Human Molecular Genetics

Human Molecular Genetics 2005 14(Review Issue 1):R59-R64; doi:10.1093/hmg/ddi117

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Imprinted X inactivation and reprogramming in the preimplantation mouse embryo

Takashi Sado^1,2 and Anne C. Ferguson-Smith^3,*

¹Division of Human Genetics, National Institute of Genetics, Research Organization of Information and Systems, 1111 Yata, Mishima 411-8540, Japan, ²PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan and ³Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK

^* To whom correspondence should be addressed. Tel: +44 1223333750; Fax: +44 1223333786; Email: afsmith{at}mole.bio.cam.ac.uk

Received January 28, 2005; Accepted February 24, 2005

	ABSTRACT

TOP ABSTRACT X INACTIVATION AND ROLES... IMPRINTED X INACTIVATION AND... X INACTIVATION, DNA METHYLATION... REFERENCES

 
X chromosome inactivation is a developmentally regulated process that causes one of the two X chromosomes in normal female mammals to become transcriptionally silenced, thus equalizing the expression of X-linked genes between the sexes. Such dosage compensation depends upon dynamic genetic and epigenetic events occurring very early in development. X inactivation is controlled by an X inactivation centre that is associated with the expression of non-coding RNAs required for the silencing. Also associated with the inactive X are repressive histone modifications and polycomb protein-mediated states, which are progressively acquired during the inactivation process. In mouse, two forms of X inactivation have been described. Random X inactivation happens in the derivatives of the inner cell mass (ICM) giving rise to embryos where the maternally inherited X(Xm) is inactive in some cells and the paternally derived X (Xp) is inactive in others. Random X inactivation occurs around the time of implantation. Imprinted X inactivation, the preferential inactivation of the Xp chromosome, occurs earlier and, although there has been some debate as to the precise timing of initiation of this event, is apparent in all cells early in preimplantation development, then is subsequently confined to the cells of the extraembryonic lineages. A picture is emerging whereby initial epigenetic asymmetry between the two parental X chromosomes is reprogrammed in a lineage specific manner resulting in a switch from imprinted to random inactivation in embryonic derivatives. Neither the underlying reason nor the full extent of these early lineage specific epigenetic changes is known, but they may be correlated with more genome-wide reprogramming events essential for normal development.

	X INACTIVATION AND ROLES FOR NON-CODING RNAs

TOP ABSTRACT X INACTIVATION AND ROLES... IMPRINTED X INACTIVATION AND... X INACTIVATION, DNA METHYLATION... REFERENCES

 
During early development of the normal female mammal, one of the two X chromosomes becomes transcriptionally silenced (1). The choice of which X chromosome to become inactivated in embryonic lineages is random; however, in the extraembryonic lineages in the mouse, the paternally derived X is chosen to be inactivated (2). This parental origin-specific inactivation is an example of genomic imprinting; a process that causes genes to be expressed from one of the two parental chromosome homologues (3). The reason for this lineage specific difference in mouse X inactivation is not known. Interestingly, in marsupial mammals, the Xp is preferentially inactivated in all lineages (4), whereas data in humans suggest that only random X inactivation occurs, although this remains debatable (5). It is possible, therefore, that imprinted X inactivation in the mouse extraembryonic derivatives represents the more ancestral mechanism. The dynamic epigenetic events that regulate X inactivation involve a number of steps including counting the X chromosomes such that all but one become repressed, choosing which X chromosome has to be inactivated, initiating the inactivation process, spreading the inactive state along the length of the chromosome and maintaining this repressive epigenetic state throughout the lifetime of the individual (6).

It has been shown that X inactivation is controlled by a cytologically identified chromosomal region called the X inactivation centre (Xic). Although the molecular basis of Xic remains obscure, two non-coding genes, Xist and its antisense transcript Tsix, mapped in Xic, play critical roles in X inactivation (Fig. 1). Tsix is expressed on the X that escapes inactivation and Xist is expressed on the X that becomes inactivated. (7–9).

