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Human Molecular Genetics Pages 1355-1361
The mouse Smcx gene exhibits developmental and tissue specific variation in degree of escape from X inactivation
Introduction
Results
   Quantitation of RNA levels for the active and inactive X Smcx alleles
   Low level partial escape in early mouse embryos
   Variable level of escape in adult tissues
   Clonally derived haematopoietic stem cell colonies exhibit partial escape
Discussion
   Variable levels of partial escape
   Mechanism of escape from X inactivation
Materials And Methods
   Mouse strains
   SNuPE analysis
   Haematopoietic colony assays
Acknowledgements
References

The mouse Smcx gene exhibits developmental and tissue specific variation in degree of escape from X inactivation

The mouse Smcx gene exhibits developmental and tissue specific variation in degree of escape from X inactivation Steven Sheardown, Dominic Norris, Amanda Fisher1 and Neil Brockdorff*

Comparative Biology Group, 1Lymphocyte Development Group, MRC Clinical Sciences Centre, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK

Received June 18, 1996; Accepted July 4, 1996

The Smcx gene is the first known example of a non-pseudoautosomal X-linked gene in mouse that normally escapes X chromosome inactivation. We have analysed the kinetics of escape at different stages of development, and in adult tissues. Our results demonstrate that Smcx exhibits partial escape from X inactivation in embryos, in extraembryonic lineages where paternally imprinted X inactivation occurs and also in adult tissues. The degree of escape in different tissues is highly variable, the level of transcript from the inactive X allele representing between 20% and 70% of the active X allele. Partial escape is also seen in clones derived from haematopoietic stem cells, suggesting that partial repression of the inactive X allele is at the level of individual cells. This contrasts with classical position effect variegation (PEV), where a given gene is either active or silent in a given cell and its clonal derivatives. We discuss the implications of these results with respect to mechanisms of X inactivation and escape.

INTRODUCTION

X chromosome inactivation (X inactivation) is the genetic silencing of a single X chromosome in female mammals, and provides the mechanism for dosage compensation of X-linked genes relative to males (1 ). X inactivation is regulated by a cis-acting master switch locus, termed the X inactivation centre (Xic), which is required both for the initiation of X inactivation in early embryogenesis, and in the propagation of inactive chromatin in cis (2 ). Recent studies have identified the Xist gene as being a key component of the Xic (3 ,4 ).

A complementary approach that has been employed in studying the molecular basis of propagation of X inactivation is the analysis of genes located in the pseudoautosomal region, and also elsewhere on the X chromosome, which escape X inactivation (5 ). There are two alternative explanations for the mechanism of escape: firstly, genes which escape X inactivation may lack sequences required for the efficient spread or maintenance of X inactivation, and secondly, genes which escape may be associated with elements that insulate from X inactivation. Studies on transgene insertions into the X chromosome (6 ), and more recently on human X-linked genes in the Xp11.2 interval (7 ), have led to the suggestion that escape is regulated at the level of chromosome domains. This idea is consistent with the observation that discrete bands on the late replicating inactive X chromosome are early replicating (8 ), and more recently the demonstration that discrete regions of the inactive X chromosome are enriched for acetylated histone H4, in contrast to the general feature of hypoacetylation of H4 on the inactive X chromosome (9 ).

The presence of a Y-linked homologue obviates the requirement for dosage compensation between males and females for genes located in the pseudoautosomal region of the X and Y chromosomes [for example the MIC2 and XE7 genes in man (10 ,11 )], and some genes that escape X inactivation which are located elsewhere on the X chromosome [for example the ubiquitously expressed human RPS4X/RPS4Y and ZFX/ZFY genes (12 ,13 )]. The mouse homologues of these genes (Rps4 and Zfx) do not have functionally equivalent Y-linked homologues (the mouse Zfy-1 and Zfy-2 genes have a restricted expression pattern), and consistent with the dosage compensation argument, do not escape X inactivation (14 ). There are nevertheless some examples of genes that escape X inactivation but have no functional Y-linked homologue [e.g. the human UBE1, KAL1, and SB1.8 genes (15 -17 )]. This suggests that failure to dosage compensate some X-linked genes has no detrimental effects. An alternative explanation is that these genes exhibit partial and/or variable escape from X inactivation. Conflicting data on escape for the UBE1 gene (15 ,18 ) suggest that this could be the case in this example. Partial escape has been demonstrated for the human STS locus (19 ), and is a feature of many X-linked genes in marsupials (20 ). Variable escape through ontogeny has been demonstrated in genetic studies on mouse loci which escape X inactivation with aging (21 ,22 ), and also on autosomal loci in cis with inactive X chromatin in X;autosome rearrangements (23 ).

