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X-chromosome inactivation: counting, choice and initiation
Author: Philip Avner ,Edith Heard
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"In mammals, dosage compensation of X-linked genes is achieved by the transcriptional silencing of one of the two X chromosomes in the female during early develop- ment ? a process known as X inactivation. The early events in X inactivation are under the control of a key regulator, the X-chromosome-inactivation centre or Xic. Initiation of X inactivation involves a recognition step in which the number of X chromosomes in the cell is counted relative to cell ploidy so that only a single X chromosome is functional per diploid adult cell. One hypothesis postulates the existence of a blocking factor that is synthesized in limiting quantities sufficient for the binding of a single Xic per diploid cell. Initiation is also thought to include a process of choosing, whereby one of the two X chromosomes in the female cell might be preferentially selected for inactivation. Examples include the imprinted inactivation of the paternal X chromosome in extra-embryonic tissues and the biased inactivation that results from allelic differences at the X- chromosome-controlling element (Xce) locus in embryonic tissues. As defined cytologically, the Xic is a roughly 1 Mb region that contains several elements thought to have a role in X inactivation (BOX 1) and at least four genes 1 (FIG. 1). One of these, the X (inactive)-specific transcript (Xist) gene, encodes a large non-coding RNA that is rel- atively poorly conserved. Xist has been shown to con- tribute to Xic function and is required for X inactiva- tion. Other elements that lie within the Xic are candi- dates for involvement in the control of Xist expression, or for the mechanisms of counting and choice. One is the DXPas34 locus, which was originally identified as a result of its unique methylation profile on the active X chromosome 2 . Another is the Tsix transcript, a non- coding transcript that is synthesized from the strand opposite to Xist and has been hypothesized to regulate the activity of Xist at the onset of X inactivation 3 . During random X inactivation, counting and choice must either precede, or be concomitant with, the onset of initiation and its earliest manifestation, the coating of the presumptive inactive X by Xist. Silencing of X-linked genes and replication asynchrony follow rapidly. Both seem to precede global histone hypoacetylation, the accumulation of a novel histone variant (macroH2A) and methylation of the inactive X, which seem to func- tion as maintenance mechanisms for X inactivation 4?6 . However, imprinted X inactivation, which occurs in cer- tain mammals and by which the paternal X is preferen- tially inactivated, might differ in some respects from random inactivation. In this article, we review recent results concerning the events that surround the initiation of X inactivation. X-CHROMOSOME INACTIVATION: COUNTING, CHOICE AND INITIATION Philip Avner and Edith Heard* In many sexually dimorphic species, a mechanism is required to ensure equivalent levels of gene expression from the sex chromosomes. In mammals, such dosage compensation is achieved by X-chromosome inactivation, a process that presents a unique medley of biological puzzles: how to silence one but not the other X chromosome in the same nucleus; how to count the number of X?s and keep only one active; how to choose which X chromosome is inactivated; and how to establish this silent state rapidly and efficiently during early development. The key to most of these puzzles lies in a unique locus, the X-inactivation centre and a remarkable RNA ? Xist ? that it encodes. NATURE REVIEWS | GENETICS VOLUME 2 | JANUARY 2001 | 59 Unit� de G�n�tique Mol�culaire Murine Institut Pasteur, 25 rue du Docteur Roux, Paris 75015, France. *Present address: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA. e-mails: pavner@pasteur.fr; heard@cshl.org REVIEWS � 2001 Macmillan Magazines Ltd 60 | JANUARY 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS inactive X chromosome, while the Xist gene on the pre- sumptive active X chromosome in male and female cells becomes silenced. The onset of X inactivation therefore seems to be intimately linked with the accumulation of Xist RNA in the cell. Xist RNA stability. Monoallelic Xist upregulation has been associated with an increase in the half-life of the Xist transcript 8,9 . Johnston et al. 10 proposed that this change in Xist transcript stability is brought about by a developmentally regulated switch in Xist promoter use. Two promoters (P1 and P2) were shown to be used for transcription of the stable Xist transcript that accumu- lates on the inactive X chromosome in somatic cells. A putative third promoter (P0) located 6.6 kb further upstream, was hypothesized to generate the unstable Xist transcripts in cells that have not yet undergone X inacti- vation. Recent findings have raised several questions concerning the existence and role of the P0 promoter. An ES cell line carrying an Xist transgene that was deleted for the P0 promoter region still gave the punctate RNA FISH signal that is normally associated with unstable Xist transcripts in undifferentiated ES cells. This indicated that P0 might not be required for the production of unstable Xist transcripts in ES cells 11 . The highly repeti- tive and poorly conserved nature of the P0 region in both voles and humans provides a further argument against this region having an important regulatory role (Tatyana Nesterova et al., personal communication). The original observations regarding the P0 promoter were probably complicated by the presence, at that time unsuspected, of antisense transcripts running from the 3? end of Xist through to, and beyond, the P1 promoter 3 , and by the presence of several ribosomal protein pseudogenes lying upstream of P1, in the proposed P0 region 11,12 (FIG. 1). Nevertheless, it cannot be ruled out that a series of alternative promoters exists in the region upstream of P1/P2 and even P0, because the more recent experiments of Warshawsky and colleagues 11 were based on transgenes truncated just upstream of Xist that might have been influenced by position effects. The issue will only be definitively resolved by either tar- geted deletion of the region or its functional inhibition. Another interesting candidate region to be consid- ered in the context of possible regulatory elements upstream of Xist is the 2.1(2)P region, which shows his- tone H4 hyperacetylation in undifferentiated female ES cells but not in male ES cells 13 . This H4 hyperacetylation disappears upon differentiation, suggesting that it might well be involved in Xist regulation before, or during, the initiation of X inactivation. Hyperacetylation of this region is substantially reduced in female ES cells carry- ing a mutated Xic (a partially deleted Xist gene) 16 ,which might reflect involvement of 2.1(2)P in counting and/or choice, both of which involve sensing the presence of two or more Xics in the nucleus 1 . Important information concerning the regulation of Xist has also come from experiments involving a full- length Xist cDNA transgene that contains the P1 and P2 promoters, but under the control of a strong inducible promoter 14 . High levels of Xist RNA derived from