Expression of X-Linked Genes Is Up-Regulated in Both Female and Male ES Cells

Expression of X-linked genes in female and male ES cells, relative to autosomal genes (X:A ratio), was assayed by co-hybridisation of labelled cDNAs to NIA15K mouse cDNA microarrays [14]. Expression levels of 180 X-linked genes (see Text S1 for filtering criteria) were distributed over a ∼200-fold range, with a close correlation between expression in female and male cells (Figure 1A and 1B). Only two genes showed clear sex-specific expression in undifferentiated ES cells, namely Xlr3b and Xlr5, both of which showed minimal expression in the CCE/R male line. Consistent over-expression of Xlr5c and Xlr5d in female ES cells relative to males has recently been reported by others [15]. On differentiation, the expression of both these genes increased progressively in male CCE/R cells and decreased in female PGK12.1 cells, such that by day 21, expression was at comparable levels in both cell types (Figure S1A). Interestingly, Xlr3b and Xlr5 are part of a cluster, some of whose members show tissue-specific imprinting [16]. For undifferentiated cells (Figure 1A), the regression line intercepts the y-axis (female) at a positive value (log₂ = 0.69, linear 1.61), showing that overall expression of X-linked genes is 1.6-fold higher in undifferentiated female ES cells, with two active X chromosomes, than in male ES cells, with only one. In differentiated cells (Figure 1B), the intercept was close to 0 (log₂ = 0.14, linear 1.10), indicating that overall expression of X-linked genes was very similar in differentiated male and female cells.

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Figure 1. Expression of X-Linked Genes Is Consistently Higher in Female than in Male ES Cells Prior to Differentiation

(A and B) Correlation in expression levels of X-linked genes between female (y-axis) and male (x-axis) ES cells. The value for each of the 249 clones (180 genes) shown is expressed relative to the expression of autosomal genes (X:A ratio, log₂ scale). Two genes, Xlr3b and Xlr5, showed female-specific expression prior to differentiation (panel A). Xist is weakly expressed overall, but relatively more strongly expressed in differentiated female ES cells than in males (panel B). Pearson product moment correlation coefficients (r values) are shown, along with estimates of the probability of chance correlation (p) corrected for false discovery rate (FDR).

(C) Box plots (linear scale) showing the distribution of expression of X-linked genes in female and male ES cells before (day 0) and after (day 15) differentiation and in lymphocytes. The box encompasses the 25th–75th percentile and the upper and lower lines represent the 10th–90th percentile. Outlying values are shown as individual dots. Data are from at least two biological replicates.

https://doi.org/10.1371/journal.pbio.0050326.g001

The distributions of expression levels of X-linked genes in male and female ES cells are shown as box plots in Figure 1C. In both males and females, expression levels are skewed towards higher expression levels (Figure S2). A similar skewing was seen when the expression of genes on individual autosomes was analysed in the same way (Figure S2). To accommodate this, we have used median rather than mean values for comparison (Figure 1C, horizontal line in each box). The median X:A ratio in undifferentiated (day 0) male ES cells is 0.81 (Figure 1C). This is significantly above the median value of 0.5 that would be expected if X-linked genes were expressed equally to autosomal genes (p = 3.2 × 10⁻³², n = 249) and demonstrates an overall 1.6-fold up-regulation of expression on the single X. Microarray expression data for three additional male ES cell lines has recently been made available (BL6.9, 129.3, and ES-D3-GL) [15], and we have used this to calculate X:A expression ratios, as above. Values ranged from 0.90 to 1.03, suggesting that an approximately 2-fold up-regulation of X-linked genes is a general property of male ES cell lines.

In XX female cells, equal expression of X-linked and autosomal genes would give an X:A ratio of about 1.0, whereas the measured ratio is 1.39, which is significantly higher (Figure 1C, p = 1.1 × 10⁻⁶, n = 249). A very similar X:A expression ratio (1.37) was given by cDNA from the hybrid, female ES cell line 3F1 (see below). Thus, X-linked genes in female ES cells are also up-regulated. After 15 d of differentiation, X:A ratios in both male and female cells were close to 1, which is comparable to the situation in adult cells (Figure 1C).