View larger version (19K): [in this window] [in a new window]   Figure 1. Genomic structures of the Xist/Tsix loci in the Xic and the effects of paternal and maternal transmission of the Xist- and Tsix-deficient allele. (A) The gene structures of Xist and Tsix and relative orientations of their transcription on the mouse X chromosome are shown. Boxes and arrows delineate exons and transcriptional orientations of each gene, respectively. Although exon1 of Tsix has been found about 30 kb downstream of the Xist gene (17), the major transcription of Tsix appears to occur in the vicinity of exon2 (17,18,42). Xite is a non-coding transcript occurring in the same direction as Tsix in the upstream region of exon2 of Tsix (43). Enox/Jpg located 10 kb upstream of Xist is a non-coding gene transcribed in an opposite direction to Xist (44). (B) Although the Xist-deficient allele is transmitted to both male and female pups from the mothers, only its paternal transmission causes embryonic lethality in females soon after implantation due to the failure of imprinted inactivation of the paternal X in the extraembryonic tissues (upper left) (16). In contrast, although the Tsix-deficient allele is transmitted to viable female pups from the fathers, its maternal transmission results in early postimplantation lethality of both male and female embryos due to functional nullisomy of the X in the extraembryonic tissues caused by ectopic inactivation of the maternal X (upper right) (17,19). Intercrosses between XTsixX and XXistY reveal that a subset of females carrying the paternal XXist and the maternal XTsix survive to term and thereafter (bottom) (17).

 
The Xist allele becomes upregulated from the X chromosome that is to be inactivated as cells differentiate. Xist RNA, thus expressed, coats the X chromosome in cis (10,11) and induces chromosome-wide inactivation by recruiting proteins involved in heterochromatin formation, such as the polycomb group proteins (12–15). Targeted disruption of the Xist gene reveals that Xist is essential for X-inactivation to occur in cis (16). Paternal transmission of the Xist-deficient X (X^Xist) results in embryonic lethality in female embryos at the early postimplantation stage due to the failure of paternal X inactivation and the subsequent presence of two active Xs in the extraembryonic tissues (Figs 1 and 2). It is noteworthy that complete lethality of these female embryos indicates that the imprint laid on the Xm to resist inactivation is very rigid in the extraembryonic tissues, as Xm is unable to activate Xist.

View larger version (75K): [in this window] [in a new window]   Figure 2. Comparative early postimplantation development of e7.5 normal female mouse embryos and embryos deficient for Xist and Tsix on paternally and maternally inherited X chromosomes as indicated. Heterozygotes deficient for the paternally inherited Xist gene fail to inactivate the paternally inherited X and die at early postimplantation stages. In contrast, heterozygotes deficient for Tsix on the maternally inherited X chromosome express Xist on that chromosome and inactivate both X chromosomes, also resulting in early postimplantation lethality. Double heterozygous females deficient in Xist and Tsix show markedly improved development and the lethality observed in the single heterozygotes is rescued at a frequency of 20% of expected females.

 
Transcription of Tsix covers a 65 kb genomic region including the entire transcription unit of Xist in an antisense orientation (17,18). Targeted disruption of Tsix reveals that Tsix has a repressive role in the transcriptional regulation of Xist in cis (8,17,19). A Tsix-deficient X chromosome (X^Tsix) is therefore able to express Xist and subsequently undergoes inactivation. Maternal transmission of Tsix deficiency causes ectopic expression of Xist from the maternal X^Tsix in the extraembryonic tissues of both male and female embryos, resulting in functional nullisomy for the X chromosome and eventual death of the embryos at the early postimplantation stage (Fig. 1). Normally, Tsix does not appear to be expressed until the blastocyst stage. Tsix transcripts have not been noted during earlier stages, and therefore, although Tsix is required for imprinted Xm activity in the extraembryonic lineages, it is unlikely to be responsible for the initial imprinted Xist repression on Xm at the earlier stages. For this, some other, currently unknown mechanism must be inferred.

