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Optical Approach Used to Visualize DNA Folding During Drosophila Development

NEW YORK (GenomeWeb) – A team of Stanford University researchers has used a new optical approach to examine DNA chromatin architecture during Drosophila development.

The researchers applied their optical reconstruction of chromatin architecture (ORCA) approach to visualize a Hox gene cluster in Drosophila embryos. Using this method, the researchers uncovered physical borders between active and Polycomb-repressed DNA as well as unexpected Polycomb-independent borders, as they reported today in Nature. Elimination of these Polycomb-independent borders, they further noted, leads to developmental defects.

"Whereas previous results have suggested that genome structure is largely static during development and across cell types, our results support an emerging view that for developmental control loci, cell differentiation is accompanied by extensive 3D remodeling of chromatin structure," Stanford's Alistair Boettiger and his colleagues wrote in their paper.

With ORCA, the researchers aimed to combine the ability of Hi-C to perform de novo mapping of topological-associated domains (TADs) and enhancer-promoter interactions with the ability of multi-color fluorescent in situ hybridization to yield single-cell resolution and tissue organization. ORCA relies on array-derived oligonucleotide probes, or Oligopaints, and tiles the region of interest with probes containing unique barcodes that are labeled with a fluorophore. The barcodes are then sequentially imaged.

Boettiger and his colleagues applied this approach to visualize the bithorax complex (BX-C) in Drosophila embryos. This group of genes governs the differentiation of the fruit fly's abdominal and posterior thoracic segments.

As the researchers noted, development relies on particular interactions between genes and their regulatory regions, and such enhancers that can be located up to hundreds of kilobases away. These interactions are influenced by how DNA folds in three-dimensional space.

With ORCA, they visualized BX-C at both 10-kilobase and 2-kilobase resolution in cryosections obtained between 10 hours and 12 hours post-fertilization.

By 10 hours of development, the researchers found that the Drosophila embryos harbored distinct cell types, which, in conjunction with RNA data, they found clustered into 18 groups with different organizational patterns. Some anterior cells, the researchers noted, had long-range interactions that were closer physically than was expected, while other short-range interactions were further apart than expected.

The closer-than-expected, long-range interactions are consistent with previous reports of Polycomb-repressed DNA being compact, the researchers noted, adding that their farther-than-expected, short-range interactions were more surprising. Still, they said that those findings were consistent with other recent imaging of Polycomb-repressed DNA and support the notion that Polycomb-repressed DNA is organized like a random coil.

The researchers also noted physical boundaries between active and Polycomb-repressed DNA that correlated with the different body segments the cells originated from. In segment T3, for instance, BX-C split into two TADs, one centromeric and one telomeric, marked by its associated H3K27me3 epigenetic state, while in segment A1 and A2, the border shifted right. This, the researchers noted, is consistent with findings from ChIP-seq and classical experiments.

Some TAD borders, though, didn't correspond with epigenetic domain borders. For instance, segment A1, the de-repressed portion of BX-C split into two TADs and in A9, there were no H3Kme3 boundaries predicted, but there were two unexpected structural regions. The abd-A and Ubx genes and their regulatory regions fused in a centromeric TAD. The researchers suggested that these boundaries provide an explanation for segment-specific enhancer and gene expression activity.

Embryos homozygous for deletions spanning some of these domains exhibited changes in gene expression as well as developmental defects, the researchers noted.

Previously, it had been posited that only vertebrates had evolved an additional layer of organization such as this. But the researchers said their findings suggest that may not be the case. "Even if these observations prove to be unique to the BX-C, they challenge the conclusion that the additional layer of genome organization is solely a vertebrate innovation," the researchers wrote.

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