Expression of X-Linked Genes in the ICM

To test whether the up-regulation of X-linked genes detected in ES cells also occurs in the cells of the blastocyst from which ES cells are derived, RNA was prepared from single ICMs from cultured male and female embryos (distinguished by presence or absence of the Sry gene, Figure 2A), amplified and used to prepare cDNA for labelling of NIA15K arrays. As with expression in ES cells, the expression of X-linked genes in female and male embryos was closely correlated (Figure 2B). The median X:A ratio in male ICMs was 0.86 (Figure 2C), very similar to the value of 0.81 obtained for male ES cells (Figure 1C) and indicative of up-regulation of X-linked gene expression. In female ICMs from the same batch of embryos, the X:A ratio was 0.89 (Figure 2C). This is consistent with up-regulation of genes on Xa in females only if one of the two Xs is inactivated in all or most of the ICM cells used in this experiment. This is certainly possible, because levels of Xist RNA were particularly high in female ICMs (around 8-fold higher than the median expression of autosomal genes) and significantly higher than the Xist RNA levels in male ICM (Figure 2D) and differentiated female ES cells (Figure 1B). Whether this represents persistence of the imprinted paternal X inactivation present from early in development, or random X inactivation, or a combination of the two [17], remains to be determined.

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Figure 2. Expression of X-Linked Genes in Male and Female ICM

(A) Gel showing presence or absence of PCR products derived from the Sry gene in trophectodermal material from single blastocysts used for ICM cDNA preparation. Lane 1, 123 bp size markers; lane 2, male embryo; lane 3, female embryo; lane 4 male ES cell line CCE/R.

(B) Correlation in expression levels of X-linked genes between female (y-axis) and male (x-axis) ICMs. The value for each gene is expressed relative to the expression of autosomal genes (X:A ratio, log₂ scale). Pearson product moment correlation coefficient (r value) and FDR-corrected probability of chance correlation (p) are shown.

(C) Box plot showing expression of X-linked genes (X:A ratio) in male and female ICMs. The box encompasses the 25th–75th percentile, and the upper and lower lines represent the 10th–90th percentile. Outlying values are shown as individual dots. Female values are from two biological replicates and male values from two technical replicates.

(D) Expression of the Y-encoded, male-specific antigen Eif2s3y and Xist RNA, relative to autosomal genes (log₂ ratio), in ICMs from female and male embryos.

https://doi.org/10.1371/journal.pbio.0050326.g002

Changes in Expression of X-Linked Genes Proceed throughout ES Cell Differentiation

The dynamics of differentiation-related changes in X-linked gene expression were determined by analysing cDNAs from male and female cells at various times of differentiation between 0 and 21 d. The X:A expression ratio in female cells showed a gradual and progressive decrease until day 15, whereas in male cells, there was little change until day 7, after which there was a progressive increase (Figure 3A). The gradual changes in X-linked gene expression contrast with the relatively early change in distribution of Xist silencing RNA detected by RNA–fluorescence in situ hybridisation (FISH) (Figure 3B) and loss of expression of the pluripotency markers Nanog, Pou5f1/Oct4, and Zfp42/Rex1 (Figure S1B). Xist RNA levels increased through differentiation from day 2 onwards (Figure S1C).

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Figure 3. Dynamics of Change in X-Linked Gene and Autosomal Gene Expression during ES Cell Differentiation

(A) Expression of X-linked genes (X:A ratio, log₂ scale) in female (red) and male (blue) ES cells at different stages of differentiation from day 0 (undifferentiated) to day 21. Each data point (± SE) is the averaged median value from 5 (day 21), 4 (day 0), 3 (day 12), or 2 (days 2, 4, 7, 15) biological replicates. (See Text S1 for details).

(B) RNA FISH showing distribution of Xist RNA (green) in undifferentiated (day 0, upper panel) and differentiating (day 5, lower panel) female ES cells, counterstained with DAPI (red). The prominent, widely spread Xist signal was detected in ∼60% of day 5 cells.

(C) Expression of genes on Chromosome 2 (n:A ratio, log₂ scale) in female (red) and male (blue) ES cells at different stages of differentiation. Replicates as in (A).

https://doi.org/10.1371/journal.pbio.0050326.g003

The observed increase in expression of X-linked genes in differentiating male cells and the decrease in female cells are unique properties of X-linked genes. Expression of genes on each of the 19 mouse autosomes, relative to all genes (designated the n:A ratio) showed no such changes. The n:A ratio varied from one chromosome to another over only a narrow range and did not change with differentiation or differ between females and males to the same extent as did the X:A expression ratio (Chromosome 2 is shown as an example in Figure 3C and all 19 autosomes are shown in Figure S3).

