Human Molecular Genetics

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Human Molecular Genetics, 2001, Vol. 10, No. 20 2225-2232
© 2001 Oxford University Press

Forty years of decoding the silence in X-chromosome inactivation

Rebecca Maxfield Boumil and Jeannie T. Lee⁺

Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, Boston, MA 02114, USA

Received July 3, 2001; Accepted July 24, 2001.

	ABSTRACT

TOP ABSTRACT INTRODUCTION THE X-INACTIVATION CENTER RANDOM X-INACTIVATION IS A... INITIATION AND ESTABLISHMENT:... MAINTENANCE OF THE INACTIVE... MODELS OF XIST ACTION THE PERPLEXITIES OF MACROH2A CHOICE: THE ROLE OF... FUNCTIONAL TSIX TRANSCRIPTION:... MODELS OF TSIX ACTION EVIDENCE FOR MORE REGULATORS... REFERENCES

 
In 1961, Mary Lyon first put forth the hypothesis that one X chromosome is inactivated in each cell of the female mammal. As we enter the new millennium and complete 40 years of study, the field of X-inactivation is rich with ideas and many contrasting viewpoints. This review will focus on the random form of X-inactivation and present the latest views on its mechanism. Much attention has been focused on the genetic parsing of X-chromosome counting, choice, silencing and maintenance. It is now known that counting is functionally distinct from choice and that initiation and establishment of silencing are distinct from maintenance. Since Xist’s seminal discovery 10 years ago, significant progress has been made towards understanding its function. Required only for initiation and establishment, Xist must act within a narrow developmental window, but its precise mode of action remains elusive. The ongoing search for Xist RNA-binding factors and effector proteins for silencing has led to members of the macroH2A family of histone variants. Finally, the recent discovery of Tsix implicates regulation of Xist expression by an antisense mechanism. Required for choice but not counting, Tsix blocks Xist RNA accumulation and hence blocks initiation of silencing on the future active X.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION THE X-INACTIVATION CENTER RANDOM X-INACTIVATION IS A... INITIATION AND ESTABLISHMENT:... MAINTENANCE OF THE INACTIVE... MODELS OF XIST ACTION THE PERPLEXITIES OF MACROH2A CHOICE: THE ROLE OF... FUNCTIONAL TSIX TRANSCRIPTION:... MODELS OF TSIX ACTION EVIDENCE FOR MORE REGULATORS... REFERENCES

 
It was 40 years ago that Mary Lyon proposed that dosage compensation takes place in mammals by silencing one of two X-chromosomes in female cells in order to achieve transcriptional balance with the XY male (1). It is now known that X-inactivation exists in an imprinted and a random form, with the imprinted form believed to be the ancestral mechanism. Marsupial mammals, such as the kangaroo, undergo non-random X-chromosome inactivation and preferentially shut off the paternal X chromosome (‘imprinted X-inactivation’) (2,3). In eutherians (placental mammals), inactivation takes place randomly in the soma so that either the paternally or maternally inherited X can be silenced (‘random X-inactivation’). In some eutherian mammals, vestiges of imprinted inactivation can be observed in extra-embryonic tissues (4,5), while random inactivation takes place in embryonic cell lineages (epiblast). Significant progress has been made in recent years towards understanding both forms of X-inactivation. As a review of imprinted X-inactivation will appear elsewhere (K.Huynh and J.T.Lee, manuscript in preparation), this essay will focus primarily on random X-inactivation. Because many excellent historical accounts already exist, we refer readers to other reviews for the older X-inactivation literature (6–9). Here, we will discuss new insights into Xist’s silencing mechanism, review our present understanding of macroH2A’s proposed role, and finally present evidence for an antisense mechanism of regulation by Tsix.

	THE X-INACTIVATION CENTER

TOP ABSTRACT INTRODUCTION THE X-INACTIVATION CENTER RANDOM X-INACTIVATION IS A... INITIATION AND ESTABLISHMENT:... MAINTENANCE OF THE INACTIVE... MODELS OF XIST ACTION THE PERPLEXITIES OF MACROH2A CHOICE: THE ROLE OF... FUNCTIONAL TSIX TRANSCRIPTION:... MODELS OF TSIX ACTION EVIDENCE FOR MORE REGULATORS... REFERENCES

 
During the mid-1990s, transgenic analyses in ES cells substantiated the decades-old concept of an X-linked ‘X-inactivation center’ (XIC/Xic) which controls the initiation of X-inactivation (10–14). Different investigators have proposed varying estimates of how much sequence is present in the Xic (Fig. 1A). Some believe that the Xic can be circumscribed by a 35 kb transgene (11), whereas others believe that many of the Xic’s properties are contained within 80 kb (14), and still others believe that the Xic might extend much farther along the X-chromosome (15). The lack of consensus reflects differing ideas about the Xic’s full range of function. While some groups describe only the silencing function (16), others include all contiguous X-linked sequences which effect or regulate X-inactivation (14,17).

