PROJECT SUMMARY/ABSTRACT Enhancers are short DNA sequences that regulate complex patterns of gene expression during development. Misregulation of enhancer activity is associated with a wide range of pathologies, from congenital disorders to cancer. However, many properties of enhancers are not well understood, particularly how enhancers act over long genomic distances. While some enhancers are located proximally to their target genes, others are located distally, activating gene expression over thousands or even millions of base pairs of genomic distance. Most in vivo techniques that assess enhancer activity make use of a transgene system that places the enhancer directly upstream of a reporter. While transgenesis is a powerful tool, it cannot assess the role of genomic distance in enhancer activity. To address this, our group developed a novel in vivo mouse enhancer reporter assay to characterize the distal activity of enhancers. Using this method, we identified a novel cis-regulatory sequence, the RC element, that is necessary and sufficient for distal enhancer activity. The overall goal of this proposal is to characterize this newly identified RC element and dissect its role in mediating distal enhancer activity. To address this goal, this proposal outlines a plan to visualize the impact of the RC element on enhancer-promoter interaction (Aim 1) and to determine what factors are critical regulators of RC element function (Aim 2). For the former, both fluorescence in situ hybridization and chromosome capture based techniques will be employed to carefully analyze enhancer-promoter co-localization and overall nuclear organization in the presence and absence of the RC element. For the latter, mutagenesis of the RC element will be used to identify critical motifs. In parallel, knockout of candidate transcription factor regulators will be used to evaluate their importance in RC element-dependent enhancer activity. Findings resulting from this proposal will help elucidate a novel mechanism coordinating enhancer activity, adding to our overall understanding of gene regulation. My sponsor (Dr. Evgeny Kvon) and co-sponsor (Dr. Ken Cho) are experts in the field of developmental genetics, and, with their guidance, I have designed a training plan to help guide my transition to independent research. My plan focuses on developing my abilities in five key areas: technical skills, scientific communication, mentoring, and career development.