the We place an emphasis on the role of Xist and the events that occur upstream and downstream of Xist expres- sion. For a comprehensive review of the older literature, readers are referred to REFS 1, 7. Xist ? regulation and function Before random X inactivation initiates in the developing embryo, Xist appears to be expressed at low levels from every Xic in the cell. Using fluorescence in situ hybridization (FISH), Xist RNA can be detected in embryonic stem (ES) cells as two small, punctate signals in female (XX) ES cells and a single punctate signal in male (XY) ES cells (FIG. 2). Female ES cells undergo X inactivation when stimulated to differentiate, and repre- sent a useful model system for the study of X inactiva- tion. Xist RNA first accumulates on and coats the future Box 1 | Xic and the elements of X inactivation Xist The X inactive-specific transcript (Xist) gene is expressed exclusively from the inactive X chromosome, producing a 17-kb spliced, polyadenylated transcript that is retained in the nucleus. The Xist transcript seems to be the primary signal for spreading the inactive state along the chromosome. But Xist itself does not seem to be involved in counting. Some of the elements lying outside Xist that influence counting and choice in X inactivation (see Xic) might be involved in regulating its expression. Xic The X-chromosome-inactivation centre (Xic) was originally defined through studies on structurally abnormal X chromosomes as a ?master control region?, the presence of which is essential for X inactivation to occur. It is responsible for initiating X inactivation in cis: an X-chromosome fragment that carries a Xic can become inactivated, whereas one in which the Xic is missing cannot. The Xic is also involved in ?counting?, whereby only a single X is kept active per two sets of autosomes in a cell, and all other Xic-carrying chromosomes are inactivated. Xce The X-chromosome-controlling element (Xce) affects the choice of X to be inactivated (or to remain active). In females heterozygous for different Xce alleles, an X chromosome that carries a strong Xce allele is more likely to remain active than one that carries a weak Xce allele, thereby leading to skewed X inactivation. The degree of skewing is rarely more than 70:30%. Refined genetic mapping using microsatellite markers indicate that the Xce locus might be distinct from Xist, lying distal and 3? to Xist 38 . TsiX TsiX is an element transcribed from the antisense strand relative to Xist. Tsix is expressed in undifferentiated ES cells and early embryos, and has been proposed to control Xist expression in cis at the onset of X inactivation 3,35 . TsiX antisense transcription spans the whole of the Xist gene, extending well over 40 kb. The 5? end and promoter of the TsiX gene has been proposed to be closely associated with the DXPas34 locus, although other (weaker) promoters might be scattered across a large region 3? to Xist 34 . Targeted deletion of the 5? end of TsiX/DXPas34 leads to non-random inactivation of the deleted X chromosome 35 and a failure of imprinted X inactivation 52. This might indicate that this locus and/or the transcript that it produces influences X-chromosome choice and imprinting of the X chromosome. DXPas34 The DXPas34 locus is a 3 kb CpG-rich region, containing a 34-mer minisatellite repeat lying roughly 15 kb downstream of the 3? end of Xist. DXPas34 is hypermethylated on the active X chromosome in somatic cells. The degree of hypermethylation was thought to correlate with allelism at the Xce locus, although Xce lies outside the DXPas34 region 2 . The prinicipal initiation site for TsiX transcription has been reported to lie within DXPas34 (REF. 3). � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | JANUARY 2001 | 61 REVIEWS dependent on continued Xist expression and can be reversed. It is not accompanied by any of the later char- acteristics of the inactive X, such as histone hypoacetyla- tion and late replication timing. In cells that have been induced to differentiate, Xist must be expressed during the first 48 hours of differentiation to initiate ectopic silencing. Once 72 hours of differentiation have elapsed, continued silencing is no longer dependent on Xist expression and the full range of secondary X inactivation characteristics is acquired. Irrespective of whether the initial reversible repression of transcription by Xist in undifferentiated ES cells is a normal step in the initiation of X inactivation in female cells (as suggested by Wutz and Jaenisch 14 and possibly by recent results with andro- genetic embryos), or is an artefact of the high levels of Xist produced by the inducible promoter in the trans- gene, the results are consistent with the idea that Xist RNA is the key factor that triggers X inactivation in cis. Clues from Drosophila. Insights into the function of the Xist transcript have come from recent studies in Drosophila melanogaster. Dosage compensation in Drosophila is ensured through the hypertranscription of the single X chromosome present in the male, and this is associated with the hyperacetylated state of his- tone H4. Despite this fundamental difference with mammals in terms of strategy, certain unifying epige- netic regulatory principles might be common to both systems of dosage compensation. In each case, for instance, it seems that one, or several, molecules bind specifically to an X chromosome and are critical in remodelling the structure of the dosage-compensated chromosome. Once remodelled, the chromatin then maintains the specific transcriptional state associated with the dosage compensated X chromosome. Dosage compensation in Drosophila depends on the presence of two small non-coding RNAs, roX2 (1.1 kb) and roX1 (3.5 kb), and the five male-specific lethal pro- teins (MSLs): maleless (MLE), MSL-1, MSL-2, MSL-3 and MOF (Males absent on the first) 22 . The MOF pro- tein, which is known to have histone acetyltransferase activity, is present in both sexes but is associated with the X chromosome only in males, in a complex composed of the MSL proteins. The MSL proteins localize together, presumably as a complex, at several hundred X-chromo- some sites along the entire length of the male X. In mutants lacking MOF, MLE or MSL function, binding is restricted to about 30 well-distributed sites. These ?core sites? have been proposed to represent assembly sites from which the MSL complexes spread to the other sites on the X chromosome. The association of MOF with the male X chromosome has now been shown to be RNase sensitive and to depend on interaction with the roX2 RNA transcript 23 . In vivo binding of MOF to roX2 is through its CHROMODOMAIN. Whereas the various MSL proteins in the dosage compensation complex seem to be held together through protein?protein interactions, two other MSL proteins, MLE and MSL-3, (the latter is also a chromodomain protein) might also be RNA-bind- ing factors 23 , indicating that RNA interaction might be a property of many chromatin regulatory molecules. cDNA transgene were stable in undifferentiated ES cells, with a similar half-life to the Xist transcript in female somatic cells. This indicates that the short RNA half-life associated with low-level endogenous Xist expression cannot be due to an absence of Xist RNA stabilizing fac- tors in undifferentiated ES cells, a result supported by studies