It’s easy to think that at this point, science has it all figured out. We’ve developed genome-wide techniques to look at chromosome structure in many different contexts: from mapping chromatin interactions (Hi-C), to observing DNA-RNA interactions (SPRITE), and even identifying higher-order DNA structures (GAM). Unfortunately, none of these techniques have the resolution to look at specific regulatory elements (i.e. interactions occurring at a singular enhancer or promoter). Not to be deterred, the lab of Jim Hughes (Oxford University, UK) has developed a new technique called “Tri-C” for observing multi-way interactions of regulatory elements at the level of a single allele!
Tri-C allows for high-resolution mapping of chromatin interactions at a specific viewpoint of interest. Tri-C piggybacks on the current chromatin capture techniques, utilizing the same formaldehyde fixation to cross-link adjacent chromatin, followed by restriction digest and random ligation. However, traditional chromatin capture methods are limited in identifying multi-way associations, due to the very low percentage of reads containing more than two interacting fragments. Tri-C overcomes this obstacle by sonicating the ligated fragments to a size of ~450bp, yielding a pool of fragments where over 50% contain multiple interactions. Fragments containing the viewpoint of interest are captured by hybridization of biotinylated oligonucleotides, and a pull-down with streptavidin beads. These fragments of interest are amplified by PCR and sequenced using the Illumina MiSeq platform. Ultimately, this method provides a high-resolution snapshot of multi-way chromatin interactions at a point of interest on a single allele.
Not content to simply develop this powerful new tool, the authors put it to the test by looking at the interaction of chromatin regulatory elements—at the single allele level—in the mouse α-globin locus. Here’s what they uncovered:
- Multiple enhancers and promoters can interact simultaneously to form a multiplex regulatory hub
- The CTCF boundary elements do NOT form higher order structures, and instead display diffuse interaction patterns, indicative of transient/dynamic interactions
- This data supports the loop extrusion model of chromatin loop formation
We’d definitely promote this technique for those looking to enhance their understanding of the multi-way interactions behind chromatin architecture and their gene regulatory effects.
To read more about the triumphs of Tri-C, head over to Nature Genetics, October 2018.