Mammalian X-chromosome inactivation and the XIST gene Andrea Ballabio and Huntington F. Willard Baylor College of Medicine, Houston, Texas and Stanford University, Stanford, California, USA X-chromosome inactivation is a unique developmental event that results in the cis-limited transcriptional inactivation of most genes on one of the two X chromosomes in female mammals. Studies in both human and mouse have demonstrated that X inactivation requires the presence in cis of a locus, the X-inactivation center, that is thought to be involved in the initiation and/or spreading of the inactivation signal in early development. Identification and characterization of a gene, XlST, which is located at or near the X-inactivation center and which is expressed specifically from the inactive X chromosome in both humans and mouse, suggests that it may be involved in X inactivation. Current Opinion in Genetics and Development 1992, 2:439-447

Introduction X inactivation is the process by which mammals compensate for the presence of an unbalanced dosage of X chromosome-linked genes between sexes. This phenomenon involves a series of events leading to the transcriptional 'switch off of genes on one of the two X chromosomes in female mammals (for recent reviews, see [1,2]). Although the mechanisms of the X inactivation process are far from being understood, studies on several mammalian species, and comparisons with different dosage compensation processes in other organisms [3,4], have helped delineate its complexity. Three aspects of this phenomenon are particularly fascinating: first, the long-range action of the X-inactivation process can spread over a hundred million base pairs of DN& turning off up to an estimated several thousand genes on a chromosome; second, the cis-action, which is the capability of distinguishing between, and acting on, only one of two apparently identical chromosomes within the same nucleus; and third, the stable transmission of the inactivation signal through mitosis during somatic development. As a consequence of this stable transmission, cells originating from a common progenitor cell after the initiation of X inactivation will have the same X c h r o m o s o m e inactivated. Conceptually, it is useful to think of X inactivation as occurring in three sequential steps: first, initiation early in development in XX embryos at a site on the X chromosome called the X-inactivation center; second, spreading of the inactivation signal along the length of the X chromosome selected to be the inactive X; and third, stabi-

lization and maintenance of the inactive state at individual gene loci on the X. While the basic tenets of the hypothesis proposed by Mary Lyon have been amply demonstrated over the past 30 years, our understanding of even the most fundamental aspects of the inactivation process remains incomplete. New revelations continue to yield sometimes surprising and unanticipated results. For many facets of X inactivation, recent advances have provided new impetus to the field and promise to reveal much about the molecular mechanisms of inactivation and their genetic control. In this review, we discuss some of these findings, concentrating on reports in the literature published within the last 15 months, up to March 1992.

Some genes on the human X chromosome 'escape' inactivation That not all genes on the X chromosome need be subject to inactivation has been appreciated for over 30 years, since the prescient prediction by Lyon [5] that genes in the pseudoautosomal pairing segment of the X and Y chromosomes might be expected to be expressed from both active and (otherwise) inactive X chromosomes, thus 'escaping' inactivation. Indeed, this has turned out to be the case, at least for the one human pseudoautosomal gene studied in depth, the MIC2 cell surface antigen gene. Even after the strictly X-linked steroid sulfatase (STS)-encoding gene was also shown to escape (at least partially) X inactivation, this was con-

Abbreviations ORF~open reading frame; RT--reverse transcribed; Xce---X-controlling element; XIC--X-inactivation center; XIST--inactive X (Xi)-specific transcript.

(~ Current Biology Ltd ISSN 0959-437X

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Mammalian genetics sidered to be an evolutionary remnant of a time when STSwas pseudoautosomal, as it physically maps relatively close to the current pseudoautosomal boundary on the human X chromosome. Thus, until recently, the concept that most, perhaps virtually all, X-linked genes would be subject to X inactivation was widely accepted, despite the fact that relatively few genes (less than ~ 20) had been critically studied with unequivocal molecular or genetic tests. Recent data, however, have called the generality of this conclusion into question, at least for the human X chromosome. In addition to MIC2 and STS, at least six other genes have been demonstrated to escape X inactivation, being well expressed from both active and inactive human X chromosomes (CJ Brown, L Carrel, HF Willard, unpublished data) {6,7.,8°,9,10]. Although several of these genes (including STS, the Kallman syndrome gene KALIG1, and GSI, which is a cDNA of unknown Amction) are tightly clustered within a few million basepairs in Xp22.3 [7.,8o], the others are widely scattered over at least the proximal long arm (q) and the distal and proximal short arm (p) regions of the X (Fig. 1). Thus, while it is clear that there are several regions along the X that can harbor genes which escape inactivation, the number of genes studied to date is too small to evaluate whether neighboring genes in these regions (at least outside of Xp22.3) will also have a greater likelihood of escaping inactivation. Further, it is by no means clear what the mechat~sm of 'escape' is; such genes may never receive the X-inactivation signal or they may be unresponsive to (or unable to maintain) such a signal (e.g. [11]). Of particular interest will be determining the nature of a likely boundary (in Xp22.3) between genes that escape inactivation and those more proximal that undergo inactivation. This boundary may correspond to an evolutionary boundary as well, because, curiously, distal genes (including STS, KALIG1, MIC2, GS1) do not appear to be conserved in rodents, whereas more proximal genes clearly are. Establishing whether the basic mechanism of X inactivation is sequence-specific or region-specific remains an important goal of current research and will require isolation, mapping and testing of m a w new genes from different regions of the X.

now strong evidence from gene mapping studies that at least much of the short arm consists of genes that were autosomal (and thus not subject to X inactivation) in a common ancestor of the current eutherian, monotreme and marsupial lineages some 150-170 million years ago [12,13]. In contrast, all long arm genes studied are Xlinked both in eutherians and in marsupials [14]. It is of interest that, of the eutherian X-linked genes mapped to an autosomal location in marsupials or monotremes, some (such as the Duchenne muscular dystrophy gene, the ornithine transcarbmnylase gene, and the DNA polymerase cz gene) are clearly subject to X inactivation in eutherians, whereas others (such as the ZFX gene, encoding a zinc-finger protein) escape X inactivation. The features of these genes (i.e. sequence or regional localization on tile X) responsible for their differential behavior with respect to X inactivation remain unknown, but of great interest.