View larger version (14K): [in this window] [in a new window]   Figure 1. The structure and function of the murine X-inactivation center (Xic). (A) The X-inactivation center and regulatory elements. The genes Xist and Tsix have been shown to regulate the silencing step and choice, respectively, during X-inactivation. Neighboring genes, Tsx, Brx and Cdx, are also shown. Proposed elements or regions for counting (17), choice (28,35,36) and the Xce (30) are indicated by colored bars below. The 35 kb cosmid transgene (11) and the 80 kb P1 transgene (10) have been proposed as the murine Xic. (B) The steps of X-inactivation. Indicated below each step are Xic genes and other factors proposed to function at each step.

 
Virtually everyone agrees that, at the minimum, the Xic contains Xist, the noncoding RNA gene first described 10 years ago by Hunt Willard’s group (18–20). Xist RNA accumulates on the inactive X (21,22) and is associated with the silencing step (23,24). Mounting evidence, however, implicates additional sequences 3' of Xist (Fig. 1A). Most notably, these include the antisense locus, Tsix (25), which harbors the differentially methylated minisatellite marker, DXPas34 (26). Tsix represses Xist expression and regulates X-chromosome choice (27,28). It remains possible that more control sequences lie even further 3' of Xist. One such regulator includes the Xce locus, an X-linked element uncovered 2 decades ago as a modifier of X-chromosome choice (a strong Xce haplotype is associated with greater likelihood of being chosen as the active X-chromosome in a hybrid background) (29). Genetic mapping data using microsatellite markers argue that the Xce resides within a 150 kb region upstream of the major Tsix promoter (30). Other genes have been found at or near the Xic, such as Tsx (31), Brx (32) and Cdx (33), but none of these genes has an expression pattern that fits the role of an X-inactivation regulator. While they lie within the proposed Xce region (30), these genes reside outside of the 35–80 kb putative Xic region (11,14).

	RANDOM X-INACTIVATION IS A MULTI-STEP PROCESS

TOP ABSTRACT INTRODUCTION THE X-INACTIVATION CENTER RANDOM X-INACTIVATION IS A... INITIATION AND ESTABLISHMENT:... MAINTENANCE OF THE INACTIVE... MODELS OF XIST ACTION THE PERPLEXITIES OF MACROH2A CHOICE: THE ROLE OF... FUNCTIONAL TSIX TRANSCRIPTION:... MODELS OF TSIX ACTION EVIDENCE FOR MORE REGULATORS... REFERENCES

 
It has been customary to divide random inactivation into the steps of counting, choice, initiation, establishment and maintenance (Fig. 1B). These steps are now known to be genetically separable and, with the exception of maintenance, appear to be controlled by the Xic (10,13,34). During the counting step, a cell measures the number of X-chromosomes relative to haploid autosome sets. In addition to autosomal loci, genetic evidence implicates sequences 3' of the Xist gene in the role of counting (17). During choice, all but one X-chromosome is committed to inactivation. Sequences within Xist (35,36), Tsix (28) and the Xce region (26) have all been proposed as regulators of choice. The initiation of silencing relies on Xist expression, but once silencing is established, maintenance of the inactive X is apparently independent of further Xic and Xist function (37,38).

	INITIATION AND ESTABLISHMENT: THE ROLE OF XIST

TOP ABSTRACT INTRODUCTION THE X-INACTIVATION CENTER RANDOM X-INACTIVATION IS A... INITIATION AND ESTABLISHMENT:... MAINTENANCE OF THE INACTIVE... MODELS OF XIST ACTION THE PERPLEXITIES OF MACROH2A CHOICE: THE ROLE OF... FUNCTIONAL TSIX TRANSCRIPTION:... MODELS OF TSIX ACTION EVIDENCE FOR MORE REGULATORS... REFERENCES

