PROJECT SUMMARY During development, a series of extracellular signals lead to the formation of different cell-types by inducing the differential expression of many genes. Although all genes experience the same signal, the expression of some genes is increased, some reduced, and others stay the same. Therefore, the gene expression profile that is characteristic of any cell-type must result from each genomic locus interpreting the signal in a distinct manner. The loss of faithful signal interpretation by genomic loci is pathogenic in many contexts including cancer and neuropsychiatric disease. Thus, an understanding of how individual genomic loci interpret extracellular signals is a fundamental yet unresolved problem. The challenge in understanding cis-regulation in response to extracellular signals is two-fold. First, any manipulation of the signal (concentration, duration, identity) leads to myriad pleiotropic effects in trans that confound interpretation. Second, multiple cis-regulatory elements (CREs) work together across large genomic windows to specify the expression of their target gene. Even in this post-CRISPR era, it has remained challenging to simultaneously manipulate multiple CREs across these large genomic regions to deconvolve their relative contributions to target gene regulation. This proposal seeks to solve these challenges using a combination of synthetic biology and single-cell sequencing. At the HoxA cluster, extracellular signals such as retinoic acid (RA) and Wnt induce the establishment of distinct transcriptional, epigenetic and topological domains that are stably inherited through cell divisions. In Aim1, we will rewire the HoxA cluster to respond to an extracellular signal that is completely orthogonal to the rest of the genome. This will enable the independent manipulation of transcription factor binding in cis and changes to the trans-regulatory environment to determine their relative contributions in establishing and maintaining the HoxA response to differentiating signals. We are unlikely to glean generally applicable principles of gene regulation from studies of a single locus. In Aim2 we will develop a technology that uses single-cells as individual experiments to massively increase the scale at which interactions between CREs can be uncovered at any locus. This technology will be applied to dissect the regulatory landscapes of genes involved in neuronal cell-type specification. We will then use the large dataset to develop a predictive model of cis-regulation at other loci.