Project Summary/Abstract Understanding the mechanisms of gene and chromatin regulation and their roles within a multicellular organism has relevance across many disciples such as synthetic biology, medicine, developmental biology and neuroscience 1–3. Large-scale efforts of the genomics community have identified many of the functional genes and gene regulatory elements (GREs) including recent atlases with the specific expression of genes and their putative regulatory regions within different cell types of complex tissues 4,5. However, it remains unclear how the 3D organization of chromatin impacts gene regulation and vice versa. To build a mechanistic understanding of the interplay between chromatin organization and gene regulation, we would ideally simultaneously measure all the key elements - DNA sequences, regulatory proteins, and the transcribed RNA - at the genomic-scale, while maintaining information about cell type identity. To address this challenge, I will develop an imaging platform that can simultaneously measure the 3D structure of DNA together with the RNA expression of the regulated genes and their interaction with key structural proteins (Aim 1). While this method can be applied to many systems, a particularly suited example is the peripheral olfactory system. Olfaction, one of the main mammalian senses, is controlled by the largest family of genes comprising more than 1000 olfactory receptors 6,7. Large networks of regulatory sequences interact across the genome to establish more than 1000 neuronal types, each expressing one and only one receptor8. I will apply this imaging method to address the longstanding question: how do different olfactory sensory neurons establish their receptor expression? These integrated measurements relating chromatin organization and regulatory protein structures to transcriptional activity will provide a model of olfactory gene regulation. Aim 2, is to dissect this model and the roles of GRE-promoter interactions in achieving cell-type specific expression using a high-throughput synthetic biology approach. I will infect the olfactory epithelium with large pools of viral vectors that combine different regulatory elements and promoters, and determine the precise cell-type expression of these vectors using multiplexed imaging. There is an additional synergy between the two aims - the first aim provides measurements of the endogenous chromatin structure-transcription relationship which will be used to design transgenic control of specific subpopulation of cells. I will explore this capability to activate/inhibit specific sub-populations of olfactory receptor neurons and determine the behavior consequences of these manipulations.