The expression of X-linked genes in differentiating female ES cells can potentially be influenced by the following three parallel processes: (i) silencing through X inactivation, (ii) up-regulation through dosage compensation on the active X, and (iii) differentiation-related expression changes that are unconnected to the dosage compensation process. Given the close correlation between expression levels of X-linked genes in male and female cells throughout differentiation (Figure 1A and 1 B), the latter two processes are likely to occur to a similar extent in both male and female cells. This being the case, changes in the female:male expression ratio of X-linked genes should reflect progression of the X inactivation process alone. With this in mind, we co-hybridised red/green labelled cDNAs from female and male cells, at the same stage of differentiation, to the same slide, and we calculated red:green or green:red ratios as a log₂ “M value”, as described [18,19]. In undifferentiated, cells the M value is around 0.65 (Figure 4A), corresponding to a linear female:male expression ratio of about 1.6. M values derived from undifferentiated cells and cells at later stages of differentiation were all normally distributed (Figure 4B). There was no detectable fall in M value for the first 7 d of differentiation, with a small increase at days 2–4. Thereafter, there was a progressive decrease, culminating in an M value close to 0, which reflects equal expression of X-linked genes in female and male cells by day 21 (Figure 4A and 4B). It seems that a net loss of expression of female X-linked genes occurs later than previously concluded on the basis of single-gene analyses [13,20].

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Figure 4. A Fall in X-Linked Gene Expression in Female ES Cells Is Detected Late in Differentiation

(A) M values (female:male expression ratio, log₂ scale, ±SE) for X-linked genes in PGK12.1 female ES cells at different times of differentiation (days 0–21). The fall in M value is attributable to the progressive inactivation of genes on one of the two female Xs. Biological replicates for PGK12.1 cells are as listed in Figure 3A.

(B) Distribution of M values in PGK12.1 ES cells at different days of differentiation (day 0, day 2, etc., as indicated) and adult lymphocytes. All distributions are normal with a progressively decreasing median.

(C) Correlation in expression levels of X-linked genes between 3F1 (y-axis) and PGK12.1 (x-axis) ES cell lines. The value for each X-linked gene is expressed relative to the expression of autosomal genes (X:A ratio, log₂ scale). Pearson product moment correlation coefficient (r value) and FDR-corrected probability of chance correlation (p) are shown.

(D) M values (female:male expression ratio, log₂ scale, ±SE) for X-linked genes in 3F1 female ES cells at different times of differentiation (days 0, 2, 12, and 15).

https://doi.org/10.1371/journal.pbio.0050326.g004

To determine the consistency of these findings between ES cell lines, we assayed X-linked gene expression in the hybrid (m. mus domesticus × m. mus castaneous) ES cell line 3F1. There is a close correlation in expression of X-linked genes between these two very different lines (Figure 4C). Further, co-hybridisation of 3F1 and male (CCE/R) cDNAs from the same stages of differentiation to the same slides showed a relatively late decrease in the expression of female X-linked genes relative to male, with no detectable decrease in female:male expression ratio (M value) after 2 d of differentiation, and complete equalisation only after 15 d (Figure 4D).

By studying the change in M value with differentiation time for individual genes, it became clear that some genes consistently showed a relatively early loss of activity in female cells, while others inactivated later, or not at all. Differences in silencing times were confirmed by real-time quantitative (RTQ)-PCR assays, in which expression levels in differentiating embryoid bodies were expressed relative to levels at day 0 (examples are presented in Figure S4). As a first test of whether genes that inactivated relatively early in one ES cell line also inactivated early in others, we co-hybridised cDNA from undifferentiated cells and cells differentiated for 7 d to the same slide and calculated the day 7:day 0 expression ratio as an M value. Genes showing reduced expression in PGK12.1 cells after 7 d of differentiation tended also to show reduced expression in 3F1 cells, with a good overall correlation between the two cell lines in the change in expression of X-linked genes after 7 d of differentiation (p < 0.001, Figure S5).