The recently described SMCX/Smcx gene (also referred to as Xe169) escapes X inactivation in man and mouse (24 ,25 ), and has a putative functional Y linked homologue (SMCY/Smcy), which has recently been shown to encode a male-specific transplantation (H-Y) epitope (26 ). Both Smcx and Smcy are ubiquitously expressed, suggesting that functional equivalence may provide dosage compensation. Smcx is the first example of a non-pseudoautosomal gene escaping X inactivation in mouse, and therefore provides an opportunity to analyse escape at the initiation stage of X inactivation early in development. In this paper we descibe analysis of the kinetics of escape from inactivation for the mouse Smcx gene. We show that at early embryonic stages, expression of the Smcx allele on the inactive X chromosome is markedly reduced relative to the active X allele. This is also the case in adult tissues but levels are variable in different tissues. Partial escape is also seen in clones derived from haematopoietic stem cells, suggesting that our observations reflect partial repression at the level of individual cells rather than a combined effect of cell clones exhibiting full repression or activation of the inactive X allele.


Figure 1.Quantitative allele specific assay for the Smcx gene. (a) Partial sequence of the Smcx gene illustrating the polymorphism between 129 (G) and PGK (C) strains, and the sequence of the SNuPE primer used to detect this polymorphism. (b) SNuPE analysis demonstrates that both Smcx alleles are detected in RNA from the PGK12.1 ES cell line (4) (ES), whilst only the appropriate single allele is detected in RNA from control PGK or 129 adult tissue.


RESULTS

Quantitation of RNA levels for the active and inactive X Smcx alleles

We have carried out this analysis based on initial observations which suggested that Smcx is inactivated following in vitro differentiation and X inactivation in XX embryonic stem (ES) cells (4 ). To investigate whether near complete X inactivation of Smcx also occurs at the time of initiation of X inactivation in vivo, we have quantitated relative expression levels of Smcx on the active and inactive X chromosome using early female embryos carrying the T(X;16)16H translocation (T16H). T16H results in complete secondary non-random X inactivation of the normal X chromosome (27 ). This occurs within 2-3 days following the initiation of random X inactivation at 5-5.5 days post coitum (d.p.c.) (27 ,28 ).


Figure 2. SNuPE analysis of 8.5-9.5 d.p.c. female embryos. SNuPE analysis of Pgk-1 was used to determine that female embryos 10.2, 10.5 and 11.3 are balanced T16H carriers showing complete non-random X inactivation of the PGK X chromosome (a = T16H allele, c = PGK allele). Embryos 10.3 and 11.2, and also control PGK12.1 ES cells (ES), show biallelic Pgk-1 expression. SNuPE analysis of Smcx shows biallelic expression in each of the T16H embryos with a lower signal from the inactive X allele (PGK = c) relative to the active X allele (T16 = g).

In order to differentiate between alleles at the Pgk-1 and Smcx loci we used pre-characterised polymorphisms between C3H/He-Pgk-1a/Ws (PGK) strain (Pgk-1a, Smcxa ) and Mus musculus domesticus (Pgk-1b, Smcxb) (4 ). To analyse relative RNA levels, we used the Single Nucleotide Primer Extension assay (SNuPE) (4 ), which provides an accurate means to quantitate relative expression levels of two alleles differing by only a single nucleotide (29 ,30 ). Figure 1 a illustrates the SNuPE polymorphism in the Smcx gene used in this analysis. A G-C transition in the PGK strain was identified at position 268 of the published mouse sequence (24 ). SNuPE analysis (Fig. 1 b) demonstrates that both alleles are expressed in RNA from the PGK12.1 ES cell line carrying a PGK and 129 strain X chromosome, whilst in RNA from control PGK or 129 strain animals only the appropriate allele was detected.

Low level partial escape in early mouse embryos

We first analysed relative expression levels in 8.5-9.5 d.p.c. female T16H embryos. To identify balanced T16H female embryos, we analysed expression of Pgk-1 alleles by SNuPE. Examples are illustrated in Figure 2 . Embryos 10.2, 10.5 and 11.3 only express the Pgk-1b allele, and are therefore balanced T16H carriers. This result clearly demonstrates that selection against cells which elect to inactivate the 16X translocation product is complete by 8.5 d.p.c. In each of the T16H embryos both alleles of Smcx are detected, but at a markedly reduced level from the inactive PGK strain allele. Embryos 10.3 and 11.2 do not carry the T16H translocation as they express Pgk-1 from both alleles. Subsequent quantitation of these results shows that the Smcx RNA level from the inactive allele is 25-30% of that seen from the active allele (Table 1 ). The mean value for the relative RNA level of the inactive X Smcx allele in embryos at this stage is 29% (Fig. 3 ).

To determine whether levels of escape increase through development we next analysed 12.5-14.5 d.p.c. embryos. In this experiment extraembryonic endoderm tissue was also isolated in order to quantitate escape from X inactivation in tissues that have undergone paternally imprinted X inactivation (the paternal PGK strain X chromosome is always inactive in extraembryonic tissues so both translocation and non-translocation bearing embryos could be used). Four of six female embryos analysed expressed the Pgk-1b allele exclusively and were therefore balanced T16H carriers. Quantitation of allelic Smcx RNA levels in the T16H embryos gave values similar to those obtained for 8.5-9.5 d.p.c. embryos, i.e. low levels of partial escape (Table 1 and Fig. 3 ). The levels of escape were also similar in extraembryonic endoderm samples where imprinted X inactivation occurs (Table 1 and Fig. 3 ).


