Reactivating Silenced X Chromosome Could Eventually Lead to Targeted Therapies, Researchers Say

Genes that are normally silenced in the X-chromosome transition from an “off’ to an “on” state at different speeds, an event that is dependent on the action of certain proteins and enzymes, according to a recent study.

These findings may one day help design a targeted therapeutic strategy for Rett syndrome and other X-chromosome-linked genetic disorders.

The study, “Dynamic reversal of random X-Chromosome inactivation during iPSC reprogramming,” was published in the journal Genome Research.

Rett syndrome is a rare genetic disorder that affects girls almost exclusively and is characterized by developmental and intellectual disabilities. The condition is caused by mutations in the MECP2 gene, located on the X chromosome, that provides instructions to make a protein called MeCP2. This protein is responsible for maintaining synapses, which are the junctions between nerve cells that allow them to communicate.

Women carry two X chromosomes — one from the mother and one from the father — while men carry only one, inherited from the mother. To ensure that all genes in the X chromosome are expressed equally in men and women, a process known as X-chromosome inactivation takes place during embryonic development, at which time one of the two X chromosomes carried by females is randomly selected to be inactivated.

A potential way to treat Rett syndrome and other X-chromosome-linked disorders is to devise a strategy to reactivate the healthy copy of the gene on the inactive X-chromosome. This reactivation would occur at the early stages of embryonic growth.

In the study, researchers at KU Leuven–University of Leuven and colleagues used a type of stem cell called induced pluripotent stem cells (iPSCs). These cells are usually derived from adult skin cells (fibroblasts) and behave similarly to embryonic cells, meaning they can give rise to nearly any type of cell in the body.

The team used adult fibroblasts from female mice and reprogrammed them back into iPSCs. Of note, both X chromosomes are active in IPSCs, making these cells a valuable tool to study the mechanisms behind chromosome reactivation.

“Working with iPS cells has numerous advantages. Most importantly, when you reprogram female adult cells into iPS cells, both X chromosomes become active again. In other words: X-chromosome reactivation starts happening right under your microscope,” Vincent Pasque, assistant professor at KU Leuven–University of Leuven and the study’s lead author, said in a news release.

“We monitored almost 200 different X-linked genes throughout the X-chromosome reactivation process. What we found is that reactivation happens gradually: different genes require different amounts of time to become active again,” said Irene Talon, one of the study’s authors.

The researchers found that some genes reactivated “early” (as early as day 8 after reprogramming), while others took longer. “Intermediate” genes were reactivated between day 8 and 10, and others between day 10 and 13 (called “late” genes), while some took longer than 13 days (which were called “very late” genes).

The reason for the different pace in reactivation, the researchers found, was that the “early” genes were located closer to genes that escape X-chromosome silencing.

Additionally, these early genes were richer in sites for the binding of transcription factors required for the transition of cells into iPSCs. Of note, transcription factors are proteins that help turn specific genes “on” or “off” by binding to nearby DNA.

Specifically, the researchers found that the reactivation timing was dependent on histone deacetylases, which are enzymes that label DNA to be silenced and work like barriers to gene activation.

“Our findings suggest that the explanation for this speed difference is a combination of the location of the gene in 3D space on the X chromosome and the role of proteins (transcription factors), and enzymes (histone deacetylases), in particular,” Talon added.

X-chromosome reactivation was accompanied by a decrease in the levels of an RNA molecule, called Xist, which is known to be involved in the silencing of the X-chromosome.

Overall, these findings suggest that reactivation of X-linked genes requires the combined action of different players — information that is key for the development of possible future therapies for Rett syndrome.

“It’s important to remember that we’re talking about very fundamental research here. Contributing to the development of a cure for Rett syndrome and similar disorders is our long-term goal, but it will take us a while to get there, and there are many hurdles to overcome,” Pasque said.

“We still need to figure out how to use the mechanism for a single gene, how to do it safely in patients, and how to target the right cells in the brain. We do not yet know how to overcome these formidable challenges but we do know that gaining a fundamental understanding of how things work is the crucial first step,” he added.

“That’s how science works: it’s a slow process.”