involving human XIST transgenes 15. One possi- bility raised by the study of Wutz and Jaenisch 14 is that stabilization of Xist RNA might depend on the levels of Xist RNA present, with low expression being associated with instability. Alternatively, the stability of the trans- genic Xist transcript could be due to the absence of the 3? or 5? sequences and intronic genomic sequences that might be involved in destabilization of the endogenous Xist transcript in ES cells. Xist function. The importance of Xist in the X-inactiva- tion process has been shown by both loss- and gain-of- function experiments. Two targeted deletions of the Xist gene showed that Xist is essential for inactivation in cis 16,17 . In gain-of-function experiments, extra copies of Xist, often with considerable amounts of flanking sequence, have been introduced as transgenes either into ES cells or, by pronuclear injection, into the mouse oocyte 18?21 . Studies involving the inducible Xist cDNA transgene 14 have been particularly insightful. In undiffer- entiated or early differentiating ES cells, inducing the expression of the Xist cDNA transgene leads rapidly (within 24 hours or about one cell cycle) to long-range transcriptional repression in cis. This inactivation is Xic DXPas34 S12 S19 2.1(2)P P0 P1/P2 Xce 10 kb Choice 1 2 3 4 5 Counting Cis-inactivation Tsx TsiX Xist Brx Cdx4 Figure 1 | The X-inactivation centre. A summary of the known elements and regions in the X-inactivation centre (Xic) thought to affect choice, counting and cis-inactivation during the initiation of X inactivation. (See BOX 1 for more information on Xist, DXPas34, Tsix and Xce.) Genes are shown in bold, with the direction of transcription indicated by the arrow. Brx (brain X-linked) 67 , Tsx (testis X-linked) 68 and Cdx4 (Caudal-4) 69 are genes that lie within the Xic that do not have, at least for the moment, any defined X-inactivation function. The 2.1(2)P region shows differential histone H4 hyperacetylation in undifferentiated female and male embryonic stem (ES) cells and has been suggested as a possible regulatory element in X inactivation. P1 and P2 are the somatic Xist promoters, and P0 is a postulated Xist promoter in undifferentiated ES cells and early embryos. S12 and S19 are ribosomal protein pseudogenes found 5? to Xist in the mouse Xic 11,12,70 . The deletions used to dissect the function of different elements in the Xic are shown as black lines and are described in REF. 16 (1) REF. 17 (2), REF. 34, (3) REFS 35,52 (4) and REF. 33 (5). Blue bars indicate the regions that have been implicated in specific functions. Effects on choice and counting have not so far been distinguished in the regions indicated by light bars. The terminal two Xist exons, lying within the 65-kb deletion, have no effect on counting or choice (C. Morey, P. A. and P. Clerc unpublished observations). CHROMODOMAIN A highly conserved sequence motif that has been identified in various animal and plant species. Chromodomain proteins seem to be either structural components of large macromolecular chromatin complexes or involved in remodelling chromatin structure. � 2001 Macmillan Magazines Ltd 62 | JANUARY 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS the MACROCHROMATIN BODY, or MCB 28 . By contrast, in undifferentiated ES cells, macroH2A is clustered away from the X chromosome 5 and seems to associate with CENTROSOMES 29 . MCB formation in differentiating female (XX) ES cells is progressive, occurring only after several days of differentiation, well after Xist RNA coating and inactivation have occurred 5,29 . These kinetics might indicate that macroH2A is unlikely to be involved in the initiation of random X inactivation and that its recruitment to the X chromosome must depend on factors other than Xist RNA alone. In fact, a recent study has shown that the association of macroH2A with the inactive X, which seems to depend on the his- tone part of the macroH2A molecule rather than the non-histone tail, might partly reflect the higher density of several histones on the inactive X 30 . As the associa- tion of macroH2A with the inactive X is disrupted upon loss of Xist RNA expression without any apparent destabilization of the inactive state 31 , it does not seem to be necessary either for maintenance of the inactive state or for the initiation of random X inactivation. However, a more important role for macroH2A in imprinted X inactivation is indicated by studies in pre- implantation embryos 32 . Counting and choice Because the Xist transcript can induce gene silencing in cis, independently of flanking and intronic DNA sequences, but cannot induce inactivation of the endogenous X chromosome in the cell 14 , the counting function of the Xic must be encoded by elements situat- ed outside of Xist. Given the central role of the Xist tran- script, such elements are likely to regulate Xist expres- sion, whether directly or indirectly. Deletion analysis is being used to define such elements. Clerc and Avner 33 created a 65-kb deletion of a region lying 3? to the main part of the Xist gene (FIG. 1). The deletion (X ?65 ) included the terminal two Xist exons, the postulated principal initiation site for the Tsix antisense transcript and the DXPas34 locus (BOX 1) on one of the X chromosomes in a female (XX) ES cell line. In undifferentiated mutant ES cells, Xist transcription from the mutated X, as visualized by RNA FISH, is markedly reduced. Upon differentiation, the deletion Recruitment of chromatin proteins by Xist. Similarly to the roX non-coding RNAs, Xist RNA might recruit reg- ulatory and chromatin proteins to the mammalian X chromosome undergoing inactivation. Recent chro- matin immunoprecipitation experiments indicate that Xist RNA might associate with chromatin that is enriched in the hypoacetylated isoforms of histones H3 and H4 (REF. 24). This is in keeping with the global hypoacetylation of the inactive X observed using immunofluorescence 25,26 and with the finding that the promoters of most genes on the inactive X chromosome are enriched for hypoacetylated histone H4 (REF. 27), as well as being hypermethylated 1 . This is suggestive of a role for the Xist transcript in targeting factors that are involved in transcriptional repression to the inactive X. However, the much later appearance of a globally hypoacetylated state and of hypermethylated promot- ers during ES cell differentiation indicates that Xist might not be directly involved in inducing such methy- lation and histone acetylation modifications. Furthermore, deletion of Xist in somatic cells does not seem to perturb the maintenance of the inactive state, implying that any direct role for Xist in recruiting chro- matin modifiers might be limited to the initiation phase of X inactivation. In agreement with this, the results of Wutz and Jaenisch 14 indicate that coating of the X chromosome by Xist RNA rapidly induces tran- scriptional silencing, whether by recruiting chromatin- modifying factors or simply by changing the environ- ment of the X chromosome, only during the first 48 hours after ES cell differentiation. The Xist-RNA-asso- ciated factors that are involved in this initial silencing must later be replaced or reinforced by other epigenetic silencing marks, such as deacetylation and methylation, that can be stably propagated and can maintain the inactive state through