Genes studied on the mouse X chromosome are all subject to X inactivation Three of the genes shown to escape X inactivation on tile human X chromosome have also been evaluated in mouse. Unexpectedly, all three - - Zfa, Rps4 (encoding the ribosomal protein $4), and Als9 (encoding the ubiquitin-activating enzyme El) - - have been shown to be subject to inactivation ill mouse [15°,16..,17,18].

The proportion of X-linked genes escaping inactivation also remains unknown. Of the human X-linked genes most recently identified and studied, at least half appear to escape inactivation. This number is, however, somewhat biased by a focus on genes on the short arm, in particular in Xp22.3. Nonetheless, given the current data, it seems reasonable to predict that a significant proportion of all X-linked genes from both the short and long arms will eventually be shown to escape inactivation. Such a finding would have significant implications for understanding patterns of expression of X-linked genes in carrier females for X-linked disorders, as counselling in such disorders is now based largely on the assumption that X-linked genes are subject to X inactivation.

While it appears likely that tile data reflect actual differences in X inactivation between human and mouse, it is also possible that they are, at least in part, a reflection of the methods used to exmnine gene expression from the inactive X. In tile case of the human X chromosome, tile conclusion that the genes escape inactivation rests largely on the demonstration of transcription in rodent-human somatic cell hybrids retaining the inactive X chromosome [6,7o,8.,9,10], confirnled in some cases by dose-dependent RNA levels in male or female sanlpies with different numbers of X chromosomes [9,10]. Only in the case of STS has escape from inactivation been definitively shown, using genetically marked alleles to demonstrate expression of both X chromosomes in female cells [19]. Because it is fommlly possible that the data on the other X-linked genes believed to escape inactivation reflect unusual transcriptional control and/or failure to maintain the inactive state in the context of rodent-human somatic cell hybrids, it will be important to confirm the expression of both active-X and inactive-X alleles in the same cell in diploid human material using genetically marked alleles. This seems particularly true for A1SgT (now known as UBE1, reflecting its identification as the ubiquitin-activating enzyme E1 gene), as data obtained in somatic cell hybrids demonstrating escape from inactivation [6] could not be confirmed by the RNA dosage approach in human material [20].

Among X-linked genes, those located on the short arm might be more likely a priori to be expressed from both active and inactive X chromosomes, as there is

In the case of the murine homologs, the X-inactivation status of genes has been assessed in Mus muscuhts x Mus spretus interstrain crosses in which the expression

Mammalian X-chromosome inactivation and the XIST gene Ballabio, Willard Genes known to be subject to X inactivation

Genes known to escape X inactivation

"~] MIC2 "~ GS1,57"5,KALIG1 PDHA E POLA C

- ] zrx

DAVIDE OTC, CYBB[~

r,Me ARAFE

--1 A,Sgr/UBel

TFE3 r'DX5423

ARC PGKI [~

RPS4X

I XlC

CLAE

HPRTC IDS C G6PD E

Fig. 1. Comparative localization of genes on the human X chromosome that are known to be subject to X inactivation (left) or are known to escape inactivation (right). In addition, the position of the X-inactivation center (X/C) is indicated. Although there is suggestive evidence for many other genes being subject to inactivation (see [2] for a review), only those with direct demonstrations of inactivation and which are, in addition, well localized on the chromosome are included. Gene acronyms include: PDHA, pyruvate dehydrogenase; POLA, DNA polymerase o~;DMD, Duchenne muscular dystrophy; OTC, ornithine transcarbamylase; CYBB,cytochrome B; TIM& tissue inhibitor of metalloproteinases; ARAF, A-raf oncogene; TFE3,immunoglobulin heavy chain enhancer-binding protein; AR, androgen receptor; PGK1, phosphoglycerate kinase 1; GLA, c~ galactosidase; HPRT, hypoxanthine phosphoribosyl-transferase; ID5, iduronate sulfatase; G6PD, glucose-6-phosphate dehydrogenase; MIC2, cell surface protein MIC2; G51, anonymous gene of unknown function; STS, steroid sulfatase; KAUG1,Kallmann syndrome gene; ZFX,zinc-finger protein; A1S9T/UBE1,ubiquitin-activating enzyme El; DXS423, anonymous gene of unknown function, RPS4X,ribosomal protein $4. Evidence regarding the genes ARAF, TFE3,IDS,DX5423 is from CJ Brown, L Carrel and HF Willard (unpublished data).

of the two alleles could be definitively monitored by using strain-specific DNA polymorphisms. For all genes examined, transcription from only the M. m u s c u l u s allele was detected in crosses in which the 34. spretus chromosome was preferentially inactivated because of an X; autosome translocation [15",16°',17,18]. These data demonstrate