We subjected the PGK12.1 M value dataset (Figure 4A) to cluster analysis using the TIGR programme [21]. The programme grouped expression data from 252 X-linked clones into selected numbers of clusters, based on the manner in which expression (M value) changed during differentiation. Figure 5A shows the results of an analysis in which the data were resolved into four clusters, each shown as a graph plotting the median value at each stage of differentiation tested. Figure 5B shows the corresponding heat maps. Clusters 1, 2, and 3 showed similar patterns of change, starting at an M value of 0.6–0.8 at day 0 (corresponding to a linear F:M ratio of 1.54–1.75) falling to around 0 by day 21, but differing in the stage at which M values first fell significantly, i.e., day 4–7 for cluster 1 (46 genes), day 7–12 for cluster 2 (74 genes), and day 12–21 for cluster 3 (64 genes). In contrast, the 21 genes in the fourth cluster behaved differently, with M values close to 0 in undifferentiated cells (median = −0.14) and generally increasing on differentiation (Figure 5A). We note that irrespective of how many clusters the programme was asked to resolve, there was always one that showed essentially the same pattern as that of cluster 4 and that stood out from the rest. The genes in cluster 4 are listed in Table S1. They include Xist, a gene known to show increased expression in female cells as they differentiate [22] (Figure S1C). Ontology analysis using Fatigo+ [23] showed that none of these four gene clusters was significantly enriched in genes associated with specific functional categories or cell lineages (unpublished results).

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Figure 5. Genes Are Silenced at Widely Differing Times during Differentiation of Female ES Cells

(A) X-linked genes on the array were separated into four clusters on the basis of their patterns of change in female:male expression ratio (M values) during ES cell differentiation. Median values (± SE) for each cluster at each time point are shown. Changes in M value are attributable to the progressive inactivation of genes on one of the two female Xs.

(B) Heat maps for each gene, with female:male expression ratio colour-coded from green (lowest) to red (highest) as shown on the coloured bar (lower right). Xist (cluster 4, arrowed) shows relatively strong expression in female cells from early in differentiation.

https://doi.org/10.1371/journal.pbio.0050326.g005

Some X-Linked Genes Show Mono-Allelic Expression in Undifferentiated Female ES Cells

One explanation (among several) for the finding that some genes are equally expressed in XX female and XY male ES cells is that they are expressed from only one allele in female cells. This could result from the failure to reactivate the paternal allele which, for most X-linked genes, is selectively silenced in the preblastocyst embryo but reactivated in the inner cell mass during blastocyst maturation [17,24]. Alternatively, it could reflect initiation of (random) X inactivation before the onset of ES cell differentiation. To explore these possibilities, we assayed the expression of paternal and maternal alleles using the hybrid ES cell line 3F1, derived from a m.mus domesticus (129/sv) × m.mus castaneous hybrid backcrossed to 129 [25]. The 129 X chromosome is maternally derived, whereas the castaneous X chromosome is paternal [26]. In 3F1 cells, the 129 X chromosome carries a loss-of-function Tsix mutation, such that when 3F1 cells differentiate, Xist is always up-regulated on the 129 X chromosome, which is therefore always inactivated [25].

We identified single nucleotide polymorphisms (SNPs) in three cluster 4 genes (Jarid1c, Gm784, and Acsl4) that distinguished the 129 and castaneous alleles and that could be selectively restriction digested so as to generate cDNAs that are distinguishable electrophoretically . Remarkably, for all three genes, expression in undifferentiated 3F1 cells was exclusively from the castaneous allele (Figure 6A). We were able to test three other genes that showed a female:male expression ratio of close to 1 at day 0, but which the TIGR programme had not placed in cluster 4. One of these, Phka2, showed expression exclusively from the castaneous allele in undifferentiated 3F1 cells; a second, Ogt, showed expression that was strongly skewed towards the castaneous allele; whereas the third, Brodl, showed bi-allelic expression (Figure 6B). Two genes whose female:male expression ratios in undifferentiated cells showed the expected female bias by microarray analysis (Pctk1 and Zfp185) showed clear biallelic expression, as did a third gene (Pgr15l), for which a suitable SNP was available but which was not present on the NIA15K array (Figure 6B, supplementary Figure S6). The conclusion from these results is that a subpopulation of X-linked genes in female ES cells is mono-allelically expressed before differentiation and that in 3F1 cells, where inactivation is 100% skewed towards the maternal X, it is always the paternal (castaneous) X that is expressed. Thus, mono-allelic expression is not due to failure to reactivate the paternal allele from its preblastocyst silent state, but instead is due to the onset of “random” X inactivation before differentiation.

