Protein may play role in sex chromosome inactivation

Researchers studying the X-inactivation process have now identified a molecule, called CTCF, which appears to be central to regulating X-chromosome inactivation. The research team, which was led by Jeannie T. Lee, a Howard Hughes Medical Institute investigator at Massachusetts General Hospital, published its findings in the December 7, 2001, issue of Science.

Researchers had long known that Xist, a gene on the X chromosome, triggers the inactivation of a single X chromosome by generating RNA that �paints� the entire chromosome and renders it inoperable. In previous studies, Lee and her colleagues had identified an antagonizing gene, dubbed Tsix, that produces an �antisense� RNA whose complementary structure might cause it to adhere to the Xist gene and block its expression. Blocking Xistprevents inactivation of the X chromosome. Both Tsixand Xistare situated on the X chromosome in a region known as the X-inactivation center.

Knowing about the two genes, however, was not sufficient to explain how inactivation is triggered, said Lee. Experiments by Lee and her colleagues on X-inactivation suggested that there is likely to be a mark at one end of the Tsixgene to which an unknown external protein would bind and work with Tsixto cause the X chromosome to remain activated. Their earlier experiments revealed a candidate region of the genome adjacent to Tsixwhere the protein would likely bind; and Lee and her colleagues reasoned that the genetic sequence of this critical region must be highly conserved throughout all mammals.

�We then took a computational strategy, in which we compared the human and mouse genome sequences in this region and asked what elements are conserved,� said Lee. Another important piece of information came from the work of other researchers who showed that the transcription factor CTCF appeared to be a regulator of imprinted genes that are selectively switched off during development. �CTCF sites are known to exist in a number of other genes, in human and mouse genomes,� said Lee. �And when we simply asked whether the conserved regions we discovered matched those CTCF binding sites, we found that they did.�

In additional studies, Lee and her colleagues demonstrated that the CTCF protein did, indeed, bind to the sites they had discovered in the X-inactivation center. While the researchers have not yet determined the precise nature of CTCF�s interaction with Tsix, their experiments hint that it serves as an activator of the gene. Alternately, said Lee, CTCF binding sites might act as an �insulator,� blocking an unidentified enhancer from attaching to the Xistgene, preventing it from inactivating the chromosome. According to Lee, discovery of CTCF�s involvement opens the way to understanding how the developing embryo chooses which X chromosome to inactivate.

�The next step is to figure out how this CTCF protein is binding to one X chromosome allele and not the other,� she said. �In other words, what�s recruiting CTCF to the anti-sense Tsixgene?� Another central mystery, said Lee, is what the control signals are that interact with the CTCF protein to make it so precisely choose to bind at the X-inactivation center.

�The problem is that the CTCF protein is ubiquitous,� said Lee. �It�s expressed early in development and late in development; it�s present in males and in females. So, how does CTCF know to go only onto one X chromosome? And how does it know to only attach at the onset of X-inactivation and not before or after? We need to understand how all these developmental decisions are made that are specific to the Tsixgene and specific to the future active X chromosome. While we currently have no idea what those factors are, now that we�ve identified one protein, CTCF, we believe it�s only a matter of time before we or our colleagues identify additional proteins in this decision mechanism,� she said.