that the Zfx, Rps4, and AIsggenes - - in apparent contrast to their human homologs - are subject to X inactivation in mouse. As discussed by Ashworth et al. [16o.], this finding may offer an explanation for the difference in viability of XO conceptuses in humans and mice. In humans, female embryos may require a double dose of genes like ZFX; RPS4X and UBEI for normal development. In the absence of a second X or Y chromosome, monosomy for X-linked genes may predispose to a functional deficit that underlies both the embryonic lethality and the symptoms of Turner syndrome in the relatively few XO conceptuses that survive to term. In mice, on the other hand, the XO condition is viable and associated with near normat fertility. Since the X-linked genes examined undergo X inactivation in mouse, the XO and XX conditions are functionally equivalent. It is, however, by no means established that all genes on the mouse X undergo X inactivation. The only genes examined thus far are those previously examined in humans. It may be that a more general screen of X-linked genes would reveal a subset of genes that escape inactivation in the mouse. In this light, it will be important to critically re-evaluate the mouse Sts gene, as conflicting data on its possible pseudoautosomal localization and possible escape from inactivation have been reported. The genetic basis for the species difference in X inactivation remains unclear. In some cases, the genes studied fall into a syntenic group that is conserved between mouse and human. Thus, altered chromosomal context appears not to be a universal explanation. Further, there is no consistent difference between the mouse and human genes with respect to the presence or absence of a functional Y chromosome-linked homolog, often proposed as the discriminating feature between genes subject to or escaping from inactivation. For example, RPS4X has a Y homolog in humans, but not in mouse, whereas the opposite appears to be true for UBE1, which has a Y homolog in mouse, but not in humans. It may be that relatively subtle differences in the timing of inactivation between the species or in the relative levels of expression from X- and Y-linked genes underlies whether an X-linked gene participates in X inactivation or not. For most genes, it is not obvious that a strong selective advantage should exist that would determine whether a gene might undergo X inactivation or not, thus raising the possibility that such a decision could be made on a 'gene-by-gene' basis through evolution. Nonetheless, that three out of three genes examined should show the same species difference suggests a more systematic basis for the X-inactivation decision.

DNA methylation, chromatin, and X inactivation It is generally accepted that DNA methylation plays a major, perhaps critical, role in at least the maintenance of X inactivation. DNA methylation~ particularly at CpG islands

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Mammalian genetics at or near the 5' end of X-linked genes, correlates with the inactivation profile, and inhibitors of DNA methylation (such as 5-azacytidine) can reactivate previously inactivated X-linked loci (reviewed in [1,2]). Several recent reports have extended our understanding of these relationships. The correlation of methylated CpG islands for X-linked genes undergoing X inactivation, based initially on work of Migeon and her colleagues (see [2]), has been extended to new genes, both on the human and mouse X chromosomes. Maestrhxi et al. [21] described a new gene tightly linked to the G6PD gene in Xq28. The CpG island associated with this gene is methylated on the inactive X and unmethylated on the active X. Similar data have been reported for the FMR. I gene associated with the fragile-X sTndrome [22], as expected if this gene is subject to X inactivation. A more general correlation has been reported on the mouse X. Norris et aL [23] studied methylation at 13 X-linked CpG islands. For 11 of these, there was clear evidence of inactive X-associated DNA methylation. In contrast, two of the islands were unmethylated on both the active and inactive X's. These may reside near genes that escape inactivation, perhaps because they are pseudoautosomal [23]. Overall, the data strongly indicate that DNA methylation at CpG islands (at least in eutherians) is an important aspect of stabilizing the inactive state after the time of X inactivation early in development. Elegant studies using the ligation-mediated genomic sequencing technique, which allows direct detection of 5-methylcytosine in genomic DNA, have provided detailed infornlation about methylation at the promoter of the PGK1 gene on the active and inactive X chromosomes. Riggs and colleagues [24,25] have shown that 60/61 potential CpG sites are methylated on the inactive X, whereas the active promoter has no methylation, both in human diploid material and in rodent-human somatic cell hybrids containing active or inactive X chromosomes. In vivo footprinting analysis demonstrated a number of protein-DNA contacts (e.g. trm~scription factor-binding sites) at the active X-chromosome PGK1 promoter. No such contacts were apparent on the inactive X [25], although the transcriptional start site does appear to be occluded by a nucleosome-sized particle specifically on the inactive X [26"]. One model consistent with these data is that the fully methylated, inactive X promoter is packaged with a methylated DNA-binding protein, such as the candidate protein described by Meehan et al. [27] that specifically binds clusters of methylated CpG's, thus preventing access to the inactive-X gene by transcription factors or by RNA polymerase [28--]. Under this model, the unmethylated promoter on the active X would be perpetuated in a transcription-competent state, whereas the methylated promoter on the inactive X would be inaccessible and transcription-incompetent [26-]. It will be important to evaluate the components of this model for other X-linked genes that are subject to or escape from inactivation. This may be achieved by using ligation-mediated genomic sequencing and active or inactive X-specific footprinting and/or by using the in vitro methylation/transcfiption and transfection assays of Boyes and Bird [28,29].