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Figure 6. Allele-Specific Analysis of X-Linked Gene Expression during Differentiation of Hybrid (129 × castaneous) Female ES Cells

(A) Electrophoretic separation of cDNA restriction fragments from the three cluster 4 genes Acsl4, Jarid1c, and GM784 reveals allele-specific expression. PCR fragments that are specific for each gene were prepared from cDNAs from undifferentiated (day 0) or differentiated (day 12) 3F1 hybrid ES cells. Fragments were uncut (U) or cut (C) with restriction enzymes, so as to reveal SNPs that distinguish m.m.domesticus (129, black arrows) and m.m.castaneous (white arrows) alleles. Small double arrows show where bands of the same size are generated from 129 and castaneous. For all three genes, the expected 129 product is missing from both undifferentiated (day 0) and differentiated (day 12) cells, showing that expression is exclusively from the castaneous allele. cDNA from the male CCE/R ES cell line provided the 129 control. Details of primers, restriction enzymes, and expected fragment sizes are given in Table S5.

(B) Electrophoretic separation of cDNA restriction fragments from four X-linked genes Ogt, Brodl, Pctk1, and Pgr15l to reveal allele-specific expression in 3F1 hybrid female ES cells at different days of differentiation (days 0–15 as indicated); labelling as for (A).

(C) In undifferentiated 16.6 hybrid female ES cells, cluster 4 gene Acsl4 (upper gel) shows monoallelic expression (castaneous allele only) while Jarid1c (lower gel) is expressed from both alleles; labelling as for (A).

https://doi.org/10.1371/journal.pbio.0050326.g006

For all genes tested that showed biallelic expression in undifferentiated cells (Brodl, Zfp185, Ogt, Pctk1, and Pgr151), allele-specific analysis confirmed the microarray data, showing that inactivation of X-linked genes occurs over a wide range of differentiation times and that individual genes have characteristic times of inactivation (Figure 6B and Figure S6).

Proximity to the X inactivation centre (Xic) Correlates with Mono-Allelic Expression in Undifferentiated Female ES Cells

In searching for possible reasons for the gene-to-gene differences in inactivation rate, we asked whether position on the X chromosome, and specifically proximity to Xist and the Xic, was of any relevance. To do this, we tabulated the distribution of genes in each cluster across seven X chromosome regions of similar gene content (Table S2). Genes in clusters 1–3 are distributed across the X chromosome with no clear enrichment or depletion in any single region, nor any clear differences between clusters. In contrast, cluster 4 showed a significant enrichment in the region (85–108Mb) that contains the Xic (8 of 21 genes, p = 0.038, Fisher's exact test). Six of these eight genes are within 6 Mb (94.8∼100.5 Mb) of the Xic (Tables S1 and S2).

The tendency of genes silenced before ES cell differentiation to be located adjacent to the Xic suggests that Xist RNA plays a role in their silencing, even before its increased expression early in differentiation. To test this, we took advantage of the finding that Xist transcript levels in undifferentiated 3F1 cells (carrying a mutation of the Tsix gene) are about 3-fold higher than in the 16.6 hybrid line, from which 3F1 was derived [25] and which has a functional Tsix gene [22]. In 16.6 cells, inactivation is 80% skewed towards the 129 X chromosome as a result of differences in strength of the Xce alleles in the parental strains [27,28]. In undifferentiated 16.6 cells, two of the four genes that are mono-allelically expressed in 3F1 cells (Gm784, Acsl4) were also expressed exclusively from the castaneous allele. However, the two that were most distant from the Xic (Phka2, Jarid1c) were bi-allelically expressed (examples shown in Figure 6C), consistent with the possibility that silencing reflects local spreading of Xist RNA.

If silencing of X-linked genes reflects the progressive spreading of Xist RNA through the X chromosome territory, then one would predict that genes that are close together on the chromosome should be silenced at similar times. To test this, we prepared a list of X-linked gene pairs separated by progressively increasing distances, and we asked whether members of each pair were found in the same cluster (i.e., any one of clusters 1–4, Figure 4) more often than expected by chance. For this analysis, the four clusters are taken as broad indicators of inactivation timing. We find that gene pairs separated by up to 40 kb (Table S3) are in the same cluster significantly more often than predicted by chance (p < 0.05, statistical procedures used are outlined in Text S1 and Figure S7). The five closest gene pairs, from 0 to 2.8 kb apart, were always present in the same cluster (p < 0.05, Table S3).

