Initiation of X inactivation in female embryos depends on the transcription of the long noncoding RNA XIST/Xist (X-inactive specific transcript) from one chromosome (which will become the inactive X (Xi)) and recruitment of a protein complex important for X-chromosome silencing and heterochromatin formation [7,8]. In humans, XIST (17 kb in size) is located in the long arm of the X chromosome, whereas in mice where there is only one arm, Xist (15 kb in size) is in the middle of the chromosome. Xist RNA spreads along the X chromosome in cis and recruits a protein complex responsible for deposition of repressive histone modifications onto the Xi [9-11]. As a result the Xi becomes heterochromatic, silent and condensed. Before implantation, X inactivation is imprinted, with the paternal X chromosome always being silenced. At the blastocyst stage, the paternal X reactivates and random X inactivation takes place (see Table ​Table11).

Although most genes on the Xi are silenced, some genes remain expressed from both the Xa and the Xi. Not surprisingly, genes that retain a Y-linked copy - for example, Kdm5c and Kdm5d (which encode histone demethylases) - escape X inactivation and thus have two expressed alleles in both male and female somatic tissues. However, not all 'escaping' genes have a Y copy, for example Car5b (carbonic anhydrase). Recent reports have shown striking differences between human and mouse regarding the identity and number of these 'escape' genes in somatic tissues [12,13]. Why are there such species differences? Structural differences between the X chromosomes may play a role as well as selective pressure to maintain sex differences.

Escape from X inactivation in other mammalian species has not been extensively characterized. Nonetheless, escape genes have been identified in marsupials, which differ from eutherian mammals in terms of key features of X inactivation - Xist is absent and the paternal X always silenced. At least four X-linked genes encoding glucose-6-phosphate dehydrogenase (G6PD), hypoxanthine guanine phosphoribosyl transferase (HPRT), phosphoglycerate kinase (PGK1), and a monocarboxylic acid transporter (SLC16A2) show incomplete silencing in a tissue- and species-dependent manner in marsupial females [16,17].

Significant differences exist in terms of the distribution of escape genes in human and mouse. In humans, most escape genes are located on the X short arm. One reason for this could be because the short arm has most recently diverged from the Y, and so these genes have only recently (in evolutionary terms) lost their Y paralogs [5,6,12]. Alternatively, the centromeric heterochromatin might exert a barrier effect that would prevent sufficient spreading of XIST RNA, which is generated from the X-inactivation center located in the long arm [18]. In contrast, escape genes are randomly distributed along the mouse X chromosome, which has its centromere located at one end [13]. In humans, escape genes are clustered (as many as 13 adjacent genes in large domains ranging in size between approximately 100 kb and 7 Mb), whereas in mouse, single genes are embedded in regions of silenced chromatin (Figure ​(Figure2a).2a). This suggests that escape from X inactivation in mouse is controlled at the level of individual genes rather than chromatin domains [12,13,19].

The fact that few escape genes exist in the mouse is consistent with the significant differences in the impact of X-chromosome monosomy in female mice and in women [13]. Genes that escape from X inactivation in humans but are subject to X inactivation in the mouse may be good candidates for genes responsible for Turner syndrome severe phenotypes. Pseudoautosomal genes may play a prominent role in these phenotypes, as already demonstrated for SHOX, and possibly for CSF2RA. Indeed, the mouse pseudoautosomal region contains only one gene, Sts (steroid sulfatase) [30], whereas all genes located in the pseudoautosomal region in humans are autosomal in the mouse and thus are not affected in X0 mice [31].

While Xist RNA coating is important in the initiation of X inactivation, many other epigenetic modifications follow to silence the X and maintain silencing. An early repressive chromatin mark, tri-methylation of lysine 27 on histone H3 (H3K27me3), is recruited by the Polycomb complex of chromatin-modifying proteins, resulting in compaction of the silenced portion of the Xi (Figure ​(Figure2a).2a). Other repressive marks include H3K9me3 and the histone variant macroH2A1, which are also enriched on the Xi (Figure ​(Figure2b)2b) [7,39]. Concomitantly, 'active' marks such as acetylation of histone H3 and H4 are lost from the silenced chromatin [7,40]. Modifications characteristic of silenced genes contrast with those within escape genes, which remain euchromatic and harbor histone H3 and H4 acetylation [7,41]. H3K4me3, another mark associated with transcriptional activity, is absent from most of the Xi except at discrete regions corresponding to areas of escape, as shown in female lymphoblasts [42] (Figure ​(Figure2b).2b). We recently demonstrated a lack of H3K27me3 at escape genes in mouse, which shows complete concordance in the cell line used to assay allelic expression [13].

The existence of discrete areas of 'escape chromatin' adjacent to silenced chromatin suggests the need for boundary elements, such as insulator sequences, that may block the spreading of heterochromatin into escape regions or prevent repressive marks from being added to escape domains (Figure ​(Figure2).2). Supporting this idea are our findings that the insulator protein CTCF (CCCTC-binding factor), which binds known insulator sequences, binds to the transition region between the escape gene Kdm5c and the inactivated gene Iqsec2 (IQ motif and SEC7 domain-containing protein 2) in mouse, whereas in humans, the corresponding region between the same genes, which both escape X inactivation, does not bind CTCF [43]. Furthermore, we have found that the CpG island at the 5' end of Kdm5c remains hypomethylated throughout mouse development, possibly because it is rendered inaccessible to DNA methyltransferases by CTCF binding (Figure ​(Figure2b).2b). CTCF-binding sites were also identified in other transition areas between escape and inactivated genes, suggesting that CTCF may play a role in the insulation of escape domains [43]. However, a subsequent study showed that insertion of CTCF-binding sites from the HS4 insulator site (from the chicken β-globin gene cluster) at each end of a short reporter gene was not sufficient to protect it from silencing when inserted within an inactivated gene on the Xi in mouse cells [44]. A more recent study reported that a bacterial artificial chromosome clone containing Kdm5c and its flanking regions retains its properties of escape even when inserted at other sites that are normally inactivated on the Xi in mouse cells [45]. CTCF-binding sites may turn out not to be sufficient for insulation, and other elements within or around escape genes may be important.

In particular, the structure of chromatin may have an important role in insulation by looping specific regions out of the condensed Xi (Figure ​(Figure2a)2a) [46]. Our recent X-chromatin profiles show a discontinuous distribution of the repressive chromatin mark H3K27me3 along the Xi, consistent with the presence of insulator elements and/or specific attachment sites for looped chromatin [13]. However, in human × mouse hybrid cell lines, where the human X can be distinguished from the rodent background, repressive chromatin marks were found to be progressively diminished in the intergenic region between the inactivated RBM10 (RNA-binding motif protein 10) and the escape gene UBA1/UBE1 (ubiquitin-like modifier activating enzyme). Specifically, H3K9me3 and another histone modification associated with gene silencing, H4K20me3, were enriched in the last RBM10 exon but were already depleted approximately 2 kb upstream of UBA1/UBE1 [41].