It has been demonstrated that the lethality of female embryos caused by the paternally derived X^Xist is rescued if combined with the maternally derived X^Tsix (Fig. 1B), although such X^TsixX^Xist survivors are found only in 20% of the expected females (17). Interestingly, ectopic expression of maternal Xist seems to occur only after implantation in X^TsixX^Xist females (unpublished data), further supporting a role for Tsix-independent imprinting of Xm activity at the earlier stages. It is tempting to speculate that the 80% lethality of the double heterozygous females might be associated with the early presence of two active Xs (due to lack of Xist expression from either X chromosome). However, despite the high loss, the development of X^TsixX^Xist females was greatly improved when compared with either X^TsixX or XX^Xist (Fig. 2). Nonetheless, in the surviving females, the Xm becomes inactivated instead of the Xp, suggesting that Tsix is, at least in part, playing a role in the imprint normally laid on the Xm to resist inactivation. Furthermore, the successful 20% rescue seen in these females also raises an interesting question about the biological significance of the inactivation of Xp during normal preimplantation development. This is because in the absence of paternally expressed Xist, both X chromosomes might be expected to stay active in Xm^TsixXp^Xist females until the blastocyst stage and the onset of random inactivation. Thus, in contrast to cells that have differentiated into the embryonic and extraembryonic lineages, dosage compensation of the X-linked genes may not be an absolute requirement during early preimplantation development. This is consistent with incompleteness of the inactivation status of the Xp at that time (described subsequently) (20–22).

	IMPRINTED X INACTIVATION AND SUBSEQUENT REPROGRAMMING

TOP ABSTRACT X INACTIVATION AND ROLES... IMPRINTED X INACTIVATION AND... X INACTIVATION, DNA METHYLATION... REFERENCES

 
The difference in X inactivation between the embryonic and extraembryonic lineages in the mouse is quite remarkable. Although either one of the two X chromosomes undergoes random inactivation in the embryo, the Xp is preferentially inactivated in the extraembryonic lineages, and therefore, X inactivation is imprinted (reviewed in 4). Recently, there has been some debate over when imprinted X inactivation begins in the mouse. This is a relevant issue because understanding this mechanism will provide insight into germline inherited silencing mechanisms in general, will facilitate mechanistic comparisons with autosomal imprinting and will allow consideration of the relationship between X chromosome reprogramming and genome-wide reprogramming events—issues important for our knowledge of the epigenetic control of genome function during mammalian development.

The prevailing view, held over the past 30 years, had stated that X inactivation occurs in mouse and humans after the blastocyst has implanted into the uterine wall (23). This is consistent with the first cytological evidence of a visible inactive X and the expression of X-linked gene products from both the maternally and paternally inherited X at earlier stages (20–22). Inactivation of the paternally inherited X was believed to occur for the first time in the trophectoderm and primitive endoderm (giving rise to the placenta and part of the extraembryonic membranes), leaving both Xs active in the undifferentiated inner cell mass (ICM) cells. Subsequently, in the ICM cells, either X became inactivated randomly regardless of parental origin. Three different recently published papers convincingly argue against this conventional view of the X inactivation process in the preimplantation embryos and shed light on the mechanisms of X inactivation during preimplantation development (24–26). These studies indicate that X inactivation is governed by imprinting in all cells during the preimplantation stages. Thereafter, reprogramming occurs in the embryonic derivatives and X inactivation becomes random.