mitosis. An association between Xist RNA and another chro- matin protein, the histone variant macroH2A1.2 has been shown using chromatin immunoprecipitation and reverse-transcriptase polymerase chain reaction (RT-PCR) on extracts of adult female cells 24 . Immunofluorescence studies on such cells had previ- ously revealed that macroH2A is enriched on the inac- tive X chromosome and forms a structure known as Figure 2 | Xist transcription in embryonic stem cells. Patterns of Xist RNA expression in female ES cells undergoing differentiation using RNA fluorescence in situ hybridization. The left panel shows that undifferentiated ES cells have two punctate Xist RNA signals, representing the presence of unstable Xist transcripts at the site of transcription on both (active) X chromosomes. The middle panel shows that, upon differentiation, Xist RNA from one of the two alleles becomes stabilized and coats the X chromosome that is to be inactivated in cis. The X chromosome that remains active continues to express Xist in its unstable form. The right panel shows that, in fully differentiated cells, Xist RNA coats the inactive X chromosome and the Xist gene on the active X has been silenced. MACROCHROMATIN BODY (MCB). Discrete accumulation of the histone variant, macroH2A, on the inactive X chromosome. CENTROSOME The microtubule organizing centre that divides to organize the two poles of the mitotic spindle and directs assembly of the cytoskeleton, thus controlling cell division, motility and shape. � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | JANUARY 2001 | 63 REVIEWS 65-kb deletion by successively adding back parts of the deleted region. Adding back the terminal two Xist exons did not change the behaviour of the deleted X ?65 chro- mosome, confirming that elements outside of the Xist gene itself are responsible for the phenotype (C. Morey, P. Clerc and P. A., unpublished observations). It has become increasingly clear that the 65-kb dele- tion covers a complex series of regulatory loci that con- trol counting, chromosome choice, the levels of Xist transcription and antisense transcriptional activity across the Xist gene in undifferentiated ES cells. Deletion of the DXPas34 locus, which lies within the 65-kb dele- tion and is associated with the principal initiation site for the Tsix antisense transcript, leads to the abolition of both antisense activity and either an absence of, or a marked reduction in, Xist transcription from the mutat- ed Xic in undifferentiated ES cells, as visualized by RNA FISH 33,34 . Upon differentiation, the deletion results in complete skewing of X inactivation, with only the mutated Xic inducing X inactivation 34,35 .However,the counting function of the Xic locus is unaffected by dele- tion of DXPas34 (REF. 35). Although counting and choice are therefore distinct functions, it is possible that control of Xist and Tsix antisense transcriptional activity could be mechanistically linked to chromosome choice through a pivotal mechanism present in ES cells both before, and at the onset of, inactivation. The situation is becoming more complicated because there are good rea- sons to think that antisense transcription in the Xic could be both more widespread and complex than initially thought 34 . Furthermore, the antisense RNA itself might not have a direct role. Instead, the act of transcription might be critical in regulating the state of a large chro- matin domain around Xist, as proposed for other loci, including the human ?-globin locus 36,37 (FIG. 3). The importance of chromatin domain formation might explain why single-copy yeast artificial chromo- some transgenes for the Xic, which are capable of syn- thesizing Xist in a correctly regulated manner in undif- ferentiated cells, cannot initiate X inactivation, unlike multicopy arrays of the same transgene 18 .Multicopy arrays are thought to create a chromatin domain that provides counting and/or choice functions, albeit to variable extents 18 . By contrast, single-copy transgenes would not be able to form such a chromatin domain. Whereas only a single region of the Xic might be involved in counting, several regions influence the choice of X chromosome that will be inactivated (FIG. 1). Apart from the DXPas34/TsiX region and the Xce locus, which lies beyond the 65 kb deletion 38 , data from the human XIC indicate that mutation of the XIST promot- er itself can lead to skewed X inactivation 39 . The pheno- type of the Xist knockout described by Marahrens et al. 40 suggests another element involved in this choice lies within the Xist gene itself. Imprinted X inactivation X inactivation is initially subject to imprinting during the early development of some EUTHERIAN (placental) mammals ? the paternally inherited X chromosome is preferentially inactivated in the first cells to differentiate, results in complete skewing of X inactivation, with only the mutated X chromosome being inactivated. Surprisingly, ES clones (derived from the original XX ?65 line) in which only the X ?65 chromosome is present, still initiated X inactivation upon differentiation, despite the absence of a second Xic. At present, this is the only example of a cell line that contains a single Xic and that undergoes X inactivation. The blocking hypothesis of X-chromosome count- ing proposes that limited quantities of a blocking factor bind to the Xic of a single chromosome per diploid cell, protecting it from inactivation. The second Xic present in a female cell nucleus remains unprotected and is therefore inactivated. This hypothesis predicts that a single Xic in a diploid cell will always be blocked and therefore will always be protected from inactivation. The blocking factor could either be a diffusible molecule or, equally, a unique nuclear compartment or attach- ment site such as the nuclear membrane. The results from the X ?65 chromosome can be explained if a bind- ing element for the blocking factor lies within the delet- ed region. In the absence of this element, the blocking factor cannot bind and the deleted X chromosome is automatically chosen for initiation of inactivation. Clerc and co-workers have refined the analysis of the a Masking Tsix transcribed Tsix silent b Interference c Repression d Chromatin opening Figure 3 | Potential roles for Xist antisense transcription. a | Masking of the Xist transcript by the antisense transcript TsiX (black dashed lines) prevents binding of factors that might be involved in the stabilization of Xist RNA or in allowing it to associate in cis with chromatin. b | Transcriptional interference between Xist and TsiX might prevent the efficient transcription across the Xist gene and thereby prevent the accumulation of Xist transcript. c | Antisense transcription might be involved in the repression of high level Xist transcription. This might be through the binding of the putative ?blocking factor? somewhere in the Xist 3? region. d | Antisense transcription might be either involved in, or symptomatic of, ?chromatin opening?, which might be required to enable the binding of developmentally regulated factors to the Xic region. EUTHERIANS Mammals that give birth to live offspring (viviparous) and possess an allantoic placenta. � 2001 Macmillan Magazines Ltd 64 | JANUARY 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS only late in differentiation (see above), whereas in early embryos (8?16-cell stages) and in extra-embryonic cells of the blastocyst, association of macroH2A with the paternal X chromosome undergoing imprinted X inac- tivation occurs rapidly 32 (FIG. 4). The early association of the paternally derived X chromosome with Xist RNA and, soon after this, with macroH2A might reflect a par- ticular chromatin state that is acquired during male gametogenesis of the paternally derived X. The observa- tion that macroH2A is associated with the SEX VESICLE in male meiosis could be compatible with such an inter- pretation 42,43 . In fact, the paternal genome as a whole is markedly different to the maternal genome in its chro- matin state, its global methylation 44 and its replication 45 during the cleavage stages of embryogenesis. Indeed, the two genomes remain compartmentalized up to, and beyond, the 4-cell stage 46 . The arrival of the paternal X chromosome in the fertilized zygote, in a partially con- densed state as a result of its passage through the male germ line, has been suggested as one way in which the X-inactivation process may initially have evolved (reviewed in REF. 1). Aspects of this state might be main- tained during the initial cleavage divisions of the embryo through the conservation of chromatin struc- ture during replication, and this might have provided the basis for selection during evolution to establish dosage compensation in the female. The idea that the arrival of the paternal X in a par- tially condensed state is one of the keys to imprinted inactivation has received indirect support from the cloning experiments of Eggan and colleagues 47 . The X chromosome selected for inactivation in extra-embry- onic tissues derived from a cloned somatic nucleus is always the one that had initially been inactive and which carried a mature Xist RNA domain and other character- istics of the inactive X at the moment of transfer. By contrast, embryonic tissues derived from the cloned adult somatic nucleus showed random inactivation. This emphasizes the specificity of the observations made in the extra-embryonic tissues and also shows the capacity of the early embryo to reverse Xist RNA coating and erase other epigenetic marks on the inactive X. Although the paternal X chromosome seems to be predisposed to X inactivation, this paternal imprint can be overridden to allow dosage compensation when two paternal X chromosomes are present. Whether the counting process can occur in extra-embryonic tissues is not totally clear, although the recent finding, that cloned nuclei from ES cells (in which both X chromo- somes are active) can give rise to extra-embryonic tis- sues showing random X inactivation 47 ,might indicate that counting does occur. However, other observations are less clear-cut. For instance, Okamato and colleagues recently produced female ANDROGENONES which survived to embryonic day 7.5 (E7.5) ? longer than in previous experiments. These androgenetic embryos showed inac- tivation (late replication) of only one of the two paternal X chromosomes in all tissues, including extra-embryon- ic tissues, again suggestive of counting 48 .However,in these experiments, both paternal X chromosomes were in fact initially coated by Xist RNA, indicating that X which later give rise to extra-embryonic tissues (FIG. 4). Such imprinted, paternal X inactivation is observed in all tissues of marsupials. It has been hypothesized 41 that this might have been the ancient form of X inactivation, with random X inactivation evolving only later in eutherian mammals. Recent studies in the mouse have revealed some important differences in the kinetics of the events that lead up to the establishment of the inactive state between the imprinted, extra-embryonic and the ran- dom, embryonic forms of X inactivation (FIG. 4).For example, Xist RNA is found to coat the paternal X from an early stage (as early as 2?4 cells), well before any sign of cellular differentiation. This coating precedes the first signs of imprinted inactivation in the TROPHECTODERM of the blastocyst by several cell divisions. This differs from the rapidity with which Xist RNA coating is followed by transcriptional inactivation in differentiating ES cells (within about one cell division 14 ), where random X inactivation takes place. Studies on the timing of appearance of macroH2A association with an X chromosome (MCB formation) during early embryogenesis have also pointed to a fun- damental difference between imprinted and non- imprinted X inactivation (FIG. 4). In ES cells, localization of macroH2A to the inactive X chromosome occurs ab X inactivation in ES cells Unstable Xist RNA expressed from paternal or maternal X Paternal X coated with Xist RNA Asynchronous replication of paternal X MacroH2A association with paternal X Inner cell mass 4-cell embryo Morula Xist RNA coating of paternal or maternal X Late replication timing Histone hypoacetylation MCB association Methylation of promoters Inactive paternal or maternal X in embryonic tissues Blastocyst Extra-embryonic tissues Paternal X inactivation Figure 4 | Imprinted versus random X inactivation. Kinetics of the events underlying X inactivation in imprinted versus random X inactivation. The different epigenetic changes associated with the X-inactivation process are shown. a | During pre-implantation mouse development, Xist RNA coating of the paternal X occurs well before (at least 4 cell divisions) the first signs of inactivation in the trophectoderm of the early blastocyst, and before the novel histone variant macroH2A accumulates and the macrochromatin body forms. b | By contrast, during random X inactivation in embryonic stem (ES) cells, Xist RNA coating of the X chromosome is rapidly followed by transcriptional silencing (within roughly 24 hours, or 1 cell cycle) and MCB formation is a much later event. TROPHECTODERM The precursor to the bulk of the embryonic part of the placenta. SEX VESICLE OR XY BODY Pairing of sex chromosomes during meiosis in male mammals is associated with heterochromatinization and occurs in the sex vesicle or XY-body, a specific nuclear structure that can be discerned morphologically. ANDROGENONE Embryo with two paternal sets of chromosomes. � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | JANUARY 2001 | 65 REVIEWS can also spread into an autosome from Xist-containing transgenes. Silencing of autosomal material therefore differs from that of the X only in degree: it is usually both less effective and less extensive, and this is associat- ed with a correspondingly limited spread of Xist RNA into the autosome 55 . Recently, Lyon 71 suggested that LINES might function to promote spreading along the X chromosome, acting as the ?way stations? or ?booster elements? originally hypothesized by Riggs 56 . LINEs were regarded as inter- esting candidates for this function in view of the LINE- 1-rich nature of the human and mouse X chromo- inactivation might have actually initiated on both X chromosomes, but by the late blastocyst stage one of the two paternal Xs had apparently lost this Xist RNA associ- ation in most cells. This might indicate that X inactiva- tion in early preimplantation embryos is reversible and indeed relatively unstable, (as in REF. 14, discussed above), and it is this instability that provides the possibility to ?override? the imprint in the event of defective dosage compensation, rather