Here is the Eureka! moment: Mary Lyons proposed a startling hypothesis in 1961: as the dosage compensation pathway has evolved to maintain gene dose equity between the heterogametic sexes, and in mammals the female embryo has twice as many copies of X-linked genes as the male, Could the dosage compensation pathway alter the expression of genes in females to create functional equivalence between males and females in terms of expression of X-linked genes? If this hypothesis is true, it would imply that all mammalian females are functionally mosaic for X-linked gene expression.

Over the years since Mary Lyon's hypothesis, a number of experiments have demonstrated its validity.

One of the most surprising observations was the discovery of the Xist RNA. The Xist RNA is a large non-coding RNA which has been shown to necessary for developmentally regulated chromosomal silencing in females.

The Xist RNA is quite larger (18 kb), is not translated, it is spliced, and polyadenylated. It is also organized into blocks of repetitive sequence. In the following diagram one can see four blocks (A, B, C, D) of repetitive sequence in the Xist RNA. This diagram was generated using a dot matrix program. The murine Xist RNA was compared against itself using a window of 20 bases and whenever 17/20 bases were the same a dot was recorded.

One of the most remarkable qualities exhibited by the Xist RNA is that it can be found to be stably associated with the silenced X chromosome. Forming an Xist body. Ectopic expression of the Xist RNA demonstrates that Xist can form a body with any (and every) chromosome from which it is expressed. In fact, the expression of Xist RNA is always CIS-limited.

The Xist body is always one of the two X-chromosomes in mammalian females, and it is always "silent" in terms of X-linked gene expression.

Xist expression constructs have been designed and constructed. They can be used in stem cells as well as in whole animal experiments to confer CIS-linked silencing.

Deletion analysis has revealed that the A-region of Xist has a very important (but uncharacterized) role in function.
Single deletions of the B, C, D regions do not prevent the binding of the Xist RNA in CIS to a chromosome. However, using PNA interference mapping, or P-IMP, PNA oligomers directed at the C-region can block the association of the Xist RNA with the X-chromosome.

When Xist binding is inhibited by PNA, the Xist RNA is still found within the cell. Despite the presence of normal levels of the RNA, no silencing occurs. Thus the binding of the RNA is essential for its function.