Calico Cats - How does X chromosome inactivation work?

The differences between males and females, what we call phenotypic sex differences, are rooted in genetics, and their impact on health is vast. To understand how the sex chromosomes affect gene expression in humans, we can look at a visual, striking example found in other mammals: the calico cat.

Calico cats are known for their multicolored fur, featuring orange and black patterns that, at first glance, appear to be random. However, many complex genomic factors are at play.

First, it’s important to know that humans have 46 chromosomes. Most humans typically have the same 45 chromosomes, but they differ in their 46th chromosome. Biological females typically have two X chromosomes (XX), whereas biological males usually have an X and a Y chromosome (XY). This difference—the number and type of sex chromosomes a person has—provides the building blocks for our understanding of sex differences.

What can be commonly misunderstood is the role of the inactivated X chromosome. In cells with two X chromosomes, the body must balance the dose of X-linked genes on the X chromosome to maintain similar expression to XY individuals, since they have one less copy of the X. To do this, one X chromosome is randomly chosen to be active (Xa), and the other becomes largely inactive (Xi). This occurs early in development, which causes “patches” of cells to have the same X chromosome inactivated, leading to the colored pattern on calico cats. 

A patch of orange fur marks cells in which the X chromosome carrying the orange X was selected as the active one, while a patch of black fur marks cells in which the X chromosome carrying the black X remained active. 

As these early cells divide, they form clusters that expand into the characteristic mosaic coat of the calico cat.

Here, we see that depending on which X chromosome is activated, either orange or black fur will grow. Meaning their multicolored fur is directly connected to its RNA!

Typically, male cats are either black or orange because of their XY genetic makeup. Whereas female calico cats could have any mixture of patterns, depending on their inactive X. There is only one X chromosome for fur color, and none on the Y. Y chromosomes also have fewer genes than X, hence the rarity of calico-colored male cats.

In cells with 2 X chromosomes, one of the X chromosomes becomes inactive. First, randomly, one of the X chromosomes makes an RNA molecule, called Xist. Xist then covers the chromosome it was made off of and then promotes the accumulation of epigenetic modifications. These modifications “silence” the inactive X. This means that the active X is able to make RNAs that will make proteins for the cell; the inactive X largely makes no RNA. Shockingly, however, there are some genes that “escape” X-inactivation, and these genes are expressed on both the active and inactive X chromosomes.

Something Scientists are still not sure about is how exactly the body knows how to differentiate the inactive X from the active X. 

X-inactivation happens in individuals with 2 or more X chromosomes. Typically, this means females, who have two X chromosomes, undergo inactivation of one of the Xs, while males do not, since they only have one X chromosome and one Y chromosome. In rare cases, you can get a male calico cat. In this case, a male cat would have to have at least 2 chromosomes. Depending on which X is inactive, it changes how the genes are expressed, like in female calico cats!

While humans look different, they share these chromosomal-level changes with Calico cats. 1 in 2000-2500 women have Turner Syndrome, a condition only found in women missing or partially missing an X chromosome. As in male cats, human males do not have this condition because they typically have 1 X and Y.

Because X-inactivation requires two X chromosomes, almost all calico cats are female (XX). On rare occasions, a male cat can be calico if he has XXY chromosomes, which is a condition similar to Klinefelter syndrome in humans. In these rare cases, X-inactivation still occurs because the cat has more than one X chromosome, allowing one X to become inactive in the same way it does in XX females.

Although the inactive X is often described as “silenced,” it is not completely turned off. Around 20% of its genes escape inactivation and remain expressed. These genes play important roles in regulating gene expression across the rest of the genome, influencing development, physiology, and disease risk.

The San Roman Lab is committed to pioneering discoveries that advance our understanding of the genetic and molecular basis of sex differences in human health. One focus of the lab’s research is understanding how the genes that escape inactivation on the Xi help shape genome-wide gene regulation and contribute to sex differences in human health and disease.