than a counting process per se. Unlike this apparent reversibility of the imprint on the paternal X, the maternal X chromosome initially car- ries a stable imprint to resist inactivation and remain active in the first cell lineages that differentiate ? the extra-embryonic tissues. Strong evidence for this resis- tance to inactivation of the maternal X has been obtained by Marahrens and colleagues 17 . Female embryos that carry a mutated Xist allele on the paternal X, and which therefore cannot inactivate this chromo- some die soon after implantation, whereas those carry- ing the Xist deletion on the maternal X grow normally, inactivating the paternal X selectively. Recent studies on embryos that are DISOMIC for the maternal X also support the idea that the maternal X is resistant to inactivation in extra-embryonic tissues, with premature embryonic death resulting from a failure to undergo X inactivation in these tissues 49 . This maternal imprint, of unknown nature, seems to be acquired relatively late during oocyte growth, as it is absent in the non-growing oocyte 50 . The actual epigenetic modifications underlying both the paternal and maternal imprint remain enigmatic. Differential methylation of the Xist promoter was one obvious candidate for such an imprint, but detailed bisulphite sequencing has failed to support early reports based on PCR?restriction enzyme analysis of methyla- tion differences 51 . Given the importance of the 3? region of Xist in counting and choice, the imprint might instead lie here. Support for this comes from the recent- ly reported partial disruption of the maternal imprint in mice carrying a knockout of the region containing the putative Tsix promoter 52 . When maternally inherited, this deletion leads to aberrant expression of the associat- ed Xist allele in a proportion of cells in early embryos and abnormal inactivation of the maternal X chromo- some in extra-embryonic tissues, resulting in post- implantation lethality in 90% of cases. This might indi- cate that the Tsix/DXPas34 region 3? to Xist carries at least part of the imprint responsible for resistance to inactivation on the maternally inherited X chromo- some. However, bisulphite analysis of sequences in the region 3? to Xist, particularly around the DXPas34 locus and the putative promoter of Tsix, have failed to reveal differential methylation in extra-embryonic tissues 53 , perhaps indicating that the imprinting mark is of another nature. A role for repeats in X inactivation X inactivation initiates from the Xic and then spreads across the entire X chromosome. Inactivation can also spread into an autosomal segment when this is attached to a Xic by translocation. Such spreading can occur over long distances ? easily 100 Mb or more 54 . Inactivation a Blocking factor? Way stations (LINEs?) b c Xist Xist RNA coating in cis d Establishment of the inactive state, asynchronous replication e MacroH2A recruitment Histone H3 and H4 hypoacetylation Figure 5 | A model for X inactivation. The model indicates speculative roles of some of the proposed players in the initiation of X inactivation. a | Before inactivation, Xist RNA is expressed in an unstable form (dotted red lines) and the postulated blocking factor(s) (red) prevents Xist upregulation and/or its association with the chromosome in cis. b | Xist RNA becomes upregulated through stabilization, transcriptional upregulation or release of the blocking factor. LINEs might participate in the spreading process in some way ? either through association with nucleoprotein complexes including Xist or by a mechanism such as REPEAT-INDUCED GENE SILENCING (RIGS). c | Stabilized Xist RNA coats the X chromosome before its inactivation. d | Transcriptional silencing of genes on the X chromosome occurs as a result of Xist RNA coating using an unknown mechanism and is rapidly followed by a shift to asynchronous replication timing of the X chromosome. e | Chromatin modifications, such as the histone deacetylation and methylation of promoters of X-linked genes, as well as the recruitment of the histone variant macroH2A, presumably transform the Xist RNA-coated chromosome into a stably inactive and condensed chromatin state. Animated online UNIPARENTAL DISOMIC An individual or embryo carrying two chromosomes inherited from the same parent LINES Long interspersed nuclear elements (such as L1 repeats) are retroelements present in over 100,000 copies in the mammalian genome. REPEAT-INDUCED GENE SILENCING (RIGS). Transgene expression in several organisms may be silenced epigenetically when repeated sequences are present. It has been proposed that interactions between homologous sequences (repeats) might lead to the formation of folded chromatin structures that attract heterochromatin-specific macromolecules � 2001 Macmillan Magazines Ltd 66 | JANUARY 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS modifications in the X-inactivation process presumably ensures stability both during evolution and within the individual. A particularly flagrant example of this is the increased relative stability of the inactive X in placental mammals, compared with that of marsupials. In placen- tal mammals, the inactive X is characterized by hypoacetylation and hypermethylation of gene promot- ers whereas only hypoacetylation is present in the mar- supial inactive X. The superficial nature of our understanding of the role and redundancy of epigenetic mechanisms in gene regulation and higher order cellular control is empha- sized by the recent discovery that several genes that are involved in a wide variety of epigenetic processes are responsible for well-defined monogenic human dis- eases. Examples include the DNMT3B gene (encoding a DNA methyltransferase) in immunodeficiency-cen- tromeric instability-facial anomalies syndrome (ICF syndrome) 65 and the MECP2 gene (encoding a methyl- CpG-binding protein) in Rett syndrome 66 . RNAs, rather than proteins, are particularly appealing as primary epigenetic signals in processes that require cis-limited gene regulation. Transcripts such as Xist might function by allowing the deposition of repressor complexes on the inactive X in mammals or, in the case of the roX RNAs, by facilitating and guiding the deposition of activator complexes onto the hypertranscribed X chromosome in Drosophila. The proteins that interact with these RNAs to form chromatin-associated nucleoprotein complexes are only just being deciphered in the Drosophila dosage- compensation system. These complexes are likely to represent an area of intense future research in mam- malian X-chromosome inactivation. Other tran- scripts, exemplified by the widespread low-level tran- scription both in the antisense and sense directions, from the Xic region, are also likely to be an integral feature of many chromatin control mechanisms, although their mode of action is less clear. In this review, we have indicated the many areas of uncertainty concerning X inactivation. The pivotal posi- tion for Xist is not in doubt, but there is much to discov- er about how Xist is controlled, and the precise sequence of events that takes place after Xist is expressed. Hints are emerging, and there will undoubtedly be parallels with other cellular processes. So X inactivation, along with other epigenetic processes, is likely to prove no more of a stranger than the rest of biology to