Localization of the X-inactivation center In both humans and mouse, there is strong evidence from the stud}, of structurally abnormal X chromosomes that initiation of X inactivation requires a c/s-acting locus on the X chromosome that is called the X-inactivation center (X/C and Xic in humans and mouse, respectively). According to current models, a chromosome retaining X/C can participate in X inactivation, whereas one missing X_/C cannot. Previous analyses had localized the inactivation centers in both species to a region close to the genes for androgen receptor (AR/Ar), ectodennal dysplasia (EDA) and phosphoglycerate kinase 1 ( PGK1/Pgk- 1). On the human X, this corresponds cytologically to Xq13, the region required for fomlation of a visible Barr body in nuclei containing an inactive X chromosome, as well as the region in which a cytological fold is apparent on the inactive, but not the active, X chromosome (reviewed in [2] ). New information on the nature of the Barr body and the significance of this fold has come from studies reported by Migeon and colleagues [30"]. Using in situ hybridization to interphase nuclei, they demonstrated that the inactivated X in the Barr body appears to consist of a looped chromosome, anchored at file nuclear periphery by its two telomeres. These data show that tile configuration of tile inactive X is distinctly non-random, but call into question the significance of the fold, which may simply reflect the mid-point of the looped X, rather than a distinct characteristic of the Xic itself. Recent studies have significantly refined tile localization of XIC/Xic on both the human and mouse X chromosomes (Fig. 2). In the mouse, Xic has been localized between two X-chromosome breakpoints, and is most likely between the tabby (Ta) locus (the murine equivalent of EDA) and the Pgk-1 locus [31]. The most refined mapping has benefitted from a new radiationinduced deletion that removes the region surrounding the Ar and Ta loci [32,33]. As the deleted X still appears to be capable of X inactivation, the deleted region can be excluded as a potential location for the Xic. The remaining Xic candidate region is also the site of the Xce (X-controlling element) locus described by Cattanach, alleles at which affect the randomness of X inactivation. It is likely, but as yet unproven, that Xce and Xic are one and the same locus. The location of the X/C locus on the human X-chromosome has also been delineated by two X-chromosome breakpoints [34"], one in an X; autosome translocation in which the distal portion of the translocated X remains capable of X inactivation, the other in a case of an isodicentric Xp chromosome that is also capable of inactivation [35]. This region lies between EDA and PGK1 [34"], consistent with the mouse data [31,36]. The X/C region on tile human X contains at least 2.5 million basepairs of DNA and probably many, genes (RG Lafreniere, HF Willard, unpublished data). Thus, it will be importam to evaluate X inactivation in additional cases of X chromosome abnormalities involving breakpoints in this

Mammalian X-chromosome inactivation and the X I S T gene Ballabio, Willard

Human

Mouse

Cen

Cen

in which an X/C allele on one X predominates over the allele on the other X.

T16H AR EDA

" RPS4X, P H K A

~'

XIC

- XIST

,' Ta25

Ta-

', deletion

Ccg- 1 -

" CCG 1

t(X;14)

At-

Rps4, Phka -

region

Xist -

idic (Xp) --D - PGKI

Pgk-1

HD3

Tel

Tel

Fig. 2. Comparative physical map of the X-inactivation center (X/C) region on the human and mouse X chromosomes with the most centromeric (Cen) and telomeric (Tel) regions shown. The XlC candidate region is shaded. The chromosomal breakpoints defining the XlC region are indicated by black arrows. In the mouse, the region removed in the Ta2s deletion can be excluded as a potential location for XIC. Although the region centromeric of the proximal Ta2s breakpoint cannot be formally excluded from the XlC candidate region, this is unlikely based on the homology with the human X chromosome. Thus, it seems reasonable to assume that the critical region in the mouse lies as indicated, between the distal breakpoint of the Ta2s deletion and the HD3 breakpoint. The order of loci between human and mouse is completely conserved in this region. Loci acronyms include: AR/Ar, androgen receptor; EDA, ectodermal dysplasia; CCG'UCcg-'/, cell-cycle progression gene; RPS4X/Rps4, ribosomal protein $4; PHKA/Phka, phosphorylase kinase; XlST/Xist, inactive X-specific transcript; PGK1/Pgk-'I, phosphoglycerate kinase, Ta, tabby; idic, isodicentric chromosome.

region in an attempt to further refine the exact location of file X/C. The presence of other genes notwithstanding, both the X/C and Xic candidate regions on the human and mouse X chromosomes contain a gene that has tile unusual (and thus far unique) property of being expressed only from inactive X chromosomes [37.°,38%39"]. This gene, called XIST/Xist(for inactive X (Xi)-specific transcript), is a candidate for the X-inactivation center.

Genetic conditions associated with possible X-inadivation variants or anomalies? The existence of the Xce alleles in mice raises tile possibility of genetic variation at the human X-inactivation center and of observable consequences of such variation. As with the Xce alleles, variants of human X inactivation might be expected to manifest non-random inactivation

Clarke et al. [40] described a case of a female ascertained because she had Hunter syndrome, an X-linked recessive trait associated with deficiency of iduronate sulfatase (IDS) activity. Unlike females expressing X-linked diseases in cases of X;autosome translocations, in whom the translocation breakpoint disrupts the X-linked gene and in whom the normal X is non-randomly inactivated (see [2] for review), this Hunter syndrome patient had two normal X chromosomes. Molecular genetic studies demonstrated that the girl was a carrier of IDS-deficiency and that manifestation of the disease was the result of non-random inactivation of the X chromosome carrying a normal IDS allele. Whether this case reflects an extreme, unbalanced version of X inactivation, as occasionally expected from a normally distributed random process, genetic variation at the X/C, or the existence of sequences near the IDS locus important for X inactivation, remains unknown. The third possibility is strengthened by the report of a second case of a deletion in Xq27 (including the IDS locus) in a mentally retarded female [41]. This case also showed non-random inactivation of the normal X, consistent with tile deleted region playing a role in X inactivation. While the above cases are unlikely to represent primary variants at the X/C locus, two other cases may. In one, Taylor etal. [42 °] described a fanlily with females in two generations affected with hemophilia B, an X-linked trait only rarely manifesting in carrier females. In the other, we have observed a family in which a mother and her daughter, each heterozygous for deletion of the STS locus, showed non-random inactivation of the deleted X (CJ Brown, HF Willard, unpublished data). As the deletion in this fan-fily is no larger than that seen in most cases of X-linked ichthyosis, the deletion itself is unlikely to be an explanation for the non-random inactivation. Such fan~ilies could represent a genetically determined nonrandom X inactivation, leading, in the case of the first family, to clinical expression of an X-linked defect in heterozygous carriers. Although the current data. do not allow one to determine tile basis for non-random inactivation, it is tempting to speculate that one or the other case could represent a genetic variant at the X inactivation center, as hypothesized by Taylor et aL [42"].