Progressive Silencing of X-Linked Genes in Differentiating Female ES Cells

Increasing the level of Xist RNA transcripts early in differentiation of female ES cells, is a key event in silencing, in cis, of X-linked genes [6,22]. The more extensive Xist signal detected by RNA-FISH, following up-regulation, has been thought to represent “coating” of the X chromosome, an event that triggers gene silencing. However, the data presented show that global silencing of X-linked genes in differentiating ES cells is not contiguous with the onset of Xist up-regulation, but is put in place progressively over several weeks of differentiation. Indeed, we find no general relationship between transcriptional silencing and any one of the chromosome-wide changes that first become apparent on Xi at specific stages of ES cell differentiation, including Xist coating and H3 lysine 9 methylation (days 1–2), histone deacetylation (days 5–7), and incorporation of the histone variant macroH2A (days 8–12) [20,29–31].

The slow, progressive fall in X-linked transcripts is not easily attributable to experimental or technical factors. Falls in transcript levels inevitably lag behind transcriptional silencing and will show a spread of values that reflects how promptly individual cells begin to differentiate, but such effects cannot account for the consistent variation from one gene to another in the stage at which transcript levels fall. Nor can differences in RNA turnover or stability account for the gene-to-gene variation in silencing time. An unstable transcript will vanish as soon as transcription stops, while a completely stable transcript will be diluted 2-fold at each cell division (i.e., every 16–18 h). Even these extreme stability differences cannot explain variations in inactivation time spread over 3 wk of differentiation. Strain-related differences in global gene expression patterns in different ES cell lines have recently been carefully documented [15], but we find no evidence that differences between ES cell lines, or the mouse strains from which they were derived, are fundamentally influencing our results. Levels of expression of the X-linked genes on the NIA15K array were very closely correlated between CCE/R (129/sv), PGK12.1 (PGK × 129/OLA), and 3F1 (129 × m.mus castaneous) ES cells, whereas both overall up-regulation of X-linked gene expression prior to differentiation and the slow decrease in X-linked gene expression during differentiation were similar in PGK12.1 and 3F1 cells.

Our observation that some genes (e.g., Jarid1c and Acsl4) are silenced on the chosen X in undifferentiated ES cells shows that up-regulation of Xist expression is not essential for silencing. However, the tendency of these early-silencing genes to lie close to the Xist locus suggests that Xist RNA may still be involved, possibly through local spreading, the extent of which is limited by the low level of Xist expression in undifferentiated cells [22]. This possibility is consistent with the finding that whereas four genes were shown by allele-specific analysis, to be mono-allelically expressed in undifferentiated 3F1 cells, only two of these show mono-allelic expression in 16.6 cells, in which Xist transcript levels are 3-fold lower [22]. The two genes that escape silencing in 16.6 cells (Phka2 and Jarid1c) lie furthest from Xist, a finding generally consistent with a local spreading model. These results also raise the possibility that Jarid1c silencing is particularly sensitive to Xist expression levels, which might help explain reports that this gene (previously known as Smcx) escapes inactivation to varying degrees depending on stage of differentiation and cell or tissue type [32–34].

Recent data suggest that up-regulation of Xist RNA early in ES cell differentiation leads to the formation of a distinct Xist domain within the X chromosome territory, into which genes are placed as they are silenced [20,35]. The results presented here are consistent with an Xist domain, but we suggest that the domain expands through the X chromosome territory as differentiation proceeds, and that genes are silenced as they come into contact with the spreading Xist RNA. The progressive increase in Xist transcript levels during differentiation of female, but not male, ES cells (Figure S1C) is consistent with an expanding Xist domain. The stage at which any gene is silenced will therefore depend on its position within the X chromosome territory relative to the Xist locus. These positions will depend on how the X chromosome is folded within its territory, and for all genes except those most proximal to Xist, the folding need bear little relationship to the gene's (linear) position on the chromosome, which we find to be the case (Table S2). We do find, as predicted by the spreading model, that genes that lie close together on the chromosome (within about 40 kb) tend to be silenced at about the same time, as measured by cluster analysis (Table S3). None of this evidence is inconsistent with the possibility that some genes at least may be actively drawn into the Xist domain [20]; it is possible that Xist spreading and gene repositioning occur in parallel.

