Abstract: Establishment of different cell types during development requires precise interactions between genes and distal regulatory elements. However, our understanding of what these interactions look like in three dimensions, how they vary across cell types in complex tissue, and how they relate to transcriptional state remain limited. I will describe optical reconstruction of chromatin architecture (ORCA), a microscopy approach to follow the path of DNA in intact nuclei in thousands of cells. We have used this approach to the study the structure of the Bithorax-Complex in Drosophila embryos with nanoscale accuracy and genomic resolution as high as 2 kilobases. Simultaneous imaging of nascent transcription allowed direct correlation of DNA folding to RNA expression in single cells. We recovered predicted cell-type-specific boundaries between active and Polycomb-repressed DNA and also observed Polycomb-independent boundaries which further partitioned the active, but not repressed, portions of the locus in a cell-type-specific manner. Deletion of these boundary regions corresponded with ectopic enhancer-promoter contacts, aberrant gene expression, and developmental defects. Together, these results illustrate a powerful approach for high-resolution, single-cell DNA domain analysis in vivo and suggest a prominent role for 3D chromatin organization in cell identity.
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