At the outset of this project, it was critical to ensure that PNA oligomers sense or antisense to Xist do not alter the catabolic fate of the RNA in living cells. Thus in this figure we see that steady state RNA levels of Xist is unchanged after administration of PNA to cells.

 

The effect of these PNA oligomers on Xist body formation was then evaluated by FISH. Using a full length DNA probe against Xist, we evaluated female (and male) cells treated with Xist -sense, -antisense and -scramble sequence PNA. Only PNA antisense (1246 &1248) to Xist interfered with the formation of Xist bodies in living cells.
 

The following table shows a complete description of PNA oligomers administered during experiments and the results of these administrations on Xist body formation.
 

The time course of the disappearance of Xist body formation was evaluated after PNA administration. We also measured the effect of antisense Xist PNA oligomers would have on the macrohistone body (MCB).
 

The next question we wanted to ask was: If one interferes with Xist body formation during early embryonic development, what happens to the dosage compensation machinery? The general experimental outline consisted of administration of PNA to murine female embryonic stem cells and then measuring Xist body formation and cis-linked silencing.

In this figure we show a schematic description of the murine X-chromosome and the location of a few key genes and their expression on the active (Xa) or inactive (Xi) X-chromosome.
 

Several predictions were made about the possible results of PNA administrations in female ES cells. In the following diagram the large circle represent an ES cell nucleus, and the small colored spots refer to a particular FISH signal, red is for Xist and yellow stands for either pgk or MeCp2, both X-linked genes.

The top of the figure is early time and the bottom represents either a 3 or 6 day interval. With out PNA treatement it is expected that one Xist signal would grow in size while one pgk or MeCp2 signal would dissapear.

We wanted to test the effect of PNA on silencing X-linked gene expression, so if PNA disruption of the Xist body prevents the development of silencing, then the pattern of expression should be similar to that seen on the right.
 

Undifferentiated (d=0) ES cell. FISH analysis shows Xist in red and MeCp2 in green.
 

Differentiated (d=6) ES cell. FISH analysis shows Xist in red and MeCp2 in green. One Xist signal has be significantly upregulated, and the MeCp2 expression from this chromosome, has been silenced.
 

Undifferentiated (d=0) ES cell. FISH analysis shows Xist in red and Pgk-1 in green.
 

Differentiated (d=6) ES cell. FISH analysis shows Xist in red and Pgk-1 in green. One Xist signal has be significantly upregulated, and the MeCp2 expression from this chromosome, has been silenced.
 

Using this kind of analysis of gene expression we determine the effect of PNA administration on the formation of the Xist body and the silencing of X-linked genes (Pgk-1).
 

Using this kind of analysis of gene expression we determine the effect of PNA administration on the formation of the Xist body and the silencing of X-linked genes (MeCp2).

Using this kind of analysis of gene expression we determine the effect of PNA administration on the formation of the Xist body and the silencing of autosomal genes (actin).
 

We further evaluated the effect of PNA administration on transcription by analyzing the observing the quantitative (by real time PCR) expression for X-linked and autosomal genes before and after differentiation.
 

The samples that were processed for Real Time PCR were also processed for RNA-FISH and the number of Xist bodies determined. The upshot was that PNA did not alter the steady state level of Xist but did disrupt the Xist body formation.
 

Future directions for P-IMP analysis of RNA structures and functions.