 
In the mouse, Xist occurs in three expression states, low, high and off (39–42). Prior to the onset of X-inactivation, cells of the embryo proper (epiblast) show low level expression from all X-chromosomes. The onset of X-inactivation coincides with upregulation of Xist to high levels on the chosen inactive X-chromosome and repression of Xist on the chosen active X. This implies a close relationship between initiation of silencing and Xist RNA accumulation along the X. In preimplantation embryos, however, the pattern of Xist expression does not seem to correlate with X-inactivation. Here, high level Xist expression can be detected from one X-chromosome of XX preimplantation embryos at the 4- to 8-cell stage (42–44), a time when many believe that there are two active Xs. This apparent uncoupling of Xist levels and silencing indicates that the regulation is more complex than present models can explain (discussed further by K.Huynh and J.T.Lee, manuscript in preparation).

What is responsible for the transition from low to high level Xist expression? Panning et al. (41) and Sheardown et al. (42) have proposed that the change in Xist levels involves increased RNA stability rather than increased transcriptional rate. Evidence for this idea includes nuclear run-off experiments which show that Xist transcription is equal in undifferentiated ES cells and fully differentiated XX cells. When cells are treated with actinomycin D to block new Xist synthesis, Xist RNA is apparently stable in differentiated somatic XX cells but is rapidly degraded in undifferentiated ES cells, consistent with differential RNA stability. Increased RNA stability might reflect binding to protective protein partners which occurs specifically at the onset of cell differentiation. Subsequently, the discovery of Tsix led to the idea that coexpression of the antisense RNA might destabilize Xist RNA in undifferentiated cells (25). Indeed, deletions of Tsix result in higher steady state levels of Xist in undifferentiated ES cells (28,45).

While evidence supports a role for RNA stabilization, the recent work of Wutz and Jaenisch (46) points to additional considerations. In this work, tetracycline induction of Xist transgene expression is sufficient to cause cis-silencing of autosomal genes. Because this silencing can take place in both undifferentiated and differentiated ES cells, the idea of a differentiation-specific stabilization factor seems less likely. One possibility is that the lack of Tsix expression from the transgene allows for stable, high level Xist expression in undifferentiated ES cells. The authors also proposed that a higher level of Xist product might itself raise the half-life of the RNA or that, contrary to the RNA stabilization hypothesis, transcription itself might be elevated.

This work also furthers the concept of a critical developmental window for Xist action (46). By inducing and withdrawing Xist expression on various days of ES cell differentiation, the study demonstrated that Xist-mediated silencing can only be established during the first 48 h of cell differentiation and that, beyond this stage, no degree of expression can cause cis-inactivation. Furthermore, once Xist-mediated silencing takes place during the first few days, withdrawing Xist expression does not perturb the inactive state.

	MAINTENANCE OF THE INACTIVE X

TOP ABSTRACT INTRODUCTION THE X-INACTIVATION CENTER RANDOM X-INACTIVATION IS A... INITIATION AND ESTABLISHMENT:... MAINTENANCE OF THE INACTIVE... MODELS OF XIST ACTION THE PERPLEXITIES OF MACROH2A CHOICE: THE ROLE OF... FUNCTIONAL TSIX TRANSCRIPTION:... MODELS OF TSIX ACTION EVIDENCE FOR MORE REGULATORS... REFERENCES

 
The idea that the inactive state can be maintained in an XIC-independent manner was first advanced by Brown and Willard (37) in work using human-mouse somatic cell hybrids. Acquired human X chromosomes lacking the XIST gene can also remain inactive in human leukemia cells (47). More recently, Csankovszki et al. (38) found the same in mice by a conditional deletion of the Xist gene in somatic cells demonstrating that loss of Xist expression did not affect the already established silent state of the inactive X. However, Csankovszki et al. (48) later reported that, although not necessary for maintenance per se, continuous Xist synthesis does contribute to stability of the inactive state. They propose a synergistic effect of Xist expression, DNA methylation and histone deacetylation on the overall inactive X stability. Synergism between deacetylation and DNA methylation is consistent with the report of Gilbert and Sharp (49), which shows that promoters of X-inactivated genes are coincidentally hypoacetylated and hypermethylated. These outcomes reinforce the concept of self-propagating heterochromatin which is hypoacetylated at N-terminal lysines of core histones (50,51), is late-replicating (52) and hypermethylated in promoter regions (53,54). In the case of the inactive X, hypoacetylation and late-replication appear to be specifically associated with the maintenance phase, as Xist-induced inactivation in undifferentiated and early differentiation ES cells are not associated with these changes (46).

