Lessons from comparative analysis of X-chromosome inactivation in mammals
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In most mammals, X-chromosome inactivation is used as the strategy to achieve dosage compensation between XX females and XY males. This process is developmentally regulated, resulting in the differential treatment of the two X chromosomes in the same nucleus and mitotic heritability of the silent state. A lack of dosage compensation in an XX embryo is believed to result in early lethality, at least in eutherians. Given its fundamental importance, X-chromosome inactivation would be predicted to be a highly conserved process in mammals. However, recent studies have revealed major mechanistic differences in X inactivation between eutherians and marsupials, suggesting that the evolution of the X chromosome as well as developmental differences between mammals have led to diverse evolutionary strategies for dosage compensation.
KeywordsX inactivation dosage compensation Eutheria marsupial evolution
Inner cell mass
Meiotic sex chromosome inactivation
The difference in X-chromosome number between heterogametic and homogametic individuals requires some form of dosage compensation to ensure equal levels of X-linked gene activity. In placental mammals (marsupials and eutherians) this is achieved through X-chromosome inactivation: the developmentally regulated transcriptional silencing of one of the two X chromosomes in females. Since its proposal by Mary Lyon (1961), X inactivation has remained an outstanding example of chromosome-wide epigenetic regulation. However, its function and the exact mechanisms are still poorly understood. Furthermore, recent studies have shown that the mechanisms underlying X inactivation, as well as the developmental dynamics of the process, vary considerably between mammals. The choice of which X chromosome is inactivated is usually random in eutherians, and the inactive state is then stably inherited, giving rise to adults that are mosaics for two cell types expressing one or the other X chromosome. However in marsupials and during early embryogenesis of some eutherians such as rodents, only the paternal X chromosome is inactivated. In mice, this imprinted form of X inactivation is subsequently reversed in cells of the embryo-proper and random inactivation of either the paternally or maternally derived X chromosome occurs. Although these two examples of imprinted XCI appear superficially similar, in fact they seem to be controlled differently. X inactivation in eutherians is controlled by a complex locus, the X-inactivation centre (Xic), which produces the non-coding Xist RNA responsible for triggering silencing in cis. Xist appears to be a eutherian invention however, having evolved after the divergence between eutherians and marsupials. In rodents, random X inactivation relies on random, monoallelic Xist up-regulation, while imprinted X inactivation appears to rely on imprinted paternal-specific Xist expression. Marsupials, on the other hand, may exploit meiotic sex chromosome inactivation (MSCI), which occurs in the male germ line (Namekawa et al. 2007, see review by Turner 2007) as a means of faciliating the silencing of the paternally-inherited X chromosome following fertilization. Thus marsupials and eutherians may have evolved quite different strategies of X inactivation to achieve dosage compensation in the 130 million years since they diverged. Such differences in X-inactivation strategies would be consistent with the major differences also found between these mammals in their sex determination and genomic imprinting pathways (Graves 2006). The nature of sex chromosome dosage compensation in monotremes, which diverged from eutherians and marsupials 170 million years ago, remains even more enigmatic, and may not require X inactivation.
In this review we will focus on recent advances in our understanding of the developmental regulation of X inactivation, the role of the Xic locus and the epigenetic marks associated with the inactive X chromosome, paying particular attention to what is known in different species.
The X-inactivation centre and the Xist gene
Evolutionary conservation of the Xic
In eutherians, it is tempting to speculate that the evolution of Xist was the basis for the evolution of random X inactivation. It is therefore interest to consider the conservation of some of the known regulatory elements of Xist, such as Tsix. In fact, although there are some sequence homologies to Tsix present in the human XIC, this region is actually rather poorly conserved (Chureau et al. 2002; Duret et al. 2006). Furthermore, antisense transcription does not cover the whole XIST gene and in particular it does not extend up to the XIST promoter (Migeon et al. 2001), unlike the situation in the mouse. This raises the interesting possibility that in humans, and presumably also in other eutherians, XIST is in fact regulated in a TSIX-independent fashion and that Tsix may have evolved for specific regulatory requirements of Xist during mouse development. Other potential Xic regulatory sequences include the recently discovered Xpr locus(Augui et al. 2007). Although the exact sequence elements within this large region have still to be defined, it contains several highly conserved sequences suggesting that this locus may be important for Xic function across species. Thus, in eutherians the appearance of the Xist gene through the pseudogenisation of Lnx3, may have been the first event in the evolution of this locus as “Xic”, or master regulator of X inactivation. This may have occurred in the context of a regulatory landscape including sensing elements such as Xpr and other as yet undiscovered sequences. Subsequently, further control elements (such as Tsix) may have evolved in some eutherians to enable the fine tuning and appropriate developmental regulation of Xist Indeed, little is known about the developmental regulation of the initiation of XCI in mammals other than the mouse.
Thus, contrary to the idea that a single X-inactivation process evolved from a common mammalian ancestor, the emerging picture is that multiple strategies have evolved to ensure dosage compensation in different mammals and that even within eutherians, the Xic is rather an evolutionarily labile locus.