The

XIST

gene

An unanticipated finding in file study of X-chromosome inactivation has been tile identification of a gene, X/ST, which is expressed only from inactive X chromosomes [37.°]. This gene was first isolated, probably in a fortuitous manner, by the immunoscreening of a human placenta cDNA expression library using anti-human steroid sulfatase antibodies. It was assigned to Xql3 by in situ hybridization, and its position was subsequently refined by hybridization to the panel of somatic cell hybrids that was used for the mapping of the X-inactivation center [32]. The results showed coincident localization of X I S T

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Mammalian genelics with the X/C, thereby making X I S T t h e first gene assigned to the X-inactivation center critical region (Fig. 2). As its nmne suggests, the most striking feature of the X / S T gene was its pattern of expression. Hybridization of the X / S T cDNA clone to RNAs of human male and female individuals, and to h u m a n - m o u s e somatic cell hybrids retaining either the active or the inactive human X chromosome, revealed that this gene was exclusively expressed from the inactive X chromosome [37"]. Screening of cDNA libraries using the original X/STcDNA clone as a probe led to the isolation of several overlapping cDNAs. Assembly of a cDNA contig was, however, complicated by the presence of an extremely high frequenw of alternatively spliced transcripts, a feature confirmed by comparing the sequence of the cDNAs with that of genomic DNA. Sequence analysis of the longest cDNA contig originally assembled (2.2 kb) did not revea! the presence of any significantly long open reading frame (ORF) or of ,any homology with previously described genes [37"]. Because the total X_IST transcript appears to be > 15 kb in length, much more extensive studies are required. It was important to check whether XIST also showed these unique features in other species. AS X inactivation had been extensively studied in the mouse ,-rod the X/C had been mapped to a specific region of tile murine X chromosome, the mouse was selected as a model. The XIST murine homolog, Xist, was isolated by screening cDNA libraries using the human probe. Two independent studies reported the isolation of several murine Xist cDNA clones [38",39"]. In both studies, detailed mapping of Xist was achieved using the DNAs of mice from interspecific backcross panels. The map location of Xist was perfecdy consistent with the location of the human X / S T gene and therefore coincident with the assignment of both murine and human X-inactivation centers (Fig. 2). Expression was checked both by PCR analysis of reverse transcribed (RT) RNA and by northern blot analysis and was found to derive exclusively from the murine inactive X chromosome. This conclusion was supported by several lines of evidence. First, Xistwas expressed only in nom~al XX female mice, but neither in normal XY male mice nor in an XO female mouse in which the X was paternally derived [38",39"], and second (and most definitively), Xist expression was only detected from the inactive X chromosome of a M. musculus x M. spretus interspecific cross in which the M. m u s c u l u s X was totally and non-randomly inactivated [39"]. Tlaerefore, like its human homolog, the murine Xist is exclusively expressed from the inactive X ch romosome. Partial sequence analysis of Xist initially revealed the presence of a 900 bp ORF [38°]. More recent data, however, seem to indicate that the previously described ORF is unlikely to correspond to a protein product. Extensive sequence analysis of at least 2 kb of the region of the Xist transcript located upstream from the ORF indicates that the ORF does not extend further upstream and that there is no other significandy long ORF (A Ballabio, G Borsani, unpublished data). No evidence for a high frequenw of

alternative splicing was found in the murine Xist, unlike in the human X/ST. The degree of conservation of the X / S T gene was studied by comparing the sequence of the human with that of the murine homolog as well as with a small region of the feline and bovine homologs [38"]. Overall, tile identity between X / S T sequences in the different species was quite low and was not higher in the region of the human and murine homologs corresponding to the ORF, again suggesting that the ORF does not correspond to a protein product. The human and murine X/STgenes share two remarkable features: assignment to the X-inactivation center region and exclusive expression from the inactive X chromosome. Although this suggests that XIST has a role in X inactivation, it is difficult to predict what this role is, as the molecular mechmaism of X inactivation is completely unknown. Furthem)ore, sequence analysis of this gene to date has not provided any clue for an understanding of its function..As discussed previously [36,37",38",39"], X/ST may be involved either in the initiation mechanism by which the cell selects the X chromosome to be inactivated at the time of the embryogenesis when X inactivation appears, or only in the spreading of the inactivation process. The possible absence of a protein product encoded by the X/STgene may indicate that the .~STencoded product is a cis-acting RNA molecule [37"], involved (among other possibilities) in the formation of the heterochromatic Barr body. Current theories postulate the presence of a 'developmental factor' responsible for initiating the process of X inactivation at the X-inactivation center, and of a 'blocking factor' responsible for preventing the inactivation of the one X chromosome that remains active (e.g. [2,31,36]). The expression of both the developmental and the blocking factors may be subject to developmental control. X / S T may play different roles in this general model. It may either be directly responsible for the heterochromatinization process, or alternatively it may cause heterochromatinization by means of interaction with the developmental factor. All additional, pessimistic view would be that X / S T is not involved causally in X inactivation at all and that its unique expression pattern is a general feature of the X-inactivation center region. A refinement of this general model is suggested by the work of Fontes and colleagues [43], who reported a male patient with a duplication of the Xql3 region, including the XIC X / S T expression was exanlined and could not be detected in this patient, indicating that two copies of the X/C on two different X chromosomes are required for X/STexpression. The presence of two copies of the X/C on the same chromosome apparently does not lead to X inactivation, even of loci that are duplicated on that chromosome [43]. One intriguing hypothesis is to postulate that it is the transcriptional status of the X / S T gene (and not the sequence of the gene itself) that is critical for the spread of X inactivation. The active transcription of either X/ST, or of the entire region of the X chromosome in which X / S T is located, may cause a change in the chromatin structure