evolution- ary tinkering, incest, mugging and bag snatching. somes. In addition, in mice there seems to be a correla- tion between the efficiency of the spread of inactivation into autosomes in X:autosome translocations and their LINE content 71 . Sequence analysis of the human X chromosome has now shown it to be twofold enriched for LINE-1 repetitive elements compared with the auto- somes, with the greatest enrichment being in a subset of younger LINE-1 elements that were active some 60?100 million years ago 57 . LINEs therefore clearly satisfy at least one of the basic criteria for boosters ? a ubiqui- tous distribution throughout the genome coupled to an increased frequency on the X chromosome. A recent global analysis of the 200 or more X- linked transcribed sequences available showed that about 15% of genes on the human X chromosome escape X inactivation 58 . Most of these are located on the short arm (Xp) of the human X. Indeed, the fre- quency of ?escapees? on Xp is similar to that observed for autosomal genes in X:autosome translocations, a reflection of the evolutionarily recent autosomal ori- gin of Xp 58 . Interestingly, significantly fewer LINE-1s are present in X-chromosome segments, particularly Xp22, which contains most genes that escape X inacti- vation. Conversely, the density of LINEs is highest in the Xq13?Xq21 region, which contains the human XIC. More detailed analysis of the XIC has shown a non-uniform LINE distribution, with a lower density of LINEs associated with the early replicating region 5? to XIST and XIST itself than the region 3? to XIST, which shows late replication 59 . The XIST transcript seems to associate preferentially with gene-rich (Giemsa-light) rather than LINE-rich (Giemsa-dark) chromosome bands 55,60 , indicating that XIST and LINEs might not interact directly. One possibility is that Xist transcripts could mask gene-rich sequences thereby increasing the propensity of LINEs towards mediating repeat-induced gene silencing (RIGS) (Mary Lyon, personal communication). Mice are thought to have a much smaller number of genes that escape X inactivation than humans 61 , and there is evidence that the human and mouse genomes differ considerably in the number of retrotransposition- competent LINE-1 elements that they contain 62 .The mouse X chromosome might therefore have accumulat- ed a greater and more uniform distribution of LINEs than the human X. However, escape from inactivation might not be due to inefficient spreading in some cases, but rather to inadequate maintenance of the inactive state, once established 63 . LINEs, with their tendency to be methylated, 64 could also contribute to this mainte- nance function. Perspectives The key characteristics of the X-inactivation process ? its extent, remarkable stability and precise developmen- tal regulation ? suggest that it involves the sophisticat- ed cooperation between several interacting molecules and factors, similar to many other epigenetic processes. This complexity is required to establish and then main- tain a clonally heritable state of transcriptional silence (FIG. 5). The redundancy between different epigenetic Links DATABASE LINKS Xce | Xist | Tsix | roX1 | roX2 | MLE | MSL-1 | MSL-2 | MSL-3 | MOF | DNMT3B | ICF syndrome | MECP2 | Rett syndrome ENCYCLOPEDIA OF LIFE SCIENCES X-chromosome inactivation | X-inactivation mechanisms � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | JANUARY 2001 | 67 REVIEWS 1. Heard, E., Clerc, P. & Avner, P. X-chromosome inactivation in mammals. Annu. Rev. Genet. 31, 571?610 (1997). A thorough review of the older X?inactivation literature. 2. Courtier, B., Heard, E. & Avner, P. Xce haplotypes show modified methylation in a region of the active X chromosome lying 3? to Xist. Proc. Natl Acad. Sci USA 92, 3531?3535 (1995). 3. Lee, J. T., Davidow, L. S. & Warshawsky, D. TsiX, a gene antisense to Xist at the X-inactivation centre. Nature Genet. 21, 400?404 (1999). This paper first described the presence of antisense transcripts overlapping that of the Xist gene. 4. Keohane, A. M., O?Neill,L. P., Belyaev, N. D., Lavender, J. S. & Turner, B. M. X-inactivation and H4 acetylation in embryonic stem cells. Dev. Biol. 180, 618?630 (1996). 5. Mermoud, J. E., Costanzi, C., Pehrson, J. R. & Brockdorff, N. Histone MacroH2A relocates to the inactive X chromosome after initiation and propagation of X- inactivation. J. Cell Biol. 147, 1399?1408 (1999). 6. Sado, T. et al. X inactivation in the mouse embryo deficient for Dnmt1: distinct effect of hypomethylation on imprinted and randon X inactivation. Dev. Biol. 225, 294?303 (2000). 7. Lyon, M. F. Some milestones in the history of X- chromosome inactivation. Annu. Rev. Genet. 26, 15?27 (1992). 8. Panning, B., Dausman, J. & Jaenisch, R. X chromosome inactivation is mediated by Xist RNA stabilisation. Cell 90, 907?916 (1997). 9. Sheardown, S. A. et al. Stabilisation of Xist RNA mediates initiation of X chromosome inactivation. Cell 91, 99?107 (1997). 10. Johnston, C. M. et al. Developmentally regulated Xist promoter switch mediates initiation of X inactivation. Cell 94, 809?817 (1998). 11. Warshawsky, D., Stavropoulos, N. & Lee, J. T. Further examination of the Xist promoter-switch hypothesis in X inactivation: evidence against the existence and function of a P0 promoter. Proc. Natl Acad. Sci. USA 96, 14424?14429 (1999). 12. Romer, J. T. & Ashworth, A. The upstream region of the mouse xist gene contains two ribosomal protein pseudogenes. Mamm. Genome. 11, 461?463 (2000). 13. O?Neill, L. P. et al. A developmental switch in H4 acetylation upstream of Xist plays a role in X chromosome inactivation. EMBO J. 18, 2897?2907 (1999). 14. Wutz, A. & Jaenisch, R. A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation. Mol. Cell 5, 695?705 (2000). An important paper describing studies with an inducible Xist cDNA transgene in ES cells, providing definitive evidence that Xist RNA is sufficient for inactivation in cis. In undifferentiated ES cells, Xist RNA coating leads to gene repression but not full inactivation. Upon differentiation, Xist RNA is initially required for X inactivation during a limited window of time, but is subsequently dispensable. 15. Heard, E. et al. Human XIST yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells. Proc. Natl Acad. Sci. USA 96, 6841?6846 (1999). 16. Penny, G. D., Kay, G. F., Sheardown, S. A., Rastan, S. & Brockdorff, N. Requirement for Xist in X chromosome inactivation. Nature 379, 131?137 (1996). 17. Marahrens, Y., Panning, B., Dausman, J., Strauss, W. & Jaenisch, R. Xist-deficient mice are defective in dosage compensation but not spermatogenesis. Genes Dev. 11, 156?166 (1997). 18. Heard, E. et al. Transgenic mice carrying an Xist-containing YAC. Hum. Mol Genet. 5, 441?450 (1996). 19. Matsuura, S.,Episkopou, V.,Hamvas, R. & Brown, S. D. M. Xist expression from an Xist transgene carried on the mouse Y chromosome. Hum. Mol. Genet. 5, 451?459 (1996) 20. Lee, J. T., Strauss, W. M., Dausman, J. A. & Jaenisch, R. A 450 kb transgene displays properties of the mammalian X-inactivation center. Cell 86, 83?94 (1996). 21. Heard, E., Mongelard, F., Arnaud, D. & Avner, P. Xist Yeast artificial chromosome transgenes functions as X- inactivation centers only in multicopy arrays and not as single copies. Mol. Cell. Biol. 19, 3156?3166 (1999). A transgenic analysis suggesting that additional, as- yet-undefined functions, other than those covered by the Xist gene and its immediate flanking regions, are necessary for counting and choice to occur. 