In contrast to histone acetylation and replication timing, a role for DNA methylation in X-inactivation has been suggested for initiation as well as maintenance. Differentiating ES cell lines lacking the Dnmt1 ‘maintenance methylase’ exhibit Xist derepression in some cells and initiate inactivation of X-linked genes in cis (40,55), indicating an additional requirement for DNA methylation in blocking initiation of silencing in ES cells. An additional role in maintenance is demonstrated by the observation that, in mouse embryos homozygous for a Dnmt1 mutation, reactivation of an X-linked marker can be detected in epiblast-derived tissues (56). The recent isolation of a third family of DNA methyltransferases, Dnmt3a and Dnmt3b, adds another dimension to the control of X-inactivation (57). Mutations in DNMT3B cause the human disease of immunodeficiency centromeric instability facial anomalies (ICF) (57–59) for which instability of the silent state on the inactive X has been reported (60). In ICF patients, the inactive X is characterized by hypomethylation of CpG islands, an advance to an early replication timing and reactivation of X-linked loci (60). Thus, maintenance of the inactive state might depend on both the Dnmt1- and Dnmt3-type methylases.

	MODELS OF XIST ACTION

TOP ABSTRACT INTRODUCTION THE X-INACTIVATION CENTER RANDOM X-INACTIVATION IS A... INITIATION AND ESTABLISHMENT:... MAINTENANCE OF THE INACTIVE... MODELS OF XIST ACTION THE PERPLEXITIES OF MACROH2A CHOICE: THE ROLE OF... FUNCTIONAL TSIX TRANSCRIPTION:... MODELS OF TSIX ACTION EVIDENCE FOR MORE REGULATORS... REFERENCES

 
Much of Xist’s mechanism of action remains enigmatic. One model postulates that mere expression of the locus is sufficient to initiate silencing (21,61). That is, independent of Xist RNA itself, a moving polymerase complex could alter the chromatin state and allow silencers to bind the chromosome. No evidence directly supports this type of mechanism. A second model argues a functional role for the RNA product in light of Xist RNA’s unique ability to ‘paint’ the inactive X (21,22,61). In this model, translocation of Xist RNA along the X-chromosome enables deposition of silencing factors in a cis-limited manner. Despite its broad appeal, however, a definitive role for the RNA has also not been demonstrated. Validation of Xist RNA’s function might ultimately depend on the isolation of Xist RNA-interacting factors. One popular approach of the past few years has been to identify differences in the chromatin composition specific to the inactive X. This approach has led to the candidate Xist RNA-interacting factor discussed in the ensuing section.

	THE PERPLEXITIES OF MACROH2A

TOP ABSTRACT INTRODUCTION THE X-INACTIVATION CENTER RANDOM X-INACTIVATION IS A... INITIATION AND ESTABLISHMENT:... MAINTENANCE OF THE INACTIVE... MODELS OF XIST ACTION THE PERPLEXITIES OF MACROH2A CHOICE: THE ROLE OF... FUNCTIONAL TSIX TRANSCRIPTION:... MODELS OF TSIX ACTION EVIDENCE FOR MORE REGULATORS... REFERENCES

 
Costanzi and Pehrson (62) introduced the possibility of H2A histone variants as possible effectors of silencing and Xist RNA-interacting factors. One member of this family, macroH2A1 (with isoforms 1.1 and 1.2), caught widespread attention by virtue of its being enriched on the inactive X in female cells. In preimplantation mouse embryos, a macroH2A1.2-dense body appears during the 8- to 16-cell stages (63), a time when high-level Xist expression becomes detectable on one X. In mouse ES cells, the behavior of macroH2A1 is particularly intriguing. In ES cells that have not yet differentiated or undergone X-inactivation, a single macroH2A1.2-dense body is found in both XX and XY nuclei (64). At this time, the macroH2A1.2 body is not coincident with the X but unexpectedly turns out to colocalize with or reside near the centrosome (65). It is hypothesized that the centrosomal region acts as a storage site for macroH2A1.2 prior to its use for the process of X-inactivation in ES cells. In differentiating XY ES cells, the macroH2A1.2 body disappears so that staining becomes diffuse throughout the nucleus (64). In differentiating XX cells, the macroH2A1.2 body apparently relocates from the centrosome to the inactive X on the seventh day of differentiation (64,65).

