Project Summary/Abstract Mammalian genomes are folded into spatial domains termed Topologically Associating Domains (TADs) spanning hundreds of kilobases. By increasing the contact probability of DNA loci inside the same TAD, TADs are thought to regulate gene expression by regulating enhancer-promoter contact. Consistently, several recent studies have demonstrated that TAD disruption can cause disease, including cancer and human neurodevelopmental disorders. Additionally, the causal regulators of TADs, CTCF and the cohesin complex, are among the most frequently mutated genes in cancer. At the same time, however, other recent studies have found little to no role for CTCF and TADs in regulating specific genes and loci. The premise of this proposal is that the current disagreements in the field are partly due to at least two limitations. First, genome organization and chromatin looping is likely inherently dynamic, but we lack the tools to capture this (Part A). Second, developmental gene regulation is selected for robustness, and therefore characterized by redundancy and complexity. Here, a bottom-up strategy is proposed to overcome the redundancy of natural developmental gene regulatory circuits (Part B). First, in Part A of this proposal, the development of an integrated set of new experimental and computational tools is proposed. Previous approaches, although powerful, relied on static snapshot approaches that are limited to dead and chemically fixed cells. Here, live-cell single-molecule imaging is proposed as a method to overcome this limitation to follow chromatin looping in live cells with nanometer resolution in space and second resolution in time. By complementing this approach with the development of new computational tools and a complementary genomics approach, this will allow the dissection of how genome folding regulates transcription in both time and space. Second, in Part B of this proposal, these tools will be applied in a bottom-up synthetic biology approach. The goal will be to build TADs, enhancers, and promoters de novo in a simple genomic context. This will make it possible to escape the complexity of natural developmental gene regulation, where redundancy makes establishing causality highly challenging. Through a bottom up approach, this proposal aims to distill out the general mechanistic principles, test a wide range of mechanistic hypotheses through perturbation experiments, and to establish causality. The overarching goal of this proposal is to elucidate how and to what extent genome organization contributes to transcriptional regulation and to develop a quantitatively predictive understanding. Ultimately, this may allow us to correct genome misfolding in disease.