PROJECT SUMMARY/ABSTRACT DNA replication is essential for the structural and functional integrity of genomes, and spatiotemporal control of DNA replication (replication timing, RT) is intimately related to 3D genome architecture and organization, however, we know little about how it is regulated. The lab has recently identified cis-acting elements that are crucial for controlling RT, chromosome architecture, and transcription in mouse embryonic stem cells (mESCs), termed early replication control elements (ERCEs). ERCEs contain many active chromatin marks, such as acetylated histone residues and are co-occupied by pluripotency factors Oct4, Sox2, and Nanog (OSN). ERCEs also contain transcription start sites (TSSs), which may account for their role in regulating transcription. ERCEs form 3D interactions independent of architectural proteins CTCF and cohesin. What is unknown is whether ERCEs are one functional unit that coordinately regulates RT, architecture, and transcription, or if they are composed of discrete, separable elements that independently regulate different nuclear functions. The hypothesis is that ERCEs are composed of multiple elements, such as OSN binding sites and TSSs, that can be uncoupled, to determine their individual roles in regulating RT, architecture, and transcription. The rationale is that RT, genome architecture, and transcription are all commonly disrupted in disease states, and so understanding how they are regulated will lead to a better understanding of the molecular and cellular basis of disease. AIM 1 will use CRISPR to genetically dissect ERCEs in order to identify the sequences harboring activity necessary for ERCE activity, such as RT and transcription. AIM 2 will transfer ERCEs or components of ERCEs to ectopic sites and assay their sufficiency to promote early replication and transcription and to alter chromatin architecture at the ectopic genomic location. This contribution will be significant because it will address the regulation of DNA structure and function and how that regulation is perturbed in disease. The proposed research is innovative because the discovery of ERCEs provide novel biological questions and approaches to investigate the regulation of chromosome structure and function.