Mammalian X-chromosome inactivation and the XIST gene Ballabio, Willard of the region that would then spread to the rest of the chromosome, thereby preventing most of the genes from being actively transcribed. This model could be consistent with the possible absence of a protein product corresponding to XIST and with the lack of strong sequence conservation between the m o u s e and human genes (in the regions reported to date), as according to dais model the X/ST transcripts need not have a specific function once expressed. This model would also be consistent with the c/s-action of the X-inactivation center, as local chromatin structure spreading from X/ST, rather than a potentially diffusible substance, would be the agent of inactivation. The full characterization t)f the X1STgene and transcripts, ,and of the entire X/C region in nlan and mr)use will be required to clarify a number of questions. Are the intriguing features of XISTlimited to it or are they sharecl by a larger genolnic region? Does X/STcorresl)ond to a single or to nlultiple transcription~ units? Are there normally tr:mslated genes in the XIC region and are they expressed by flae inactive X chromos()nle? Expression studies should be able to detennine al what slage of embD:onal development XIST transcription starts. If .Y/ST expression is the "initiating factor' of X inactivation, then the onset of X1ST expression ratty be coincident with the developmental timing of X inactivation. Alternatively, X/ST expression may not be developmentally regulated, but m:l\' still be neccsszu-v to initiate X inactivatit)n by means ()f interactions with a developmentally regulated factor. Finally, two t3q~es of approaches, one 'negative' and t)ne 'positive', may allow testing the potential role of XIST:md, more generally, of the X-inactivation center. In the 'negative' approach, experiments aimed :it destrt)ying X/ST function by gene knock-out in either cultured cells or in embr3'onal stem cells for the creation of transgenic aninmls should reveal if the absence of XJST transcription corresponds to the absence t)f X inactivation. In the 'positive' apt)roach , insertion of X/ST in a transgenic animzd may cause selective inactivation of large regions of the genome. &s the extent of the functional X-inactivation center is still unknown, y e a s t artifickd c h r o m o s o m e s may serve as suitable vectors for the tmnsfection of the X/STgene or perhaps of the entire X-inactix'ation center region.

Acknowledgements We are grateful to G Borsani and C Brown for helpful discussions and comments and for providing the figures. We acknowledge support from the National Institute of Health (GM46970 to A Ballabio and GM45441 to HF Willard).

References and recommended reading Papers of particular interest, puMished within the annual period of review, have been highlighted :LS: • of special interest oe of ~,)utstanding interest I.

GtL.\NTSO, CIIAPbIANV: Mechanisms of X-chromosome Regulation. : l n n u Rev Genet 1988, 22:199-233.

2.

BROWNCJ. \";qLL,~RD Ill:: Molecular and Genetic Studies of Human X C h r o m o s o m e Inactivation. Ach, Dev Bfol. in press.

3.

VII.I.ENI-UVF AM, MEYI'R BJ: The Regulato W Hierarchy Controlling ~ x Determination and Dosage Compensation in Caenorhabditis elegans. A d v Genet 1990, 27:117-188.

q.

KIRODAMI, KEIC,:AN MJ, KRFBI'R R, G,LNH-ZKY B, BAKER BS: The Malelcss Protein Associates with the X C h r o m o s o m e to Regulate Dosage Compensation in D r o s o p h i l a . Cell 1991, 66:9Y~ 9-17.

"3.

lh'()N MF: ~ x Chromatin and Gene Action in the Manlmalian X-chromosome. Am .I H u m Genet 1962. 14:13q-lq'3.

O.

BR(3X";NCJ, \Y!II.IARD HF: Noninactivation of a Selcctable Human X-linked Gone that Complements a Murine Temperature-sensitive Cell Cycle Defect. Am .1 Plt#n Genet 1989. 45:592-598.

7. •

FIL-LNt;OB. Ot'lOU S, PI',AGI.R)IA A. IN('I:RTI B, BAREX)NI B, TONI.ORENZIP,, CARRt)7~.O R, MAI-.'s'n(INIg.., PII-RE'I'I'IiX'l, TAII.LONMII.IJZI,: P. I~1 .4L: A Gene Deleted in Kallmann's Syndrome Shares Homology with Neural Cell Adhesion and Axonal Path-finding Molecules. N(ltttrt, 1991, 353:529-536. This paper denlt)nstRUes that tile Kallnlan syndronle gene, I,L4LIGI. escapes inactivation..tvs this gene maps to vdthin a few million h:Lsepairs (ff the $7S gene, it supports the concept of a cluster of genes in Xp22.3 that escapes inactivation. 8. •

YFN PII. EI.LISON.], SAIJIX) EC, MOHANI)tLST, SHAPIRO L: Isolation of a N e w Gene from the Distal Short Arm of the Human X C h r o m o s o m e that Escapes X-inactivation. Hum Mol Genet 1992, 1:47-52. This paper descrihed a new gene oF unkno',Xql fi.mctk)n and is significant because this gene also maps to the cluster in Xp22.3. Thus, at least three contiguous genes all escape inactivation, suggesting coordinated regulation at the level of a large chromosomal region. 9.

FISHEREMC, BEER-ROMF.RO P, BROWN LG, RIDt.EY A, MCNEIL JA, LatWRENCEJB, WII.IARD HF, BIEBER FR, PAGE DC: Homologous Ribosomal Protein Genes on the Human X and Y Chromosomes: Escape from X Inactivation and Possible Implications for Turner Syndrome. Cell 1990, 63:1205-1218.

10.

SCHNEIDER-GI~DICKEA, BEER-ROMERO P, BROWN LG, NUSSBAUM R, PAGE De: ZFX has a Gene Structure Similar to ZFY, the Putative Human Sex Determinant, and Escapes X Inactivation. Cell 1989, 57:1247-1258.

11.