In the model we propose, the pattern of gene silencing through differentiation is critically dependent on the configuration of the X chromosome territory, specifically the positioning of the Xist locus and of other loci relative to it. Differential reconfiguration of X chromosome territories in female cells prior to the onset of X inactivation, or even changes in their intranuclear location, may be a crucial initial step in the X inactivation process [22,36]. The fact that XY male ES cells express low levels of Xist RNA prior to differentiation, but do not inactivate genes proximal to Xist (e.g., Gm784, Acsl4, Figure 6), indicates that Xist RNA is not the sole determinant of inactivation. Perhaps configuration of the X chromosome territory or chromatin conformation in undifferentiated male ES cells is such as to preclude contact between Xist RNA and critical X-linked loci. Our previous observation that X-linked genes in female ES cells carry levels of histone modifications associated with transcriptional activity that are higher than those in males [37] raises the intriguing possibility that chromatin modifications might help determine susceptibility to Xist silencing. In this respect, it is interesting that the dosage compensation complex in D. melanogaster, which includes roX RNAs, preferentially targets transcriptionally active genes, possibly through their distinctive histone modifications [3,4,38].

Evolutionary Considerations

Dosage compensation is a rapidly evolving process, and the mechanisms by which it is accomplished vary from one organism to another [1,10,38]. It is interesting to ask whether evolution is driven predominantly by a need to equalise overall X-linked and autosomal expression levels, or whether transcript levels of key individual genes exert the major selection pressure. Recent studies on expression of Z-linked genes in birds, in which females are heterogametic (ZW) and males homogametic (ZZ), throw some interesting light on this [39]. For a representative group of genes in various tissues in two species (zebra finch and chicken), the expression of Z-linked genes was consistently and significantly higher in ZZ males—where the Z:autosome expression ratio was around 1—than in ZW females—where the Z:autosome ratio ranged from 0.7 to 0.9 depending on the tissue. These findings indicate that dosage compensation is incomplete in birds, and that higher eukaryotes can tolerate significant overall differences in gene expression between the sexes and between X-linked and autosomal genes.

It now seems that the three model organisms commonly used to study dosage compensation: fruit fly (D. melanogaster), mouse (Mus musculus), and the nematode worm Caenorhabditis elegans have all adopted up-regulation of X-linked gene expression in XY (or XO) males as a means of balancing X:autosome expression levels [1,38]. In mouse and C. elegans [40,41] there is also an overall suppression of X-linked transcription in XX females/ hermaphrodites. The extra complexity of the mammalian, and worm, mechanisms is likely to reflect their evolutionary histories. It is generally accepted that the gene-poor Y chromosome is the evolutionary result of progressive degeneration of one of two originally homologous chromosomes, one of which (the proto-Y) carried a sex-determining allele [42,43]. Restricted crossing-over at and around the sex-determining locus, which is necessary to prevent the formation of intersex states, allows the progressive spread of mutations and the loss of functional genes along the proto-Y by reducing the selection pressure to which they are subjected [8]. For many mutated genes, selection pressure will favour up-regulation of the remaining functional allele to restore the original transcript levels. The magnitude of this selection pressure will depend on the sensitivity of the gene product's function to transcript level. If in mammals (and C. elegans), unlike Drosophila, the newly evolving up-regulation mechanism were expressed from the beginning in both males and females, then a female-specific silencing mechanism would need to evolve in parallel to suppress damaging overexpression [38,42]. The fact that the up-regulation of X-linked genes in Drosophila is male-specific, whereas that in the mouse is not, suggests that the mechanisms by which up-regulation is achieved may be fundamentally different in the two organisms, despite the presence in mammals of homologues of several of the Drosophila dosage-compensation complex components [44,45]. Unravelling the up-regulation mechanism in mammals and defining how it interacts, if at all, with Xist-mediated silencing to optimise expression of X-linked genes are now questions of particular interest.

Cells and cDNA preparation.

The mouse ES cell lines PGK12.1 (129 × PGK hybrid) [46], CCE/R (129/sv) [47], and 3F1 (129/sv × castaneous hybrid) [25] were cultured as previously described [37]. Differentiation was induced by replating on nonadherent plastic dishes in the absence of leukaemia inhibitory factor (LIF). Adult control cells were thymic lymphocytes from 4-wk-old Balb/c mice. ICM cells were prepared from cultured Balb/c mouse embryos at the early blastocyst stage by the immunosurgery procedure of Solter and Knowles [48], as previously described [49]. Embryos were sexed by testing (by PCR) the trophectodermal material remaining after immunosurgery for presence of DNA encoding the male-specific, Y-linked antigen Sry.