Because the convergence of macroH2A1.2 with the inactive X occurs well after the onset of high level Xist expression in ES cells, it seems unlikely that macroH2A1.2 participates in the initiation of silencing. However, a potential role for macroH2A1.2 in some aspect of establishment or maintenance remains to be investigated. For this purpose, macroH2A1.2 could be recruited directly by Xist RNA. Both genetic and biochemical evidence points to there being some association between the two. First, chromatin immunoprecipitates of macroH2A1.2 contain Xist RNA, arguing that Xist RNA and macroH2A1.2 exist in a ribonucleoprotein complex (66). Secondly, localization of histone macroH2A1.2 to the inactive X is disrupted when Xist is conditionally deleted, implying that Xist expression is required for deposition of macroH2A1.2 (38). However, whether Xist RNA interacts directly with macroH2A is not answered by these studies. Moreover, loss of neither macroH2A1.2 nor Xist RNA on the inactive X leads to reactivation. Therefore, macroH2A1.2 cannot be strictly required for maintenance and its role remains an open issue at present.

Since the original description of macroH2A1’s association with X-inactivation, several other H2A-variants have been shown to have a non-random distribution on the active and inactive Xs. A human macroH2A gene, macroH2A2, was recently identified with 68% amino acid identity with human macroH2A1.2 (67,68) and is significantly enriched on the inactive X (Fig. 2A). In contrast, yet another H2A variant, H2A-Bbd, is relatively deficient on the inactive X (69) (Fig. 2B). These studies further the idea of there being functional differences in histone composition between active and inactive Xs.

View larger version (25K): [in this window] [in a new window]   Figure 2. Distribution of variant histone H2A proteins on the inactive X. (A) Localization of GFP-tagged MacroH2A2 in human fibroblast cell lines of 46XY, 46XX, 47XXX, 48XXXX and 49XXXXX individuals as indicated. The number of macroH2A2-dense bodies is one fewer than the number of sex chromosomes. (B) Indirect immunofluorescence showing that the myc-tagged H2A-Bbd protein (red) is relatively deficient on the inactive X as marked by macroH2A1 (green). [Figure by Chadwick and Willard (67)].

 
While the data support the involvement of macroH2As in the X-heterochromatin, the story may turn out to be much more complex. Perche et al. (70) demonstrated that many histones show more intense staining on the inactive X because of the higher degree of chromatin compaction. This study suggests that the inactive X is intensely stained not just for macroH2A1.2 but also for H2A, H2B and H3, implying that macroH2As might have the appearance of being enriched on the inactive X only because the chromosome is more densely packed or is somehow more easily targeted by the antibody. If so, the protein might not be any more relevant for X-inactivation than any other histone. However, conflicting data have also been presented by Chadwick and Willard (67) who show that epitope-tagged H2A and H2B are not more intensely stained on the inactive X. The contrasting results could be explained by differences in transfected constructs, whether or not epitope tags are used, levels of transient expression and the timepoint of analysis. Given the potential significance of variant histone involvement, a resolution of these contrasting data will be important in the future.

	CHOICE: THE ROLE OF TSIX

TOP ABSTRACT INTRODUCTION THE X-INACTIVATION CENTER RANDOM X-INACTIVATION IS A... INITIATION AND ESTABLISHMENT:... MAINTENANCE OF THE INACTIVE... MODELS OF XIST ACTION THE PERPLEXITIES OF MACROH2A CHOICE: THE ROLE OF... FUNCTIONAL TSIX TRANSCRIPTION:... MODELS OF TSIX ACTION EVIDENCE FOR MORE REGULATORS... REFERENCES

 
Our laboratory first found evidence of an antisense transcript in the Xist locus when studying an Xist transgene that apparently lacks an Xist promoter but which nonetheless yields a transcript from the locus (25). Strand-specific analyses revealed that most of the transcription actually originates from a prominent CpG island located 15 kb downstream of Xist and extends across Xist off the opposite DNA strand. Named ‘Tsix,’ the 40 kb gene has no conserved open reading frames and produces an antisense RNA that appears to be exclusively nuclear. Several features immediately suggested Tsix as an antagonist of Xist action. First, the Tsix locus contains DXPas34, a CpG-rich minisatellite marker which was reported in one study to show differential methylation on the active and inactive Xs (26). Secondly, its expression is dynamically associated with Xist expression during the process of X-inactivation (Fig. 3). In undifferentiated XX ES cells, Tsix is expressed together with low-level Xist on both X-chromosomes. In cells undergoing differentiation, Tsix RNA is turned off on one X-chromosome but persists on the remaining X. Intriguingly, the loss of Tsix expression correlates with upregulation of Xist on the future inactive X-chromosome, whereas its persistence correlates with inhibition of Xist induction on the future active X. In fully differentiated cells, Tsix is also turned off on the active X. This expression pattern suggests that Tsix is only expressed during the ‘reversible’ window of Xist expression and that Tsix might act as a repressor of X-inactivation.