Keeping the silence: epigenetic marks underlying the inactive X chromosome
What is known about epigenetic marks on the inactive X chromosome and their actual role in maintaining the inactive state? Our knowledge in eutherians is largely based on studies in mice, where Xist RNA coating is the first event in a cascade of changes on the X chromosome during development. The detailed kinetics of these changes during ES cell differentiation and early mouse development are outlined in Figs. 2 and 3. One of the earliest events to occur following Xist RNA coating is the exclusion of transcription factors from the Xist-coated core of the X chromosome (Chaumeil et al. 2006). Subsequently, the induction of chromatin modification, such as H3K27 tri-methylation, via Ezh2 of PRC2 complex (Plath et al. 2003; Silva et al. 2003), H2A K119 ubiquitination, via Ring1a/b of the PRC1 complex (de Napoles et al. 2005) and H4K20 mono-methylation, via Prset7 (Oda et al. 2009), as well as the incorporation of the histone variant macro-H2A take place on the inactive X chromosome.
The recruitment of both PRC1 and PRC2 complexes to the Xi is at least partly dependent on Xist (Plath et al. 2003; Kohlmaier et al. 2004; Schoeftner et al. 2006). However, this is independent of Xist-mediated silencing. PRC1 complex recruitment may also involve binding, via its chromodomain, to the H3K27me3 mark induced by Ezh2 of PRC2 (Bernstein et al. 2006). Although Polycomb group proteins are known to be generally involved in altering chromatin structure and perpetuating repressed states during development, their exact role in XCI still remains unclear. Certain studies suggest that the PRC2 complex might be important for the establishment of a chromosomal memory induced by Xist RNA, but may not participate in the memory itself (Kohlmaier et al. 2004; Schoeftner et al. 2006). It is nevertheless clear, that a lack of PRC2 activity (and associated H3K27me3) results in some degree of reactivation of X-linked genes, particularly in extra-embryonic tissues (Wang et al. 2001; Silva et al. 2003) as they differentiate (Kalantry et al. 2006). In the embryonic lineage, the inactive state seems to be less perturbed by a lack of PRC1 and PRC2 (Eed) (Wang et al. 2001; Schoeftner et al. 2006). A logical explanation for this could be that the lack of promoter DNA methylation in extraembryonic lineages renders them more susceptible to perturbations in Polycomb-related epigenetic marks.
In the embryonic lineage of eutherians, DNA methylation eventually becomes recruited to the promoters of genes on the inactive X chromosome. As this is a relatively late event during the onset of X inactivation, it does not seem to be an immediate consequence of gene silencing. DNA methylation is clearly important for the stable maintenance of gene silencing on the Xi in embryonic tissues, as Dnmt1-/- mutant embryos display increased reactivation rates of X-linked genes (Sado et al. 2000). Recently, some new proteins that appear to be involved in maintenance of XCI have been identified. One of these, appears to act upstream of DNA methylation as SmcHD1 mutants show a loss of DNA methylation at the promoters of some X-linked genes. The relatively late lethality of Scmchd1 female embryos suggests a defect in maintenance rather than initiation of XCI. Intriguingly, SmcHD1 contains a structural maintenance of chromosome hinge domain (Blewitt et al. 2008), suggesting parallels with cohesin-like proteins in X chromosome dosage compensation in C. elegans (Chuang et al. 1994). Another new factor possibly involved in propagating or maintaining the inactive state is the alpha thalassemia/mental retardation X-linked (ATRX) protein, which is a chromatin remodeling protein belonging to the SWI/SNF2 ATP-dependent helicase family (Baumann and De La Fuente 2009). ATRX binds to the Xi during late differentiation and it appears to affect maintenance of imprinted XCI although its exact role in XCI remains to be found.
In addition to epigenetic marks at the chromatin level, spatial segregation (nuclear compartmentalization) of the inactive X chromosome, as well as its temporal segregation (asynchronous replication timing), may be two rather universal features of the inactive X chromosome and both may be involved in the stable maintenance of the inactive state and its cellular memory. Late (or asynchronous) replication timing correlates well with the presence of an inactive X in both eutherians and marsupials (Sharman 1971). It has recently been demonstrated that the inactive X (Xi) is targeted transiently to the nucleolus in an Xist-dependent manner during mid-to-late S phase (Zhang et al. 2007). Thus spatial and temporal segeregation of the inactive X chromosome, together with various types of chromatin modifications could provide multiple alternative layers of stability, depending on species, lineage or developmental contexts.
In marsupials, little is known about the epigenetic marks that are associated with the inactive X chromosome other than that are particularly late replication timing, global histone hypoacetylation and loss of active histone modification marks (H3K9 acetylation and H3K4 dimethylation) (Cooper et al. 1993; Wakefield et al. 1997; Koina et al. 2009) as well as X-linked promoters appear to be unmethylated in marsupials. This may be one reason why much higher frequencies of sporadic reactivation in these mammals compared to eutherians (for review, see Heard et al. 1997). The lack of Xist RNA and the chromosomal modifications that it can induce might also be a cause of this lability. However, clearly there must be some mechanisms- whether via an imprint or another non-coding RNA — that allow for overall heritable gene silencing in marsupials.