Using a range of different techniques, the three studies show that Xp has already been inactivated at the preimplantation stages in a manner that is dependent on the expression and accumulation of Xist RNA on the X chromosome. The transcriptional repression is progressive and is associated with the accumulation of repressive histone modifications and other proteins known to be enriched on the inactive X. The precise timing of the onset of Xp inactivation is the subject of debate and has been outlined elsewhere (27), and more experiments are required to settle the issue. Two models have been proposed. In the first model, the paternal X enters the egg during fertilization in a preinactive state (24). Upon activation of the zygotic genome at the two-cell stage, Xist transcripts accumulate, but in all other respects, the paternal X, in general, remains transcriptionally inactive. Transcriptional silencing of the XY bivalent during male meiosis is a well-established phenomenon consistent with the possibility that the paternal X may be inherited by the zygote in this repressed state (28). The second model suggests that the paternal X is transcriptionally active at the two to four cell stage accumulating Xist transcripts and, thereafter, becomes progressively transcriptionally silent (25). Regardless of whether either of these models is correct, it is clear that imprinted X inactivation takes place several cell divisions earlier than the blastocyst stage, in contrast to the original belief.

Okamoto et al. (25) and Mak et al. (26) also showed that Xist RNA disappears from the Xp in the epiblast lineage at the blastocyst stage with a concomitant loss of inactive X-specific histone modifications or association of polycomb group proteins members, suggesting reactivation of the hitherto inactive Xp. Intriguingly, such an event apparently takes place in the cells that become positive for Nanog, a marker for pluripotent undifferentiated cells (29,30), as early as the morula stage (26). It is likely that this population of cells is the precursor of ICM cells. During this time, it is well established that genome-wide DNA methylation reprogramming is occurring, contributing to pluripotency. It appears, therefore, that in this lineage, the X chromosome also undergoes reprogramming, causing reactivation of the Xp. This coincident reprogramming of autosomal and X chromosomes may have removed parental imprints on the X, resulting in the second wave of X inactivation being random. During this preimplantation period, and in contrast to what happens to Xp in embryonic progenitors, all autosomal germline methylation imprints tested to date seem to retain the memory of their parental origin and appear resistant to aspects of the reprogramming process (see Morgan et al., this issue).

A similar X chromosome reprogramming event also happens in primordial germ cells (PGCs) that have completed migration into the genital ridge (31). It seems likely that reactivation of the inactivated X in PGCs is an essential process for the further development of the germ cells. Genome-wide reprogramming also occurs in PGCs and at this time, autosomal imprints also get reprogrammed. Therefore, X chromosome reprogramming appears at least temporally coordinated with two other genome-wide reprogramming events integral to normal development—in PGCs and in embryonic progenitors at preimplantation stages. Therefore, it seems reasonable to speculate that the regulation of autosomal and X chromosome reprogramming might be related. However, because autosomal imprints are resistant to this reprogramming in preimplantation embryos (though not in PGCs), this implicates a difference in the mechanism of imprinted X inactivation and autosomal imprinting in the embryonic progenitors. Furthermore, on the basis of non-reprogrammable inactivation of the paternal X in the extraembryonic derivatives, one might extrapolate that genome-wide reprogramming may not occur to the same extent in cells giving rise to trophoblast derivatives compared with the epiblast.

Another key question regarding imprinted X inactivation is what prevents the maternally inherited X chromosome from accumulating Xist RNA and becoming inactivated. Although a role for Tsix is implicated in random inactivation, its absence in preimplantation embryos and the failure to inactivate the Xm in Tsix mutants suggest that the maternally inherited X carries an imprint preventing Xist expression on that chromosome. The nature of this imprint is not known (as described subsequently).

	X INACTIVATION, DNA METHYLATION AND AUTOSOMAL IMPRINTING

TOP ABSTRACT X INACTIVATION AND ROLES... IMPRINTED X INACTIVATION AND... X INACTIVATION, DNA METHYLATION... REFERENCES

 
In female somatic cells, the promoter of the transcriptionally repressed Xist allele on the active X is highly methylated, whereas the transcriptionally active one on the inactive X is not (32). Similarly, a region composed of some repetitive sequences 15 kb downstream of the Xist gene, which is called DXPas34, is methylated on the active X but not on the inactive X in somatic cells (33). DXPas34 is located in the vicinity of the major transcription start site of Tsix. These results suggest that differential methylation may play some role in transcriptional repression of the Xist gene on the active X and the Tsix gene on the inactive X.

