Epigenetic Control Of Xchromosome Inactivation

XCI is characterized by an ordered series of epigenetic events (Figure 5.2). Both imprinted and random XCI are prefaced by the expression of the X-linked nonprotein coding X (inactive)-specific transcript (Xist) RNA from the prospective Xi (Heard, 2005). Xist transcription in cis is required to initiate silencing along the X chromosome. During imprinted XCI, Xist is expressed as early as the two-cell stage, and the RNA visibly begins to coat the Xp at the four-cell stage, preceding the transcriptional silencing of X-linked genes. The progressive spreading of Xist along the Xp correlates with the gradual silencing of genes on either side of the Xist locus, both of which are largely complete by the blastocyst stage.

FIGURE 5.2 The epigenetics of X-chromosome inactivation (XCI) during preimplantation development. XCI during embryogenesis occurs twice and is presaged by the expression of the Xist RNA. The first wave of XCI results in the preferential inactivation of the paternal X chromosome (Xp), which is referred to as imprinted XCI. At the late two-cell stage, the Xp begins to express the Xist RNA. At the four-cell stage, Xist RNA starts to coat the Xp (Huynh and Lee, 2003; Okamoto et al., 2004). Subsequently, a series of chromatin modifications characterizes the inactive X chromosome (Xi), and this is concomitant with the initiation of the transcriptional silencing of genes along the Xp (Costanzi et al., 2000; de Napoles et al., 2004; Mak et al., 2004; Okamoto et al., 2004; Silva et al., 2003). The Xi becomes hypomethylated at lysine 4 and hypoa-cetylated at lysine 9 of histone H3 at the eight-cell stage, when the silencing of X-linked genes is first detected (Huynh and Lee, 2003; Okamoto et al., 2005) Then, at the morula stage, the following are enriched on the Xi: the Polycomb group proteins EED and EZH2; the histone modification that they mediate, trimethylation of lysine 27 of histone H3 (H3-3 mK27); and the histone variant macro H2A. It should be noted that Polycomb group enrichment on the Xp occurs during the window that XCI initiates, because the spread of gene silencing along the Xp may not be complete until the blastocyst stage (Huynh and Lee, 2003). At the late blastocyst stage, cells of the inner cell mass, which will give rise to the embryo proper, reverse imprinted X inactivation (Mak et al., 2004; Okamoto et al., 2004). Xist RNA coating and the chromatin modifications are lost from the Xp. These cells then undergo random X inactivation, which results in the silencing of either the maternal or the paternal X chromosome shortly after implantation (Plath et al., 2002). Light gray boxes indicate the approximate timeframe of the appearance of proteins or posttranslational histone modifications on the X chromosome. Epigenetic changes indicated in the dark gray box are listed in the order of appearance during random X inactivation. (From Nusinow and Panning, 2005.)

PcGs have also been implicated in the initiation of XCI. PRC2 proteins and H3-3mK27 accumulate on the Xi soon after or coincident with Xist RNA coating during both imprinted and random XCI, which correlates with the spreading of silencing along the Xi (Heard, 2005). Moreover, the misex-pression of Xist results in the concomitant accumulation of PRC2 proteins and H3-3mK27 (Plath et al., 2003; Silva et al., 2003). Furthermore, in the embryo as well as in ESCs and trophoblast stem cells (TSCs), PRC2 and PRC1 proteins—along with the histone modifications they catalyze—are mostly found enriched on the Xi early during differentiation (Heard, 2005). ESCs and TSCs are model cell types for the study of random and imprinted XCI, respectively (Plath et al., 2003; Silva et al., 2003). The temporal pattern of the accumulation of PcGs on the Xi coincides with the gradual silencing of the X-linked genes, and it has led to the suggestion that the PcGs contribute to the initiation and/or establishment of XCI (Plath et al., 2003; Silva et al., 2003).

A role for PcGs in XCI was first suggested by the reactivation of the Xi in embryos mutant for the mouse PcG gene Embryonic ectoderm development (Eed) (Wang et al., 2001). In Eed~'~ mutant females, XCI is initiated but not properly maintained (Kalantry et al., 2006; Wang et al., 2001). Subsequently, Eed~'~ embryos (both male and female) were also shown to harbor defects in the silencing of some imprinted genes (Mager et al., 2003). EED is an essential noncatalytic component of the PRC2 complex; when it is mutated, the entire complex is disabled, and histone methylation catalyzed by the PRC2 complex is lost (Montgomery et al., 2005). The exact biochemical role of EED is unclear, but its WD-40 motifs are thought to mediate protein-protein interactions. Importantly, EED is the only mouse PcG gene described to date to be required for XCI; mouse mutations in other PRC2 components exist, but they have not been analyzed for XCI defects. The absence of EED leads to the severe downregulation of both the core PRC2 components (EZH2 and SUZ12) and the PRC2-mediated histone methylation (Montgomery et al., 2005).

Although EED is preferentially enriched on the Xi early in XCI, it is the differentiated extra-embryonic trophoblast cells that normally do not enrich EED on the Xi in wild-type embryos, which exhibit the reactivation of the previously silenced Xp in Eed~'~ embryos (Kalantry and Magnuson, 2006; Kalantry et al., 2006; Wang et al., 2001). This observation raised the question of how the transient enrichment of Eed on the Xi in extra-embryonic progenitor cells contributes to the stable transcriptional silencing of the Xi in differentiated cells derived from these progenitors.

To resolve this apparent paradox, Eed~'~ TSCs were derived and analyzed for XCI defects (Kalantry et al., 2006). TSCs originate from the extra-embryonic trophectoderm and exhibit the exclusive inactivation of the Xp as a result of imprinted XCI. The Xi in Eed~'~ TSCs and in cells of the trophectoderm-derived extra-embryonic ectoderm in Eed~'~ embryos was found to remain transcriptionally silent, despite lacking the PcG-mediated histone modifications (and all other known Xi factors, including Xist RNA coating) that normally characterize the Xi heterochromatin (Kalantry et al., 2006). Although undifferentiated Eed~'~ TSCs maintained XCI, reactivation of the Xi occurred when these cells were differentiated (Figure 5.3). These results indicate that PcG complexes are not necessary to maintain the transcriptional silencing of the Xi in undifferentiated stem cells.

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