how one of the X chromosomes of female embryonic stem cells is silenced

In most mammals, females have two X chromosomes and males have one X chromosome and one Y chromosome in each of their cells. To avoid a double dose of X-linked genes in females, one of the Xs is silenced early in the developmental process. This silence is essential, but how it happens is relatively mysterious. Two new UM studies reveal more about this silencing process and insights that could improve stem cell research.

Human embryonic stem cells (hESCs) hold tremendous promise for early development research as well as regenerative medicine for diseases ranging from type 1 diabetes to Parkinson’s disease. Yet biologists working with female hESCs in the laboratory often encounter a phenomenon in which the normally inactivated X chromosome loses this deletion when grown in a culture dish.

“If you can’t maintain hESCs exactly as they are in culture, you can’t use them for downstream application,” said Sundeep Kalantry, Ph.D., associate professor of human genetics. He, along with Marissa Cloutier, Ph.D. intern, and their team sought to determine why X-knockout erodes under certain experimental conditions over time.

Their main suspect was the substance used to grow cells in culture, called medium. Cells are grown in media that provide them with chemical instructions called growth factors. These growth factors signal stem cells to keep dividing. One popular medium, called mTeSR1, appeared to be correlated with the loss of a key regulator of X inactivation, a non-coding strand of RNA called XIST. Another medium, called Xenofree, did not cause loss of X inactivation.

“We looked at the differences in the composition of these two media and identified lithium chloride as being present in mTeSR1 but not in Xenofree,” Cloutier said.

Lithium chloride is sometimes included in media to promote stem cell proliferation, however, it is known to interfere with many cell signaling pathways by inhibiting GSK-3 proteins. (GSK-3 protein inhibitors have been used to treat several diseases, and lithium, used to treat bipolar disorder, was one of the first natural GSK-3 inhibitors discovered.)

To confirm that lithium chloride was the culprit, they added the compound to Xenofree medium and found a loss of X inactivation. Their paper is published in Nature Communication.

“Overall in terms of our understanding of X inactivation, our study provides a possible new model for the regulation of this process,” says Cloutier. Kalantry adds that their study suggests researchers need to be a little more careful about using GSK-3 inhibitors like lithium. “They may not only interfere with X inactivation, but also with other modes of epigenetic transcriptional regulation across the genome.”

Role of ancient X-linked gene in X inactivation

A separate article, also published in Nature Communication by Kalantry and colleagues, provides insight into the evolution of X inactivation in mammals. The premise of the study was that knockdown of X is triggered by one or more of a subset of X-linked genes that, paradoxically, escape knockdown of X once knockdown of X started. These genes are expressed from both X chromosomes in females compared to a single X in males and, therefore, are more highly expressed in females than in males. Kalantry’s lab, working closely with the lab of Shigeki Iwase, also at the University of Michigan, discovered that due to the higher expression in females compared to males of such a gene linked to ‘X, Kdm5cit induces Xist and therefore X-inactivation selectively in females.

Deletion Kdm5c on both X chromosomes in a female cell turned off X inactivation almost completely. Conversely, when researchers introduced Kdm5c in male mouse cells, they were able to initiate X-knockout, which under normal circumstances would not occur.

Kdm5c is an ancestral X-linked gene shared by all mammalian species. The authors tested and found that Kdm5c from evolutionarily distant lineages of marsupial and monotreme mammals (e.g., the egg-laying platypus), which split off from placental mammals more than 150 million years ago, could also remarkably induce Exists and X-inactivation in male mouse cells. This finding suggests that Kdm5c retained an ancestral function to induce Xist and X-inactivation in mice and humans.

“If you express X-linked genes at higher than normal levels in a female cell, that cell will often suffer and die. In males, if you reduce the expression of X-linked genes beyond normal levels by inactivating that single X, the cells also suffer and die,” Kalantry said. “The right level of genes on the X chromosome is really, really important for the health of the cell.”

The team then hopes to explain the random process that determines which X in a female cell is silenced: the mother’s or the father’s. Determining this basic mechanism, Kalantry says, could in the future be applied to reactivate X-linked genes at will for potential therapeutic purposes.

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