View larger version (34K): [in this window] [in a new window]   Figure 3. The dynamic relationship between Xist and Tsix expression during ES cell differentiation. Strand-specific RNA FISH showing changes in expression of Xist RNA (fluorescein-labeled, green) and Tsix RNA (Texas-red labeled, red) at the onset of cell differentiation and X-inactivation. [Figure adapted from Lee et al. (25), with permission from Nature Genetics.]

 
The hypothesis that Tsix regulates X-inactivation has been strengthened by recent genetic studies (27,28,45,71). Knocking out the putative Tsix promoter and its associated CpG island (CpG) has distinct effects on both random and imprinted X-inactivation (effects on imprinted X-inactivation described by K.Huynh and J.T.Lee, manuscript in preparation). In cells that normally undergo random X-inactivation such as ES cells (and epiblast-derived somatic cells), X-chromosome counting is not affected because –/Y male and –/+ female cells still show proper dosage compensation (28). Thus, the deletion has no phenotype in –/Y male ES cells, presumably because of an operational counting mechanism that blocks the subsequent steps of X-inactivation. In –/+ female ES cells, a single X-chromosome is properly inactivated. Interestingly, however, X-chromosome choice is dramatically skewed towards inactivating the targeted X-chromosome. These results indicate that deleting Tsix does not disrupt counting but does affect choice, thereby functionally separating these two steps. In –/+ female cells, Xist is upregulated almost exclusively from the mutant X-chromosome, demonstrating that Tsix is indeed a repressor of Xist. Non-random inactivation of the Tsix-deficient chromosome has also been achieved by Debrand et al. (27) in a transgene deletion of the DXPas34 element and by Sado et al. (45) in a second targeted Tsix knockout.

Thus, these genetic studies attribute X-chromosome choice to the 5' CpG-rich domain of Tsix and raise the possibility that one aspect of Tsix’s mechanism relies on a CpG-rich DNA element (DXPas34). Indeed, Courtier et al. (26) have reported differential methylation on the active and inactive Xs of somatic female cells. However, the more recent work of Prissette et al. (72) has not found differential CpG methylation in early mouse embryos and gametes by bisulfite sequencing. Therefore, if DXPas34 acted as a DNA element, it may not be regulated by DNA methylation.

	FUNCTIONAL TSIX TRANSCRIPTION: REPRESSION OF INITIATION AND XIST RNA ACCUMULATION

TOP ABSTRACT INTRODUCTION THE X-INACTIVATION CENTER RANDOM X-INACTIVATION IS A... INITIATION AND ESTABLISHMENT:... MAINTENANCE OF THE INACTIVE... MODELS OF XIST ACTION THE PERPLEXITIES OF MACROH2A CHOICE: THE ROLE OF... FUNCTIONAL TSIX TRANSCRIPTION:... MODELS OF TSIX ACTION EVIDENCE FOR MORE REGULATORS... REFERENCES

 
The work of Stavropoulos et al. (73) provides evidence that antisense transcription also plays a functional role. By knocking in the constitutive human EF1a promoter into one Tsix allele in an XX background, the authors show that high level persistent Tsix transcription is sufficient to block the accumulation of Xist RNA on the same X-chromosome. In a differentiating population, Xist expression is markedly skewed towards the unaltered X-chromosome, giving rise to a non-random pattern of X-inactivation. In contrast to the Tsix knockout phenotype, the non-random inactivation in the knock-in is interpreted to be independent of X-chromosome choice. Indeed, consistent with a preservation of choice in this mutant background, Stavropoulos et al. (73) observe two subpopulations under differentiation conditions: In the first, X-inactivation is achieved and cells differentiate normally, but high level Xist expression originates from the normal X-chromosome. In the second, cells maintain two active X-chromosomes and differentiate poorly. The authors propose that this subpopulation consists of cells choosing the modified X for inactivation but cannot execute this decision because of high level Tsix expression in cis. Thus, high level antisense expression is sufficient to block Xist upregulation on the same X-chromosome, implicating transcription itself or the antisense RNA as an important aspect of regulation. The results also show that Tsix transcription operates downstream of choice.

