That wasn't the last surprise that awaited Lee and colleagues as they delved into Tsix's actions. Because strands of Tsix and Xist RNA are complementary, if they meet they'll stick together. A reasonable hypothesis is that Tsix RNA prevents X chromosome inactivation by grabbing and disabling the Xist RNA.

Reasonable but wrong, Lee and colleagues concluded in a 2006 Molecular Cell paper. Inside the cell, a chromosome's DNA wraps around spool-like proteins called histones. To shut down a specific gene, cells typically coil the DNA strands tighter, denying access to essential DNA-reading proteins. But to silence the Xist gene in fruit flies, Lee and colleagues found, Tsix appears to do the opposite; it loosens the DNA. Cinching up the DNA, by contrast, turns on Xist. Only a few fruit fly genes are known to operate in this way, she says.

In retrospect, the seemingly backward mechanism makes sense, she adds. To close down an entire X chromosome, a cell might compress all the chromosome's DNA. But if Xist were a conventional gene, that tightening would also shut it down—and once Xist stopped working, other X chromosome genes might start up again. So the unusual method for switching off Xist might permit the gene to remain active even when the rest of the chromosome is silenced, Lee says.

Thanks to her team's report two years ago in Science, researchers have another oddity to ponder that involves Tsix. Lee's group discovered that before one X is inactivated, the two X chromosomes in female mouse cells line up and briefly touch at the X-inactivation center. Such pairing doesn't normally occur once X inactivation is complete, and it requires at least three genes, including Tsix. Without the contact, the cell can't figure out how many X chromosomes it contains or which of them to inactivate, so it might shut down both or neither. During their brief dalliance, the two chromosomes appear to be communicating. What information passes between them “is something we're vigorously pursuing,” Lee says.

Findings by Lee and others also raise concerns about the safety of embryonic stem cells. Researchers have high hopes that these flexible cells, which can specialize into heart cells, liver cells, or any of the body's other cell types, can be directed to repair or replace damaged tissue and organs.

But only if X inactivation proceeds normally. Lee's group assessed the amount of Xist RNA, viewed as an indicator of chromosome shutdown, in 11 stem cell lines being maintained in the lab. The samples included “approved” lines that scientists can study with federal government money and other lines provided by HHMI investigator Douglas A. Melton, co-director of Harvard's Stem Cell Institute, that had been developed without federal funds.

“When we looked at their X inactivation state, they were all over the map,” Lee says.

As her team revealed this March in the Proceedings of the National Academy of Sciences, two of the stem cell lines carried out X inactivation just fine. But in six lines, after one X was inactivated the cells stopped producing Xist RNA. Although the team found no evidence that an entire X chromosome reawakened in these cell lines, it's possible that some—perhaps many—genes on the X could fire up again. The scientists have already found evidence that this happens in mouse stem cells.

Reactivation might just kill the cells, but it could spell trouble for another reason. Some tumor cells carry an extra X chromosome, so it's not unreasonable to wonder whether a partially reactivated X might prompt similar abnormal growth. “It's extremely disconcerting,” says Lee. “There's nothing we can do to restore X inactivation once reactivation occurs.” The findings, she says, indicate that researchers need to do more experiments to determine whether stem cells induce tumors if they are transplanted into patients.

Other stem cell experts praise this work. Although researchers have previously pinpointed X inactivation mishaps in stem cells, “this is the most thorough study” to date, says Renee Reijo Pera, director of the Center for Human Embryonic Stem Cell Research at Stanford University. “It definitely raises a red flag,” though we need more information about X inactivation in the early embryo to judge how serious the problem is, she says.

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