The methylation status of these two differentially methylated regions has been studied in preimplantation mouse embryos. Earlier studies by PCR-based analyses suggested that the 5' region of Xist is maternally methylated in the early cleavage stage embryos, and this differential methylation underlies imprinted expression of the paternal Xist (34,35). A later study using bisulphite genomic sequencing showed that this region stays unmethylated until the blastocyst stage (36). The reason for this discrepancy is unknown at the moment. Recent work by Sado et al. (37) in mouse embryos deficient for de novo DNA methyltransferases shows that differential methylation at the 5' region of Xist is not the primary mechanism for the differential induction of Xist at the onset of random X inactivation because Xist remains monoallelic despite its promoter being unmethylated on both alleles. This suggests that the parental imprint for paternal expression of Xist does not necessarily rely on DNA methylation. For Tsix, the methylation profile of DXPas34 analysed by bisulphite genomic sequencing indicates that this region is not methylated on both parental alleles during preimplantation development (38), suggesting that imprinted expression of Tsix in the extraembryonic tissues does not depend on DNA methylation at the DXPas34 region. It has been shown that, whatever its nature, an imprint is placed on the Xm during oocyte maturation in the prophase of meiosis I (39). Most recently, Kaneda et al. (40) found that DNA methylation mediated by the de novo methyltransferase Dnmt3a in both parental germlines can confer parental marks required for appropriate expression of the autosomal imprinted genes. It is therefore particularly interesting to consider whether Dnmt3a is also involved in the resistance of the Xm to inactivation. If the imprint on the Xm is not established in maternal germline mutants for Dnmt3a, then one would expect to see random X inactivation in the extraembryonic tissues of female embryos. However, results indicate that the paternal X is always inactive and the Xm stays active in the extraembryonic tissues of those female embryos derived from oocytes null for Dnmt3a (41), indicating that Dnmt3a-mediated DNA methylation does not confer an imprint on the Xm in the maternal germline to resist inactivation. This difference between imprinted X inactivation and autosomal imprinting suggests that there may be significant mechanistic differences between the two processes.

In conclusion, the assessment of the dynamic epigenetic changes occurring on the two X chromosomes before fertilization and in the early embryo provides valuable insight into heritable silencing mechanisms involved in this important dosage compensation mechanism. The role of genomic imprinting in the early part of this process and, subsequently, in the mouse extraembryonic derivatives is intriguing and may provide a link with the more ancestral mechanism occurring in all somatic cells of marsupial mammals. This has important implications for our understanding of the evolution of the imprinting mechanism in general. A resolution of the debate surrounding the precise timing of the onset of imprinted paternal X inactivation is also important from a mechanistic perspective. The early reprogramming events that result in a switch from imprinted to random X inactivation in embryonic progenitors and their derivatives provide a useful comparison with other reprogramming events. For example, erasure of the inactive X and autosomal imprints in PGCs might be regulated concurrently with the more genome-wide reprogramming events occurring during germ cell development. Similarly, we have suggested that genome-wide and X chromosome reprogramming might be part of the same process in ICM progenitors. However, although many investigators have, over the years, focused on similarities between autosomal imprinting and X inactivation and made mechanistic comparisons, here we draw attention to some striking differences. First, the X chromosome imprint is not resistant to genome-wide reprogramming at preimplantation stages like the autosomal imprints are. And secondly, at least one part of the machinery that confers maternal germline methylation imprints on autosomes does not contribute to imprinting on the maternally inherited X chromosome. Therefore, although germline reprogramming events that serve to erase epigenetic marks may not discriminate between X chromosomes and autosomes, evidence suggests that significant aspects of the re-establishment and maintenance of X-linked and autosomal imprints are different.

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TOP ABSTRACT X INACTIVATION AND ROLES... IMPRINTED X INACTIVATION AND... X INACTIVATION, DNA METHYLATION... REFERENCES

 

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