PROJECT SUMMARY / ABSTRACT DNA replication follows a defined spatiotemporal program in which different parts of the genome replicate at different times during S phase. The DNA replication timing program interfaces with genome regulation while also influencing the genome’s stability and mutational landscape. Abnormal replication timing is associated with genetic diseases and oncogenesis. Despite the importance of DNA replication regulation to cell and organismal biology, we have very limited understanding of the DNA sequences and molecular mechanisms that specify the eukaryotic DNA replication timing program. Similarly, it remains unclear how replication timing control intersects with the regulation of gene expression and with the epigenome in general. My lab has, and continues to, develop novel experimental and computational approaches for measuring genome-wide replication timing. We also pioneered a unique approach of linking replication timing to human genetic variation in order to reveal the sequence elements that control replication timing and study their mechanisms of action (Koren et al., Cell 2014; Ding et al., Nature Communications 2021; NIH Director’s New Innovator Award DP2- GM123495). In this MIRA application, we will: 1) generate a new dataset of human replication timing, the largest so far, and use a computational approach to link replication timing to gene expression and epigenomic regulation in a novel and powerful way. 2) Utilize our previous and concurrent replication timing genetic mapping in order to experimentally edit DNA sequences controlling replication timing. This will be followed by several epigenomic assays and genetic experiments that will reveal the mechanisms and consequences of replication timing regulation. As part of this, we will also develop a new approach, “perturb-RT”, to systematically screen for novel sequence elements controlling replication timing. 3) Continue our recent success of profiling replication timing in single cells by jointly measuring replication timing and gene expression in the same single cells. We will use this approach to study how replication timing and gene expression co-vary during cellular differentiation. Our work will substantially contribute to the field of DNA replication timing and beyond, by providing unprecedented genomic resources, introducing novel genetic and genomic approaches, deciphering the genetic basis of human replication timing, and revealing new insights into replication timing regulation and its consequences.