) genome organization is required to regulate gene function and its alterations are associated with many diseases. The genome is organized into compartments that align with the temporal order of DNA replication (replication timing – RT). However, little is known about the mechanisms underlying RT control and 3D genome organization. Recently, we identified a novel class of cis regulatory elements that control RT and 3D genome organization: early replicating control elements – ERCEs. Our long-term goals are to study what are the regulatory elements of genome organization in human differentiated cell types, investigate how trans-acting factors control these elements, and define how 3D genome organization is remodeled during development and evolution. To achieve these goals, we will delete and insert candidate ERCEs into the genome of human differentiated cell types and test their effect on RT, 3D genome organization and gene expression. We will track ERCE activation using highly-synchronous human embryonic stem cells differentiation systems. Finally, we will analyze 3D genome organization evolution using primary cells derived from different species. We will use the recently developed technologies for cell purification based on chip- enclosed microfluidic flow cytometry to isolate live, single cells to establish clonal cell lines carrying ERCE deletions and insertions. Moreover, we will use this technology for precise identification and isolation of cell populations in specific cell cycle stages to systematically investigate the connections between RT, genome architecture and gene expression. Overall, we anticipate that the application of microfluidic flow cytometry will allow us to optimize our cell line development, as well as our sample purification for downstream genome-wide analyses. We expect that our work will contribute significantly to understand the fundamental principles of genome organization and its relationship to gene function.