MOI-Im'4D,VST, GELLER RL YEN PH, ROSENDORFFJ, BERNSTEtN R, YOSHIDA A, SHAPIRO LJ: Cytogenetic and Molecular Studies on a Recombinant Human X Chromosome: Implications for the Spreading of X C h r o m o s o m e Inactivation. Proc N a i l A c a d Sci USA 1987, 84:4954--4958.

12.

SPENCERJa, SINCLAIR All, WATSON JM, GRAVES JAM: Genes on the Short Arm of the Human X C h r o m o s o m e are not Shared w i t h the Marsupial X. Genornics 1991, 11:339-345.

Conclusion The developmental mid genetic mechanisnas underying X inactivation in manlmals remain unknown, some 30 years after the original observations. Nonetheless, recent advances give cause for optimism that the secrets of flais fascinating chromosome-level regulatory system will finally yield to experimental investigations at a number of related levels - - molecular, genetic, chrornosomal and developmental.

445

446

Mammalian genetics 13.

14.

WATSONJM, SPENCERJA, RIGGSAD, GRAVESJAM: Sex Chromosome Evolution: Platypus Gene Mapping Suggests that Part of the Human X Chromosome was Originally Autosomal. Proc Natl Acad Sci USA 1991, 88:11256-11260. SPENCERJA, WATSONJM, GRAVESJAM: The X Chromosome of Marsupials Shares a Highly Conserved Region with Eutherians. Genomics 1991, 9:598--604.

15. •

ADLERDA, BRESSLERSL, CHAPMANVM, PAGE DC, DISTECHE CM: Inactivation of the Zfx Gene on the Mouse X Chromosome. Proc Natl Acad Sci USA 1991, 88:4592-4595. Together with the paper by Ashworth et al. [16*'], demonstrates that a gene which escapes inactivation in humans is subject to inactivation in mouse. 16. ..

suggests that the methylated promoter on the inactive X is inaccessible to RNA polymerase. 27.

MEEHANRR, lEWIS JD, MCKAY S, KLEINEREL, BIRD AP: Identification of a Mammalian Protein that Binds Specifically to DNA Containing Methylated CpGs. Cell 1989, 58:499--507.

28. o•

BoYEsJ, BIRD A: DNA Methylation Inhibits Transcription Indirectly via a Methyl-CpG Binding Protein. Cell 1991, 64:1123-1134. Investigates tile effect of DNA methylation on the expression of four model gene constructs and demonstrates that meth),lation4nediated repression is carried out by a methyl CpG-binding protein. This protein may be im,olved in maintaining both tile inaccessibility ,and gene inactivity at methylated CpG islands and may, therefore, be highly relevant to X inactivation.

ASHWORTHA, RASTAN S, LOVEtL-BADGE R, KAY G: X-chromosome Inactivation May Explain the Difference in Viability of XO Humans and Mice. Nature 1991, 351:406-408. A high b, significant paper that demonstrates, using a mouse inter-cross system, that two genes (ZFX and RPS4X) previously identified as escaping X inactivation in humans are both subject to inactivation in mouse. This suggests that there is a systematic difference between human and mouse X inactivation that may help to explain at least some of the clinical differences between mice and humans with XO karyot39es.

BOVESJ, BIRD A: Repression of Genes by DNA Methylation Depends on CpG Density and Promoter Strength: Evidence for Involvement of a MethyI-CpG Binding Protein..~ltBO J 1992, 11:327-333. Demonstrates, using in vitro methylation and transcription, :ks well as band-shift experiments, that the ability of the methyl CpG-binding protein to repress genes is correlated with the density of CpG's in their promoters. This finding may have implications for genes that, partially or completely, escape X inactivation.

17.

30.

KAY GF, ASHWORTH A, PENN'," GD, DUNLOP M, SWIFT S, BROCKDORFF N, RASTAN S: A Candidate Spermatogenesis Gene on the Mouse Y Chromosome is Homologous to Ubiquitin-activating Enzyme El. Nature 1991, 354:486-489.

18.

ZINN AR, BRESSLERSL, BEER-ROMERO P, ADLERDA, CHAPMANVM, PAGE DC, DISTECHE CM: Inactivation of the Rps4 Gene on the Mouse X Chromosome. Genomics 1991, 11:1097-1101.

19.

SHAPmOLJ, MOHANDAST, WEL%S R, ROMERO A: Noninactiration of an X Chromosome Locus in Man. Science 1979, 204:1224-1226.

20.

ZACKENHAUSE, SHEININR: Molecular Cloning, Primary Structure and Expression of the Human X.linked AlS9 Gene cDNA which Complements the tsAIS9 Mouse L Cell Defect in DNA Replication. F~qBO .I 1990, 9:2923-2929.

21.

MAESTRINIE, RIVEU.A S, TRIBIOU C, ROCCHI M, CAMERINO G, SAm'ACHtWa-BENEReCE'rn S, PAROUN~ O, NOTARANGELO LD, TONIOI.O D: Identificatioo of Novel RFLPs in the Vicinity of CpG Islands in Xq28: Application to the Analysis of the Pattern of X Chromosome Inactivation. Am J H u m Genet 1992, 50:156-163.

22.

PIERETrlM, ZHANG F, FU Y-H, WARRENST, OOSTRA BA, C,VSKEY CT, NEtsON DL: Absence of Expression of the FMR-I Gene in Fragile X Syndrome. Cell 1991, 66:817-822.

23.

NORRISDP, BROCKDORFF N, RASTAN S: Methylation Status of CpG-rich Islands on Active and Inactive Mouse X Chromosomes. M a m m Genome 1991, 1:78-83.

24.

PFEIFERGP, STEIGERWALDSD, HANSEN RS, GARTLER SM, RIGGS AD: Polymerase Chain Reaction Aided Genomic Sequencing of an X Chromosome-linked CpG Island: Methylation Patterns Suggest Clonal Inheritance, CpG Site Autonomy, and an Explanation of Activity State Stability. Proc Natl Acad Sci U S A 1990, 87:8252-43256.

