Specialized Embryonic Stem Cells Yield Insights into X Chromosome Inactivation

Please don’t be intimidated by the title of this post! By the end of this blog, you’ll be well versed in X chromosome inactivation, and you’ll understand why you should care about this topic.

Males and females are different in countless ways, but the underlying cause of these differences originates with chromosomes. Women have two X chromosomes while men have an X and a Y. The X chromosome is much larger than the Y chromosome, and consequently it harbors a larger number of genes (there are about 1000) with very important functions. Female cells have evolved to inactivate or silence one of their X chromosomes so that both male and female cells receive the same the same “dosage” of X chromosome genes.

Calico Cat.

Calico cats are a result of X-inactivation.

A great example of X-inactivation in nature is a cat with a calico coat. Did you notice that most calico cats are female? This is because there are two different versions of the fur color gene (orange and black) located on different X chromosomes. In calico cats, some patches of fur turn off the X-chromosome with the black gene while others turn off the one with the orange gene. The result is the beautiful and crazy patchwork of orange and black.

The process of X chromosome inactivation is extremely important for many reasons other than feline coat color. Think about that time you ate an extra-large pizza by yourself. That was pushing your limits right? Well imagine if you actually ate two of those pizzas. Your stomach would likely explode, and you would meet an untimely end. Apply this somewhat disturbing analogy to female cells with two active X chromosomes. You can now imagine that having double the dosage of X chromosome genes could be toxic and result in dead or very unhappy cells.

How X-inactivation works
The jury is still out on the full answer to how X-inactivation works; however, some pieces of the puzzle are known.

The major player in X-inactivation is a molecule called Xist. Xist is produced in cells with two X chromosomes, and its job is to inactivate one of these X’s. During X-inactivation, hundreds of Xist molecules swarm and attach to one of the two X chromosomes. Xist then recruits other molecule buddies to join the silencing party. These other molecules are thought to modify the X chromosome in a way that inactivates it.

This theory is where the field is at right now. However, a study published recently in Cell Reports by Dr. Anton Wutz’s group at ETH Zurich found another piece to this puzzle: a new molecule that’s critical to X-inactivation.

New Study Sheds Light on X-inactivation

Specialized haploid embryonic stem cells engineered to produce the X-inactivator Xist upon drug treatment. (Cell Reports)

Specialized haploid embryonic stem cells engineered to produce the X-inactivator Xist upon drug treatment were used to identify genes important to X-inactivation. (Cell Reports, Montfort et al. 2015)

The Wutz lab used a novel and powerful mouse embryonic stem cell (ESC) model that was engineered to have only one of each chromosome, and therefore only one X instead of two. These “haploid” ESCs were also manipulated to produce copious amounts of the X chromosome silencer Xist when treated with a specific drug. Thus, when these haploid ESCs received the drug, Xist was turned on and inactivated the only X chromosome in these cells, causing them to die.

In an example of brilliant science, Wutz and colleagues used this haploid ESC model to conduct a large-scale screen for genes that work with Xist to cause X-inactivation. Wutz and his colleagues identified genes whose loss of function (caused by mutations made in the lab) saved the lives of haploid ESCs treated with the Xist-inducing drug.

In total, the group identified seven genes that they think are important to Xist function. Their most promising candidate was a gene called Spen. When they mutated the Spen gene in their specialized ESC model, the ESCs survived treatment with the Xist-inducing drug. Further studies revealed that Spen directly interacts with Xist and recruits the other molecules that cause X-inactivation.

Big Picture
But why does this research matter? From a scientific standpoint, it highlights the power of embryonic stem cells as a model for understanding fundamental human processes. In terms of human health, it’s important because X-inactivation is actually a defense mechanism against diseases caused by mutations in genes on the X chromosome (X-linked genes).

In women with that have a disease-causing mutation in only one copy of an X-linked gene, X-inactivation of the chromosome with the mutation will prevent that woman from getting the disease. However, sometimes X-inactivation can be incomplete or biased (favoring the inactivation of one X chromosome over the other), both of which could cause activation of X chromosomes with X-linked disease mutations.

These events are hypothesized to be the cause of some cancers (although this hypothesis is still under speculation), mental impairment, and X-linked diseases such as Rett’s syndrome and autoimmune disorders. Therefore, a better understanding of X-inactivation may one day lead to treatments that prevent these diseases.