Researchers at Massachusetts General Hospital have uncovered new insights into our understanding of X-inactivation in female mammals.
Classical microscopy studies have shown that mammalian chromosomes fold into large-scale structures and occupy discrete territories within the nucleus. Researchers have proposed that chromosomes fold into topologically associating domains (TADs) that are fundamental units of 3D nuclear organisation. Cohesin and CTCF are two factors that are critical in the regulation of chromosome architecture. Cohesin forms a ring-like structure around chromatin and extrudes chromatin loops. Meanwhile, CTCF insulates enhancers from promoters and typically anchors long-range loops.
Researchers suspect that 3D genome structures play a key role in gene regulation. A series of loss-of-function studies targeting CTCF, cohesin component RAD21, and cohesin release factor WAPL have shown a dramatic alteration in 3D genome structure but only modest effects in gene expression. This begs the question of what precise role 3D organisation plays during epigenetic regulation.
An epigenetic phenomenon: X-inactivation
In this study, published in Molecular Cell, researchers revisited the role of cohesin-mediated architecture using a model epigenetic phenomenon – X-inactivation. In this process, female mammals silence one of their X chromosomes to balance the dosage of X-linked genes with males. The process is mediated by Xist, a noncoding RNA that is transcribed from and spreads across the inactive X chromosome. This triggers the recruitment of repressive factors such as Polycomb repressive complexes 1 and 2 (PRC1/2). This ultimately leads to the formation of heterochromatin and gene silencing. The inactive X chromosome shows greatly weakened TAD structure and appears to be devoid of A/B compartmentalisation.
To investigate cohesin imbalances, the team perturbed cohesin levels through targeted degradation of RAD21 and WAPL in embryonic stem cells from female mice. They found that cohesin loss disrupted the inactive X chromosome superstructure but had minimal effect on gene repression. Whereas, forced cohesin retention markedly affected inactive X chromosome superstructure, compromising the spread of Xist RNA-Polycomb complexes and attenuating its silencing effects. This demonstrates that there needs to be a balance between eviction and retention of cohesin during X chromosome inactivation.
Next, the team explored the effects of manipulating cohesin in an active X chromosome. They saw that insufficient cohesin resulted in the formation of ‘superloops’ that are usually only seen in inactive X chromosomes. In addition, when there was excess cohesin, the team saw the formation of ‘megadomains’ which are ordinarily unique to inactive X chromosomes.
These findings suggest that the shape and structure of the X chromosome plays a key role in enabling Xist to spread along the chromosome and achieve inactivation. Developing our understanding of silencing within the X chromosome may provide opportunities to find ways to reactivate it and treat conditions like Rett syndrome.
Image credit: By gr8effect – canva.com