25.

26. ••

PFEIFERGP, TANGUAYRL, STEIGERWAI.DSD, RIGGSAD: In Vivo Footprint and Methylation Analysis by PCR-aided Genomic Sequencing: Comparison of Active and Inactive X Chromosomal DNA at the CpG Island and Promoter of Human PGK-1. Genes Dev 1990, 4:1277-1287.

PFEIFERGP, RIGGS AD: Chromatin Differences between Active and Inactive X Chromosomes Revealed by Genomic Footprinting of Permeabilized Cells Using DNase 1 and Ligation-mediated PCR. Genes Dev 1991, 5:1102-1113. Establishes clear differences in chromatin between active-X and inactive-X promoters for a gene, PGK1, that is subject to inactivation. Demonstrates transcription factor footprints only on the active X and

29. •

WALKERCL, CARGILE CB, Ft.ov K M, DELANNOY M, MIGEON BR: The Barr Body is a Looped X Chromosome Formed by Telomere Association. Proc Natl Acad Sci U S A 1991, 88:6191-6195. Using fluorescence in situ hybridization and structurally abnormal X chromosomes, tile authors demonstrate that the inactive X appears to be attached to the nuclear periphery at both telomeres. The work is of interest as it provides, at least in part, an explanation for the condensed nature of the inactive X as the Barr body. •

31.

RAS'rANS, BROWN SDM: The Search for the Mouse X-chromosome Inactivation Centre. Genet Res 1990, 56:99-106.

32.

CATrANACHBM, RASBERRYC, EVANS EP, DANDOLO L, StMMLER MC, AVNERP: Genetic and Molecular Evidence of an X-chromosome Deletion Spanning the Tabby (Ta) and Testicular Feminization (Tfm) loci in the Mouse. Cytogenet Cell Genet 1991, 56:137-143.

33.

BROCKDORFFN, KAY G, CA'VI'tXNACHBM, RASTAN S: Molecular Genetic Analysis of the Ta25H Deletion: Evidence for Additional Deleted Loci. Mannn Genome 1991, 1:152-157.

34. •

BROWNCJ, LMaRENIERERG, POWEKSVE, SEB,XSTIOG, BALLABIOA, PE'Iq'IGREW AL, LEDBETI'ERDH, LE\~" E, CRAIG IW, WtUARD HF: Localization of the X Inactivation Centre on the Human X Chromosome in Xql3. Nature 1991, 349:824~4. Relines the localization of the X-inactivation center to a relatively small region in band Xql3 of the human X chromosome. Since the XISTgene maps within this region, this finding lends support to the hypothesis that XISTis involved in X inactivation.

35.

PE'I'lqGREWAL, McC,'.BE ERB, ELDFRFFB, LEDBEI'FERDH: Isodicentric X Chromosome in a Patient with Turner Syndrome - - Implications for Localization of the X-inactivation Center. H um Genet 1991, 87:498--502.

36.

BROWNSDM: XIST and the Mapping of the X Chromosome Inactivation Centre. Bioessays 1991, 13:607-612.

37. •.

BROWNCJ, BALLABIO A, RUPF.RT JL, LAFRENIERE RG, GROMPE M, TONI.ORENZI R, WII-LM~.Dl-IF: A Gene from the Region of the Human X Inactivation Centre as Expressed Exclusively from the Inactive X Chromosome. Nature 1991, 349:38-44. Describes a novel gene that is expressed only from inactive X chromosomes. This is significant because it suggests a role in the X inactivation process. Absence of an extended ORF suggests the possibility of a functional RNA molecule. 38. •

BORSANIG, TONLORENZI R, SIMMLER MC, DANDOLO L, ARNAUD D, CAPRAV, GROMPE M, PI7..ZUTIA, MUZNY D, LAWRENCEC, ET at.: Characterization of a Murine Gene Expressed from the Inactive X Chromosome. Nature 1991, 351:325-328.

Mammalian X-chromosome inadivation and the XIST gene Ballabio, W i l l a r d Confirms the female-specific expression of the X/STgene in mouse and maps the mouse homolog, Xist, to the region of the X-inactivation center. Together with the paper by Brockdorff et al. [39*], supports the possibility that X/STplays a role in X inactivation. 39. •

BROCKDORFF N, ASHWORTH A, KAY GF, COOPER P, SMITH S, MCCABE VM, NORRIS DP, PENNY GD, PATEL D, RASTAN S: Conservation of Position and Exclusive Expression of Mouse Xist from the Inactive X C h r o m o s o m e . N a t u r e 1991, 351:329-331. Establishes the inactive X-specific nature of Xist transcripts in mouse and maps XL~t to the region of the X-inactivation center. Significant because this paper demonstrates the capacity of the mouse inactive X to support transcription of at least one gene. 40.

CLARKE JTR, GREER WL, STRASBERG PM, PEARCE RD, SKOMOROWSKI /VIA, RAY PN: Hunter Disease (Mucopolysaccharidosis Type 1I) Associated with Unbalanced Inactivation of the X C h r o m o s o m e s in a Karyotypically Normal Girl. A m J Hunt Genet 1991, 49:289-297.

41.

SCHMIDTM, CERTOMAA, DU SART D, KALITSIS P, [.E\q~ILSHAM, FOWLER K, SHEFFIELD L, JACK l, DAN~S DM: Unusual X Chrom o s o m e Inactivation in a Mentally Retarded Girl with an

Interstitial Deletion Xq27: Implications for the Fragile X Syndrome. H u m Genet 1990, 84:347-352. 42. •

TAYLORSAM, DEUGAU I