X inactivation during early embryogenesis
An important issue concerns potential differences between mammals in the exact timing and mechanism of X-inactivation during early embryogenesis. Recent studies have revealed that X-linked gene silencing happens in a far more heterogeneous manner than originally thought, with some genes being silenced rapidly and others being silenced much more slowly during pre-implantation mouse development (Patrat et al. 2009) and during ES cell differentiation (Lin et al. 2007). This suggests regional differences across the X chromosome in the onset of XCI. Several genes also show a certain degree of escape from XCI. This was believed to be an intrinsic property of some X-linked loci although it seems to be lineage dependent in some cases.
Although random X inactivation is the norm in the embryonic lineages of eutherians, X inactivation is imprinted in pre-implantation embryos and extraembryonic tissues of mice (Takagi & Sasaki 1975; Okamoto et al. 2005; Patrat et al. 2009,) (Fig. 4). However, the paternal X chromosome does not undergo imprinted inactivation in human embryos and extra embryonic tissues (Daniels et al. 1997; Ray et al. 1997; Zeng and Yankowitz 2003). In bovine embryos, some studies suggest that Xist expression is imprinted and that Xp inactivation may be found in placenta (Fig. 4) (Xue et al. 2002), although definitive studies have not been performed. In marsupials, where XCI is always imprinted and where there is no Xist gene, it has been proposed that this may be a carry-over effect on the Xp chromosome of meitotic sex chromosome inactivation (MSCI) (Namekawa et al. 2007). Indeed a popular idea has been that meiotically induced inactivity of the Xp could somehow remain associated with the Xp after fertilization and that this might have been selected for in ancestral mammals thanks to the dosage compensation it could confer in an XX embryo (Monk and MacLaren 1981; Huynh and Lee 2003). However, there is no evidence for this to date in marsupials. Indeed, the degree to which chromatin marks on the paternal genome can be transmitted to the fertilized egg without being lost during the remodeling step at the end of spermatogenesis, whereby protamine replace histones (Fig. 4) remains obscure. In rodents it seems that imprinted XCI is Xist-dependent (Marahrens et al. 1997) and is largely regulated via Xist imprinting (Tada et al. 2000; Okamoto et al. 2005). The situation in other eutherians remains to be discovered.
In mice, following the initiation of imprinted XCI in early cleavage stages, the Xp is maintained in the inactive state in the trophectoderm of blastocysts but is reactivated in the inner cell mass (ICM) (Mak et al. 2004; Okamoto et al. 2004). The mechanism of reactivation is not clear but in Nanog-expressing cells, Xist appears to be down-regulated, chromatin marks such as H3K27me3 are reversed and X- linked genes are reactivated in mice (Fig. 3). Whether such reactivation of the inactive X occurs in the ICM of other eutherian mammals remains to be found. Indeed, it is not clear at what stage X inactivation initiates during early development in other mammals. In marsupials, the Xp is also believed to be silenced by the blastocyst stage (Johnston and Robinson 1987). However there is no ICM in marsupial blastocysts suggesting that there may not be a reactivation step in these mammals and that this could account for the maintenance of imprinted inactive Xp in all tissues of marsupials.
In conclusion, given the diversity of situations for imprinted X inactivation found between marsupials, mice, cows and humans, it is now evident that it will be critical to examine the Xp activity status during early embryonic develeopment; In marsupials this will require an analysis of X-linked gene activity and X-chromosome chromatin changes during early embryogenesis and particularly at the time of zygotic genome activation, in order to assess whether MSCI may underlie XCI in these mammals. Studies of Xist expression patterns and X-linked gene expression in other eutherians should also help us to understand whether Xist-dependent X inactivation initially evolved in eutherians in a random or imprinted form and also how imprinted X inactivation evolved in mammals.
In this review we have discussed early developmental aspects of X-chromosome inactivation, putting particular emphasis on the evolutionary diversity that recent findings have uncovered in this process. The discovery that in marsupials, X inactivation must be initiated in an Xist-independent manner is a clear illustration that X inactivation can be triggered in very different ways in different mammals. It has been suggested that in marsupials there is a link between meiotic sex chromosome inactivation and early female X inactivation, although this still needs to be proven. In eutherians, both imprinted and random X inactivation are clearly Xist-dependent processes, and the underlying imprint is at the level of the Xist gene. The Xic, in addition to containing Xist, is a complex regulatory locus, ensuring several functions of random X inactivation, including sensing, counting and choice, as well as Xist-dependent cis-inactivation. There are even further species-specific differences in the content and regulation of the Xic. The emerging picture is that the evolutionary origins of X inactivation are multiple, which is perhaps not surprising given the complexity of the selective pressures that are likely to act on X inactivation during early development. Clearly studies investigating the early events and players in X inactivation in species other than mouse will be important to address this issue in the future.
The authors wish to apologise for any omissions in references that may have been made. The work by our group described in this review was funded by the CNRS, EU Integrated Project HEROIC, the Fondation pour la Recherche Mediacale, and the ANR
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