In the context of previous work (27,28,45,71), this work supports a hypothesis in which silencing of Tsix is a prerequisite for Xist upregulation on the future inactive X. This is in agreement with published expression profiles of Tsix showing that Tsix is repressed before or around the same time that Xist RNA accumulates in cis (25,27). The work also argues that, conversely, Tsix transcription on the future active X is necessary to prevent high level Xist expression and the associated chromosome-wide changes. Indeed, expression profiles also show that Tsix persists for some time on the active X after the onset of X-inactivation (25,27). In the future, it will be important to determine whether Tsix-mediated repression requires increased Tsix expression or whether mere persistence of expression is sufficient to block Xist upregulation on the future active X.

	MODELS OF TSIX ACTION

TOP ABSTRACT INTRODUCTION THE X-INACTIVATION CENTER RANDOM X-INACTIVATION IS A... INITIATION AND ESTABLISHMENT:... MAINTENANCE OF THE INACTIVE... MODELS OF XIST ACTION THE PERPLEXITIES OF MACROH2A CHOICE: THE ROLE OF... FUNCTIONAL TSIX TRANSCRIPTION:... MODELS OF TSIX ACTION EVIDENCE FOR MORE REGULATORS... REFERENCES

 
How does Tsix expression block Xist RNA accumulation at the molecular level? Several potential mechanisms have been proposed (25) (Fig. 4). One possibility is that the action of RNA polymerase complexes moving in the antisense orientation is sufficient to inhibit production of the sense Xist transcript. A second model postulates a role for the antisense RNA itself. In this case, Tsix RNA might base-pair with sense RNA and block binding sites for Xist RNA-interacting proteins. Alternatively, duplex RNA formation could destabilize Xist RNA and effectively prevent its translocation across the designated inactive X-chromosome. Finally, the genetic findings are also consistent with a role for CpG-rich DNA elements at the 5' end of Tsix. These potential mechanisms are not mutually exclusive and, in fact, may work together to repress Xist expression.

View larger version (17K): [in this window] [in a new window]   Figure 4. Models of Tsix-mediated repression of Xist. (A) Tsix DNA sequence itself could function as a long-range silencer to repress or block the transcription of the Xist gene. (B) Transcription of Xist may be prohibited by the processivity of RNA polymerase in the antisense orientation. As RNA polymerase proceeds along the Tsix DNA, the ‘melting’ of the complementary DNA strands and the presence of the large polymerase complex along the template may serve as an obstacle for the transcription of Xist in the opposite direction. (C) Sites along the Xist RNA to which proteins bind may be blocked by the base pairing of sense and antisense RNA. These sites may not be functional as double-stranded RNA either through changes in secondary structure of the RNA or, alternatively, the Xist binding proteins may only bind to single-stranded RNA. (D) The formation of duplex RNA may render Xist RNA unstable. In this model, the duplex RNA may be more susceptible to degradation or may act to prevent the mobility of the Xist RNA along the inactive X chromosome or the duplex may prohibit the formation of stable Xist RNA that is necessary to initiate inactivation.

 

	EVIDENCE FOR MORE REGULATORS TO COME

TOP ABSTRACT INTRODUCTION THE X-INACTIVATION CENTER RANDOM X-INACTIVATION IS A... INITIATION AND ESTABLISHMENT:... MAINTENANCE OF THE INACTIVE... MODELS OF XIST ACTION THE PERPLEXITIES OF MACROH2A CHOICE: THE ROLE OF... FUNCTIONAL TSIX TRANSCRIPTION:... MODELS OF TSIX ACTION EVIDENCE FOR MORE REGULATORS... REFERENCES

 
Transgenesis experiments have shown that the Xic participates in many steps of X-inactivation including counting as well as choice and silencing. So far, elements involved in counting have been elusive. In a step towards identifying potential counting elements, Clerc and Avner (17) have shown that a 65 kb deletion immediately downstream of Xist (including the 5' end of Tsix) might harbor such elements. Similar to the Tsix knockouts, the 65 kb deletion also manifests non-random X-inactivation due to loss of X-chromosome choice, but it has one contrasting phenotype. When the deletion is present in a derivative cell line carrying only one whole X-chromosome, this single mutant X also undergoes inactivation, implying that something within the 65 kb region might be required for the correct calculation of the X:autosome ratio. One caveat of this experiment, however, is that the derivative cell line is not truly ‘XO’ but also contains fragments of the wild-type X-chromosome in addition to the whole mutant X. Therefore, it is formally possible that counting remains intact and the cell senses two X-chromosomes. Additional mutagenesis and analysis will be required to identify candidate counting elements in this region.