Total RNA was extracted from ES cells using the RNeasy mini kit (Qiagen). For ICM, RNA was extracted with the RNAqueous-Micro kit and amplified with the MessageAmp II aRNA amplification kit (both from Ambion). cDNA was prepared with RT-Superscript-III (Invitrogen), purified with the Qiagen PCR purification kit, and labelled with Cy3 or Cy5 (Amersham) using Invitrogen Bioprime labelling kits (see Text S1 for details).

cDNA microarrays.

The NIA 15K mouse cDNA library [14,50] was purchased through the UK Medical Research Council and printed in duplicate onto glass slides by the Genomics and Proteomics Laboratory, University of Birmingham (http://www.genomics.bham.ac.uk) using an Advalytix Automated Hybrydization Station. The library contains 15,247 cDNA clones with an average insert size of 1.5 kb. The ES cell data presented here are derived from 252 X-linked clones (corresponding to 180 named genes) and 6,945 autosomal clones (corresponding to 5,085 named genes) that consistently gave above-background signals with ES cell cDNAs. cDNAs from female and male ES cells at the same stage of differentiation were labelled with Cy3 and Cy5, and equal amounts (80∼120 pmol) were co-hybridised to arrays overnight at 42 °C. After labelling, slides were washed and then scanned using a GenePix 4000A scanner. PMT settings were set so as to balance overall signal in the Cy3 and Cy5 channels. Scans were automatically aligned using GenePix (version 6.0) software and then “cherry-picked” manually to eliminate abnormal spots. Microarray data was extracted by Genepix (version 6.0) and normalised by Gepas software. Clustering analysis used the TIGR MultiExperiment Viewer, TMEV [21] (http://www.tigr.org/tdb/tgi). Detailed analytical procedures can be found in Text S1.

Real-time PCR and SNP analysis.

Expression patterns of four genes (Maoa1, Prps1, Ssr4, and Smc1l1) were quantified by real-time PCR using SYBR Green PCR master mix (ABI) and an ABI 7900 Detection System. The primer sequences are listed in Table S4. Allele-specific quantification of Zfp185 was by radioactive PCR with two forward primers (Table S4), specifically recognising 129 and castaneous alleles. ActB was used as a control. The PCR reaction comprised 5 μl 2× buffer, 1 μl cDNA, and 2.5 pmol each of primers in a total volume of 10 μl.

SNPs distinguishing m.m.domesticus (129) and m.m.castaneous X-linked genes were identified using Ensemble SNPView. Allele-specific expression was analysed by restriction enzyme digestion following amplification of cDNA from undifferentiated (day 0) 3F1 cells by PCR. Primers, enzymes, and expected products are listed in Table S5 and detailed procedures are given in Text S1.

RNA FISH.

RNA FISH was carried out as described by Okamoto et al. [17]. Briefly, cells were cytospun to glass slides and fixed in 3.5% paraformaldehyde in PBS for 30 min at room temperature and permeabilised with 0.5% Triton X100 in PBS + 2 mM vanadyl ribonucleoside complex (Biolab) for 10 min on ice. Cells were then dehydrated, hybridised, and counterstained with DAPI. The 6-kb GPT16 Xist probe [51] was labelled with Spectrum Green-dUTP (Vysis) by nick translation, according to the manufacturer's protocol.

Accession Numbers

The National Center for Biotechnology Information (NCBI) unigene cluster IDs (http://www.ncbi.nlm.nih.gov) for the genes mentioned in the text are as follows: Acsl4 (Mm.391337), Brodl (Mm.100112), Gm784 (Mm.298000), Jarid1c (Mm.142655), Nanog (Mm.440503), Ogt (Mm.259191), Pctk1 (Mm.102574), Pgr151 (Mm.336164), Phka2 (Mm.350712), Pou5f1/Oct4 (Mm.17031), Sry (Mm.377114), Tsix (Mm.435573), Xist (Mm.435573), Xlr3b (Mm.336117), Xlr5 (Mm.435653), Xlr5c (Mm.255790), Xlr5d (Mm.435653), Zfp42/Rex1 (Mm.285848), and Zfp185 (Mm.1161).