22. Stuckenholz, C. Kageyama, Y. & Kuroda, M. I. Guilt by association: non-coding RNAs, chromosome-specific proteins and dosage compensation in Drosophila. Trends Genet. 15, 454?458 (1999). 23. Akhtar, A., Zink, D. & Becker, P. B. A chromodomain-RNA interaction targets MOF to the Drosophila X chromosome. Nature 407, 405?409 (2000). Interesting data on the role of non-coding RNAs in anchoring members of the dosage compensation complex in Drosophila (including the MOF protein, which has acetyltransferase activity) to the male X chromosome. 24. Gilbert, S. L., Pehrson, J. R. & Sharp, P. A. XIST RNA associates with specific regions of the inactive X chromatin. J. Biol. Chem. 275, 36491?36494 (2000). 25. Jeppesen, P. & Turner, B. M. The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression. Cell 74, 281?289 (1993). 26. Boggs, B. A., Connors, B., Sobel, R. E., Chinault, A. C. & Allis, C. D. Reduced levels of histone H3 acetylation on the inactive X chromosome in human females. Chromosoma 105, 303?309 (1996). 27. Gilbert, S. L. & Sharp, P. A Promoter-specific hypoacetylation of X-inactivated genes. Proc. Natl Acad. Sci USA 96, 13825?13830 (1999). 28. Costanzi, C. & Pehrson, J. R. Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals. Nature 393, 599?601 (1998). 29. Rasmussen, T. P. et al. Dynamic relocalization of histone macroH2A1 from centrosomes to inactive X chromosomes during X inactivation. J. Cell Biol. 150, 1189?1198 (2000). 30. Perche, P. -Y. Concentrations of histone MacroH2A in the Barr body are correlated with higher nucleosome density. Curr. Biol. 10, 1581?1534 (2000). 31. Csankovski, G., Panning, B., Bates, B., Pehrson, J. R. & Jaenisch, R. Deletion of Xist disrupts histone macroH2A localization but not maintenance of X inactivation. Nature Genet. 22, 323?324 (1999). Shows that Xist RNA coating is necessary for macroH2A accumulation on the inactive X in somatic cells, but neither Xist RNA nor macroH2A are necessary for the maintenance of the inactive state. 32. Costanzi, C., Stein, P., Worrad, D. M., Schultz, R. M. & Pehrson, J. R. Histone macroH2A is concentrated in the inactive X chromosome of female preimplantation mouse embryos. Development 127, 2283?2289 (2000). This paper describes the unexpectedly early association of macroH2A with the X chromosome during imprinted X inactivation, which contrasts with its much later association during random X inactivation. 33. Clerc, P. & Avner, P. Role of the region 3? to Xist exon 6 in the counting process of X chromosome inactivation. Nature Genet. 19, 249?253 (1998). Provides the first molecular evidence for counting element(s) that are localized in a region lying 3? to the mouse Xist gene. 34. Debrand, E., Chureau, E., Arnaud, D., Avner, P. & Heard, E. Functional analysis of the DXPas34 locus: A 3? regulator of Xist expression. Mol. Cell. Biol. 19, 8513?8525 (1999). 35. Lu, N. & Lee, J. T. Targeted mutagenesis of Tsix leads to nonrandom X inactivation. Cell 99, 47?57 (1999). 36. Gribnau, J., Diderich, K., Pruzina, S., Calzolari, R. & Fraser, P. Intergenic transcription and developmental remodeling of chromatin subdomains in the human ?-globin locus. Mol. Cell 5, 377?386 (2000). 37. Travers, A. Chromatin modification by DNA tracking. Proc. Natl Acad. Sci USA 96, 13634?13637 (1999). 38. Simmler, M. C., Cattanach, B. M., Rasberry, C., Rouguelle, C. & Avner, P. Mapping the murine Xce locus with (CA)n repeats. Mamm. Genome 4, 523?530 (1993) 39. Plenge, R. M. et al. A promoter mutation in the XIST gene in two unrelated families with skewed X-chromosome inactivation. Nature Genet. 14, 353?356 (1997) 40. Marahrens, Y., Loring, J. & Jaenisch, R. Role of the Xist gene in X chromosome choosing, Cell 92, 657?664(1998). 41. Graves, J. A. Mammals that break the rules: genetics of marsupials and monotremes. Annu. Rev. Genet. 30, 233?260 (1996). 42. Richler, C., Dhara, S. K. & Wahrman, J. Histone macroH2A 1. 2 is concentrated in the XY compartment of mammalian male meiotic nuclei. Cytogenet. Cell. Genet. 89, 118?120 (2000). 43. Hoyer-Fender, S., Costanzi, C. & Pehrson, J. R. Histone macroH2A1.2 is concentrated in the XY-body by the early pachytene stage of spermatogenesis. Exp. Cell Res. 258, 254?260 (2000). 44. Mayer, W., Niveleau, A., Walter, J., Fundele, R. & Haaf, T. Demethylation of the zygotic paternal genome. Nature 403, 501?502 (2000). 45. Ferreira, J. & Carmo-Fonseca, M. Genome replication in early mouse embryos follows a defined temporal and spatial order. J. Cell Sci. 110, 889?897 (1997). 46. Mayer, W., Smith, A., Fundele, R. & Haaf, T. Spatial separation of parental genomes in preimplantation mouse embryos. J. Cell Biol. 148, 629?634 (2000). 47. Eggan, K. et al. Non-random and random X chromosome inactivation in cloned embryos. Science 290, 1578?1581 (2000). The first use of somatic nuclear transfer to explore facets of the biology of X inactivation. 48. Okamoto, I., Tan, S. S. & Takagi, N. X chromosome inactivation in XX androgenetic mouse embryos surviving implantation. Development 127, 4137?4145 (2000). 49. Goto,Y. & Takagi, N. Maternally inherited X chromosome is not inactivated in mouse blastocysts due to parental imprinting. Chromosome Res. 7, 101?109 (1999). 50. Tada, T. et al. Imprint switching for non random X- chromosome inactivation during mouse oocyte growth. Development 127, 3101?3103 (2000). 51. McDonald, L. E., Paterson, C. A. & Kay, G. F. Bisulfite genomic sequencing-derived methylation profile of the Xist gene throughout early mouse development. Genomics 54, 379?386 (1998). 52. Lee, J. T. Disruption of imprinted X inactivation by parent- of-origin effects at TsiX. Cell 103, 17?27 (2000). 53. Prissette, M., El-Maarri, O., Arnaud, D., Walter, J. & Avner, P. Methylation profiles of the DXPas34 locus during the onset of X?inactivation. Hum. Mol. Genet. (in the press). 54. White, W. M., Willard, H. F., Van Dyke, D. L. & Wolff, D. J. The spreading of X inactivation into autosomal material of an X;autosome translocation: evidence for a difference between autosomal and X-chromosomal DNA. Am. J. Hum. Genet. 63, 20?28 (1998) 55. Duthie, S. M. et al. Xist RNA exhibits a banded localization on the inactive X chromosome and is excluded from autosomal material in cis. Hum. Mol. Genet. 8, 195?204 (1999). 56. Riggs, A. D. Marsupials and mechanisms of X chromosome inactivation. Aust. J. Zool. 37, 419?441 (1990) 57. Bailey, J. A., Carrel, L., Chakravarti, A. & Eichler, E. E. Molecular evidence for a relationship between LINE-1 elements and X chromosome inactivation: the Lyon repeat hypothesis. Proc. Natl Acad. Sci. USA 97, 6634?6639 (2000). 58. Carrel, L., Cottle, A. A., Goglin, K. 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Horn, J. H. & Ashworth, A. A member of the caudal family of homeobox genes maps to the X-inactivation centre region of the mouse and human X chromosomes. Hum. Mol. Genet. 4, 1041?1047 (1995). 70. Rougeulle, C. & Avner, P. Identification of an S19 pseudogene lying close to the Xist sequence in the mouse. Mamm. Genome 7, 606?607 (1996) 71. Lyon, M. F. X-chromosome inactivation: a repeat hypothesis. Cytogenet. Cell Genet. 80, 133?137 (1998). � 2001 Macmillan Magazines Ltd "
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