Water hydrates and is essential for life—for plants, humans, and even fish. So, it’s fitting that a new technique called HiDRO takes high-throughput FISH to the next level to identify factors that help genomes fold in 3D at high throughput in single cells.
Cohesin helps DNA form loops, which can then form topologically associated domains (TADs). To figure out the regulatory proteins involved in this process across thousands of individual cells at a time, Eric Joyce’s lab (U Penn) waded into the deep end and developed HiDRO (high-throughput DNA or RNA labelling with optimized Oligopaints). This approach combines high-throughput FISH and improved oligo probes, or Oligopaints, as well as automated image analysis. Here’s the details about the method:
- Oligopaints are single-stranded DNAs that generally have 32-40 bases of homology with the nucleic acid they will label, or paint, but the team lengthened the homology region in this case to 80 bases to increase labeling efficiency
- They also indirectly labeled the oligos by incorporating amino-allyl nucleotides when the Oligopaints were made
- This meant that fluorophores could easily be added after synthesis, which can be more efficient than direct labeling during synthesis (labeled nucleotides are not always added as easily as unlabeled ones)
- After these probes were made, the team performed high-throughput FISH
Testing HiDRO across many TADs, the team could label as much as 90% of the domains with much less probe than with the regular Oligopaint protocol. They also could label RNA.
Next, they performed a tsunami level of analyses with just a drizzle of effort to identify proteins that regulate folding. They used HiDRO with an siRNA library against the druggable genome, or enzymes that respond to small molecules. Here are the numbers:
- Analyzed 1.5 million nuclei, screened more than 3,000 genes, conducted over 7,000 FISH assays
- More than 300 knocked-down genes affected the spatial organization of chromatin
- Knocking down 58 of these genes had similar effects to disrupting NIPBL, a cohesin loader, or WAPL, a cohesin unloader
- In a secondary screen, they validated 43 hits, and found that they were involved in ubiquitin ligation, calcium signaling or GSK3 kinase signaling
With further experiments, they showed that GSK3A, their top hit, affected folding and is a serine/threonine kinase. Looking deeper, the team found that the kinase’s protein folding function didn’t overlap with its WNT signaling function. Depleting GSK3A boosted inter-TAD overlap; short-range interactions decreased and more long-range contacts were made. There was a gain in some types of loops, which appeared to be existing loops that were extended rather than brand-new ones. In addition, GSK3A recruited WAPL to cohesin, which would remove the complex from chromatin.
The team says that HiDRO screening could make a splash with other nuclear features in the future to get a better understanding of chromatin and how the genome is arranged.
For the next wave of your research, check out Nature, August 2023.