Researchers Offer New Insights into X Chromosome Inactivation

Science has revealed some of the secrets of the X chromosome, but many remain. In humans, female cells contain two X chromosomes (XX) and male cells contains one (XY). In each female cell, one X chromosome must be inactivated—crumpled up in such a way that the genetic code it contains cannot be read out. RNA plays an important role in this inactivation process. Specifically, Xist RNA, a long noncoding RNA molecule, is responsible for crumpling up one copy of an X chromosome in each female cell. Researchers have known that Xist is critically important; without it, an embryo will not develop. But what prevents Xist from inactivating other chromosomes in the cell? A new paper published in Cell from researchers at Mass General Brigham and Tsinghua-Peking Center for Life Sciences offers insights on the forces that restrict the RNA’s movement, with implications for how to treat X-linked diseases in the future.

“Xist is important for human disease—if we can alter its expression it could have implications for how we treat rare diseases that are linked to the X chromosome,” said corresponding author Jeannie Lee, MD, PhD, a physician-investigator and chair of the Department of Molecular Biology at Massachusetts General Hospital (MGH), a founding member of the Mass General Brigham healthcare system. “Basic discoveries like the one we describe in this paper are allowing us to slowly peel away the layers of silencing on the X chromosome, which locks in the suppressed state of X. Knowing what we do now, we have a much better chance of being able to reactivate it.”

Lee and her colleagues have theorized that in cases where genetic disorders are the result of a mutation on the X chromosome, the inactivated X chromosome could serve as a “backup copy,” and that restoring it could potentially reverse the disease state. Perhaps Xist could help release this backup; but if Xist were to diffuse into other chromosomes in the cell, it would inactivate them as well. The researchers set out to understand what keeps Xist from spreading by examining the biophysical and molecular forces in the cell that appear to keep it caged.

In a fruitful collaboration, Lee’s team at Mass General Brigham and Pilong Li’s team at Tsinghua-Peking University found that Xist RNA works with a protein partner that has special biophysical properties. The protein HNRNPK forms a kind of “oil droplet.” Normally HNRNPK forms a stiff droplet, but when it is exposed to Xist RNA, the droplet pulls on Xist and internalizes it. Once internalized, Xist “softens” the HNRNPK droplet and makes it more flexible, allowing the droplet to spread and encapsulate the X-chromosome. Xist in turn becomes entrapped in this droplet, making it harder for the RNA to move to other chromosomes in the cell. Lee uses the analogy of salad dressing to describe this sort of “phase separation.”

“If each of the molecular players is represented by the ingredients in a lemon vinaigrette, HNRNPK is an olive oil, and Xist is a lemon oil that mixes with the HNRNPK oil and is dissolved,” said Lee. “All the other chromosomes form the vinegar. Oil and vinegar do not mix no matter how you shake them. Thus, Xist is phase separated from the other chromosomes.”

The work sets the stage for exploring new approaches to treating certain types of genetic disorders. Co-first authors, Mingrui Ding, PhD, in Pilong Li’s lab (Tsinghua-Peking) and Danni Wang, PhD, in Jeannie Lee’s Lab (MGB), worked together to complete this analysis.

“Our study sheds light on a longstanding question about Xist RNA, and it would not have been possible without our wonderful collaborators in Beijing,” said Lee. “We’re grateful that, by working together, we could solve one piece of this mystery.”

 

Authorship: In addition to Lee, Mass General Brigham authors include Danni Wang, Barry Kesner, Niklas-Benedikt Grimm, Uri Weissbein, Anna Lappala, and Carlos Rivera. From Tsinghua-Peking, additional authors include Mingrui Ding, Hui Chen, Jiying Jiang, Jizhong Lou and Pilong Li.

Disclosures: Lee is a cofounder of Fulcrum Therapeutics, an Advisor to Skyhawk Therapeutics, and a Non-Executive Director of GSK. Li is a cofounder of NuPhase Therapeutics.

Funding: This work was supported by grants from National Science Foundation of China (32125010 and 32330024, 32100547), National Key Research and Development Program of China (2023YFF1204703), and the U.S. National Institutes of Health (R01-HD097665).

Paper cited: Ding M et al. “A biophysical basis for the spreading behavior and limited diffusion of Xist” Cell DOI: 10.1016/j.cell.2024.12.004

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