Abnormal temporal control of replication is observed in many diseases but causal linkages are unknown. This gap will remain incomprehensible until the mechanisms regulating replication timing during normal development are understood. The long-term goal is to understand the relationship of replication timing to cellular epigenetic states and disease. The immediate goal is to identify cis-acting DNA/chromatin elements that regulate changes in replication timing during differentiation of mouse embryonic stem cells (ESCs). Mouse ESCs are an ideal experimental system due to the availability of chromosome engineering tools, directed cell differentiation systems, and comprehensive genome-wide maps of replication timing and transcription. These maps have identified the molecular coordinates of programmed changes in replication timing that occur in 400-800kb units termed replication domains. The central hypothesis is that discrete identifiable chromatin or DNA sequence features dictate the boundaries of replication domains and the developmentally induced changes in their replication time. The rationale for this proposal is that identifying DNA/chromatin elements regulating replication timing is the essential next step in elucidating mechanisms regulating replication timing and its relationship to disease. Aim1 will test the hypothesis that replication domains are fundamental units of chromosome structure and function that can be transferred to an ectopic location. Large pieces of cloned genomic DNA from a developmentally regulated replication domain will be introduced into a region of constitutive replication timing. Repli- cation timing of the insert and flanking DNA will be monitored during differentiation to identify the minimal sequences constituting a unit of regulation. Aim2 will distinguish between models in which specific boundary elements punctuate temporally distinct domains vs. models of boundaries as passively replicated chromatin between actively programmed domains. Nested deletions will be engineered in developmentally controlled replication timing transition regions and the effects of deletions on the regulation of replication timing will be determined. Aim3 will test the hypothesis that transcription within a silent late replicating domain initiates a switch to early replication. Promoter and regulatory elements controlling transcription within a developmentally regulated replication domain will be deleted, replaced with an inducible promoter, and the effects of such manipulations on the regulation of replication timing will be analyzed. Studies described here will identify cis-acting elements regulating the developmental control of replication timing. This contribution is significant because identifying regulatory elements of replication timing control is a pre-requisite to understanding the role of replication timing in chromosome-based diseases. The work proposed here is innovative in that it proposes a novel combination of chromosome engineering and directed embryonic stem cell (ESC) differentiation to address the mechanisms eliciting developmentally programmed changes in replication timing. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Accurate duplication of chromosomes during each cell division is essential to normal growth and development. The proposed project is important for public health because abnormalities in the temporal order in which chromosome segments are duplicated have been detected in many diseases and are expected to reflect the origins of these diseases, yet we have a poor understanding of how replication timing is regulated and why it is disrupted in disease states. Thus, the proposed experiments are relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will increase our understanding of the pathogenesis of disease, suggest novel treatments, and reduce the burdens of human disability.