If you’ve ever seen a polydactyl cat you might (understandably) live in fear of cats with thumbs. If we want to prevent the takeover by our feline friends we need to understand the cause of this limb anomaly. Luckily, researchers led by Stefan Mundlos have done just that by asking if 3D chromosome structure data could help shed light on how genomic rearrangements in non-coding regions establish pathologic conditions.
Genome-wide chromosome interaction studies have shown that the genome is partitioned into megabase-scale topologically associated domains (TADs), which, like a good house, are “gated” by boundary regions, marked by CTCF binding.
The authors focused on several limb malformation diseases (including brachydactyly, F-syndrome and polydactyly) that affect the region of chromosome 2 containing the genes WNT6, IHH, EPHA4 and PAX3. These genes are organized into three respective TADs. At the embryonic stage E11.5 the TADs of mouse limbs resemble those of human cells, meaning the team were able to use a CRISPR–Cas9 approach to delete the Epha4-containing region in the mouse embryos to model human disease. They generated mouse models with a large deletion or an inversion of chromosome 2 could be successfully established. Using these models they:
- Recapitulated the phenotype of human disease.
- Found that the genes that were rearranged (Wnt6, Pax3 and Ihh) assumed the expression pattern of the gene Epha4, suggesting that the genetic position was important for expression.
- Observed that the aberrantly expressed genes established new interactions with the Epha4 TAD in both mice- and patient-derived fibroblasts.
- Identified a minimal common region between the rearrangements, which contained enhancers for Epha4. This was subsequently validated to be active during limb development and to interact with Wnt6, Ihh and Pax3 in the genetic disease models.
- Rule out the involvement of a distance effect in the pathogenesis, since by leaving intact the TAD boundaries, marked by CTCF peaks, they managed to restore normal limb development.
This study paves the way to similar investigations involving genetic diseases and copy number alterations and provides functional relevance in disease to chromosome gating.
For those wanting to learn more about how good boundaries might prevent the catpocalypse head over to Cell, May 2015.