While genetic analysis clearly indicates that the Tsix locus is required for X-chromosome choice, it also implicates additional X-linked sequences in this decision. No element has been pinpointed for the Xce function (29), although Simmler et al. (30) have mapped it to a 150 kb region upstream of Tsix. It will be of interest to learn whether the Xce is a modifier of Tsix expression. Plenge et al. (35) have also associated a primary skewing of X-inactivation choice with a point mutation in the human XIST promoter in two unrelated families, suggesting that XIST itself participates in the decision of selecting X-chromosomes. Finally, Marahrens et al. (36) believes that a region within mouse Xist exons 1–5 controls choice. This is based on a phenotypic difference between the Xist knockout of Penny et al. (23), which yields secondary non-random inactivation, and that of Marahrens et al. (36), which is thought to yield primary non-random inactivation. The delineation of precise candidate elements is of major current interest.

To date, only cis-acting X-linked elements have been identified as regulators of X-inactivation. But for counting to work, trans-acting autosomal factors must participate in these decisions. This is deduced from the observation that ploidy affects the number of X-chromosomes chosen for inactivation (74,75). These autosomal factors are presumed to act at the Xic and to modulate expression of downstream target genes such as Xist and Tsix.

In addition to trans-acting factors for counting and choice, trans-acting factors must also exist for the silencing step. What do they bind and how does Xist RNA fit into this step? A hint can be found in the observation that autosomes as well as Xs can bind Xist RNA and be silenced (12–14,46). On the eve of the 40th anniversary of X-inactivation, Mary Lyon (76) has proposed yet another intriguing hypothesis, that LINE repeats could serve as way stations for the spread of silencing from the Xic. These long-interspersed repetitive elements are enriched on the X-chromosome and near breakpoints of X-autosome translocation chromosomes on which Xic-mediated silencing spreads into the autosome readily. Moreover, they are densely clustered at the human XIC and are relatively deficient in large domains which escape X-inactivation (77,78). The LINE hypothesis, however, is at odds with the study of Duthie et al. (79) which shows that rodent Xist RNA preferentially localizes to LINE-poor bands of the X. One way to reconcile these apparently contradictory findings is to argue that Xist RNA preferentially binds LINE-poor regions but recruits silencers to LINE-rich sequences. On the 40th anniversary of the original Lyon hypothesis, the question of how silencing emanates from the Xic to encompass the entire X-chromosome remains one of the most tantalizing problems in X-inactivation.

Thus, while the past 4 decades have witnessed major advances in understanding of how dosage compensation takes place in mammals, the mechanism of X-inactivation continues to baffle investigators. As is often true in rapidly moving fields of study, the X-inactivation community sees a fair share of disagreement and uncertainty in addition to excitement and triumph. To the larger community of biologists, it has contributed new ways of thinking about gene regulation and chromatin structure, not the least of which involves the sheer scale of silencing, the role of unusual histones, and the mysterious workings of noncoding RNA genes. The coming decade of research seems certain to bring more intriguing viewpoints and novelty to this classic genetic problem.

	ACKNOWLEDGEMENTS

 
We thank Khanh Huynh and Nicholas Stavropoulos for critical reading of this manuscript and other members of the laboratory for helpful discussion.

	FOOTNOTES

 
⁺ To whom correspondence should be addressed. Tel: +1 617 726 5943; Fax: +1 617 726 6893; Email: lee@frodo.mgh.harvard.edu

	REFERENCES

TOP ABSTRACT INTRODUCTION THE X-INACTIVATION CENTER RANDOM X-INACTIVATION IS A... INITIATION AND ESTABLISHMENT:... MAINTENANCE OF THE INACTIVE... MODELS OF XIST ACTION THE PERPLEXITIES OF MACROH2A CHOICE: THE ROLE OF... FUNCTIONAL TSIX TRANSCRIPTION:... MODELS OF TSIX ACTION EVIDENCE FOR MORE REGULATORS... REFERENCES

 
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