Raw Images for Ogiyama et al., Mol Cell 2018

These data were multi-colour FISH experiments to analyze the 3D relationship of Drosophila regulatory elements in wild type strains versus strains in which a locus was engineered with CRISPR technology, so as to delete or modify Polycomb binding sites or to prevent long-range 3D interactions between these binding sites. The FISH experiments were performed in order to test whether these gene technology manipulations affect 3D architecture and the expression of the underlying genes. The summary of the main results follows below: Interphase chromatin is organized into topologically associating domains (TADs). Within TADs, chromatin looping interactions are formed between DNA regulatory elements, but their functional importance for the establishment of the 3D genome organization and gene regulation during development is unclear. Using high-resolution Hi-C experiments we analyze higher-order 3D chromatin organization during Drosophila embryogenesis and identify active and repressive chromatin loops that are established with different kinetics and depend on distinct factors: Zelda-dependent active loops are formed before the midblastula transition between transcribed genes over long distances. Repressive loops within Polycomb domains are formed after the midblastula transition between Polycomb response elements by the action of GAGA factor and Polycomb proteins. Perturbation of PRE function by CRISPR/Cas9 genome engineering affects Polycomb domain formation and destabilizes Polycomb-mediated silencing. Preventing loop formation without removal of Polycomb components also decreases silencing efficiency, suggesting that chromatin architecture can play instructive roles in gene regulation during development.