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.
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> 2-colour DNA FISH for embryo FISH procedure for embryo sample DNA FISH probes were prepared using FISH Tag™ DNA Multicolor Kit with PCR products as templates. Probes were designed so that it covers 10 kb sequential region. The primer sequences used for probe preparation are listed in TableS1. DNA FISH was performed by using embryos 0-4hrs after egg lying as previously described (Bantignies et al., 2011). At the last step after DAPI staining, embryos were washed with 1XPBS and were mounted on glass slide with vectashield antifade mounting medium. > Microscopy and Image Analysis Images were collected by using a Zeiss LSM780 confocal microscope with 63X Plan Apochromat 1.4 NA oil DIC lens. Z planes are taken every 0.30 µm. The centroid of each FISH signals was taken to measure the distances of two loci by using Volocity software (version6.1.1, PerkinElmer). To detect embryonic cycles, number of nuclei in 500 x 500 pixels, distance between each centroid of nucleus, and relative position of nuclei in embryo were used. For three color DNA-FISH, distance was taken if all the probed loci are detected within 1.5µm to avoid taking distance of two loci that are in different homologs, since homologous chromosomes are separated each other before cycle14 embryo. For the FISH in spalt loci, we selected the FISH distances from each nucleus that are in the specific region of the embryo, based on the patterns of salm expression in cycle14 (images from BDGP insitu homepage, http://insitu.fruitfly.org/cgi-bin/ex/insitu.pl). From the several examples of salm expression image, we calculated the relative positions for “salm expressed” and “salm not expressed” in the cycle14 embryo. FISH distances are selected based on these relative locations in the embryo and plotted separately.