Random XInactivation

Female ES cells do not carry an imprint that determines which X chromosome will be active and which will be silenced. However, it seems likely that some type of epigenetic mark is used to distinguish the future active X from the future inactive X in embryonic cells which are about to initiate X-inacti-vation. While no differences between the two Xa's in female ES cells have been reported, both the X chromosomes share a number of unusual chromatin features that may be important for X-inactivation to occur upon differentiation. A hotspot of H3-2 mK9 can be detected on the single X chromosome in male ES cells and both X chromosomes in female ES cells. It is approximately

100 kb in length and centered 55 kb upstream of the Xist promoter (Heard et al. 2001). When differentiation is induced, H3-K9 methylation appears to spread from this site, closely following Xist RNA coating of Xi (Heard et al. 2001), suggesting that this hotspot may serve as a nucleation center for the spread of H3-2mK9 during differentiation. These data suggest that this hotspot is an unusual regulatory element that directs alterations in chromatin structure in cis. Several key mechanistic questions about the hotspot remain: How is it established? Does Xist RNA play a role in its establishment? And how is the spread of H3-2 mK9 blocked on the active X chromosome?

There is an intriguing possibility that ES cells can sense whether they have one or two X chromosomes and that this information is used to direct the pattern of chromatin modifications on the X chromosomes. ChIP experiments suggest that each X chromosome in female ES cells is distinguished from the single X chromosome in male ES cells by an increase in H3-K4 methylation and a decrease in H3-K9 methylation (O'Neill et al. 2003). In addition, each X chromosome in female cells displays an increase in acetylation on all four core histones relative to the single X chromosome in males (O'Neill et al. 2003). These differences in histone methylation and acetylation may act to designate the cell as female so that X-inactivation can occur upon differentiation.

A recent study indicates that H3-2 mK4 is a modification that precedes monoallelic expression of X-linked genes. H3-2 mK4 is enriched at the promoters, but not the transcribed region, of X-linked genes in male and female ES cells (Rougeulle et al. 2003). In contrast, most biallelically expressed genes exhibit a more uniform distribution of H3-2 mK4 at the promoter and in the coding sequences. Interestingly, several imprinted genes, which, like X-linked genes, show monoallelic expression in somatic cells, are also enriched for H3-2 mK4 at their promoters in ES cells. In addition, Smcx, an X-linked gene that escapes X-inactivation, does not show promoter-specific H3-K4 methylation in ES cells. These results indicate that the enrichment of H3-2 mK4 exclusively at the promoter in ES cells may mark genes for monoallelic expression upon differentiation. H3-K4 methylation is generally associated with transcriptional activity in differentiated cells (Strahl et al. 1999), suggesting that this modification may have different roles before and after differentiation of pluripotent stem cells.

The Xist gene also shows monoallelic expression in differentiated cells, but does not exhibit promoter-specific enrichment of H3-2mK4 in ES cells. Instead, Xist is enriched for H3-2 mK4 in both its promoter and transcribed sequences (Rougeulle et al. 2003; Morey et al. 2004). Therefore the Xist gene is unusual among the genes that are monoallelically expressed in differentiated cells in that its pattern of H3-2 mK4 enrichment in ES cells is more similar to that exhibited by biallelically expressed genes. In ES cells the Xist locus is transcribed in both the sense and antisense orientations, generating Xist and Tsix transcripts (Lee and Lu 1999; Mise et al. 1999). These two non-coding RNAs are simultaneously expressed at low levels from both Xa's. Upon differ entiation, Xist RNA coats and silences the Xi, and Tsix expression is extinguished on this chromosome (Lee and Lu 1999). Tsix and Xist continue to be expressed from the Xa in a brief window after X-inactivation is initiated, and Tsix is thought to block Xist-mediated silencing of the Xa during this period (Huynh and Lee 2001; Lee and Lu 1999). Tsix is implicated in regulating the H3-2 mK4 distribution at the Xist locus in undifferentiated ES cells, as the Xist gene body enrichment of H3-2 mK4 in ES cells requires the Tsix promoter region (Morey et al. 2004). Therefore, this non-coding RNA may regulate chromatin structure at the Xist locus in ES cells. In yeast, elevated levels of H3-2mK4 result in part from the association of the Set1 H3 HMTase with elongating RNA polymerase (Krogan et al. 2003; Ng et al. 2003), raising the possibility that the Tsix promoter may direct assembly of a transcription complex containing a mammalian H3-K4 HMTase.

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