Figure 3. Smcx displays a variable level of partial escape from X inactivation in embryos and adult tissues. The figure shows mean values ( S.E.M.) of Smcx inactive X RNA levels expressed as a percentage of active X levels. Values were calculated from the data in Table 1. Embryo results were grouped into 8.5-9.5 d.p.c. and 12.5-14.5 d.p.c. embryos (Emb), and 12.5-14.5 d.p.c. extraembryonic endoderm (End.). Student's t-test shows a statistically significant elevation in levels in adult liver, spleen and lung compared with 8.5-9.5 d.p.c. embryos (p = 0.01, 0.02 and 0.01 respectively).

Variable level of escape in adult tissues


Figure 4. (a) SNuPE analysis of haematopoietic stem cell colonies isolated from female (PGKx129)F1 mouse. Monoallelic Pgk-1 expression was used to identify clonally derived colonies (129 allele = a, PGK allele = c). Colonies 8, 10, 11 and 13 are clonal and have an inactive 129 derived X chromosome, whilst clonal colony 9 has an inactive PGK derived X chromosome. Samples 12 and 14 represent colonies of non-clonal origin which were eliminated from subsequent analysis. Smcx SNuPE shows biallelic expression in all clones (c = PGK allele, g = 129 allele), with a reduced level from the inactive X allele relative to the active X allele. (b) Inactive X Smcx RNA levels expressed as a percentage of active X levels in clonal haematopoietic stem cell colonies. Colonies 9 and 21 inactivated the PGK strain X chromosome, and all other colonies the 129 strain X chromosome. This is consistent with the skewed random X inactivation expected as a result of heterozygosity at the Xce locus (4). Mean value S.E.M. for all colonies is 62.3 +- 16.5%.


The low level of escape seen in early mouse embryos is consistent with our previous observations indicating (near) complete inactivation of Smcx upon initiation of X inactivation in differentiating XX ES cells (4 ). We went on to analyse levels of escape in liver, spleen, lung and kidney from six week old adult female T16H animals (Table 1 and Fig. 3 ). In adult liver we observed significantly elevated levels of Smcx RNA from the inactive X allele relative to 8.5-9.5 d.p.c. embryo samples. The mean value was almost double that seen in embryo samples (56 +- 9 %, Fig. 3 ). Values obtained for spleen and lung were also significantly elevated relative to 8.5-9.5 d.p.c. embryos, but to a lesser degree than liver (40 +- 3.7% and 45 +- 8% respectively, Fig. 3 ). The value obtained for kidney was not significantly different to embryo samples (34 +- 10%, Fig. 3 ). These results, together with those reported by Carrel et al. (see accompanying paper) (41 ) indicate considerable variation in levels of escape, both between different tissues and between individual animals. In our experiments the level of escape is generally higher in the adult tissues compared with 8.5-9.5 d.p.c. embryos. It is not clear whether this reflects increased escape through ontogeny or stochastic effects in the limited number of adult tissues analysed.

Clonally derived haematopoietic stem cell colonies exhibit partial escape

The variable levels of Smcx RNA from the inactive X allele could be achieved via two contrasting mechanisms. Firstly, at the initiation of X inactivation individual cells may either fully express or fully repress Smcx. Variable inactivation in combined tissues would then reflect different proportions of clones of the progenitor cells (i.e. akin to classical PEV). Alternatively, each individual cell may exhibit partial escape that is subject to developmental and tissue specific regulation. To differentiate between these possibilities we analysed Smcx RNA levels in clonally derived haematopoietic stem cell colonies isolated from female (PGKx129)F1 animals. Assuming a similar level of escape to that seen in other adult tissues, we reasoned that if the first hypothesis was correct, approximately 50% of individual clones, as defined by mono-allelic Pgk-1 expression, would show no expression of the inactive X allele of Smcx. If on the other hand the second hypothesis were correct, we would expect to see bi-allelic Smcx expression in all clones, with a reduced level (approximately 50%) from the inactive X allele.

We analysed a total of thirty four colonies comprising 20-100 cells. Some examples are illustrated in Figure 4 a. Colonies 12 and 14 expressed Pgk-1 from both alleles and were therefore excluded as being non-clonal. Colonies 8, 10, 11 and 13 only expressed the Pgk-1a allele, whilst colony 9 expressed only the Pgk-1b allele. Smcx is expressed from both alleles in all of the clonal colonies, the relative strength of the signal always being weaker from the inactive X allele. A total of 12 colonies displayed clear mono-allelic expression of Pgk-1. Smcx was expressed bi-allelically in all of these colonies and the RNA level from the inactive X allele was similar to that seen in adult tissues (Fig. 4 b). This result suggests that the second hypothesis is correct, and that the Smcx allele on the inactive X is partially repressed at the level of the individual cell.

DISCUSSION

We have quantitated the level of Smcx RNA transcribed from the inactive X chromosome relative to the active X chromosome. In early mouse embryos, escape from X inactivation for Smcx occurs at a low level, approximately 30% of that seen from the active X allele. In extraembryonic endoderm, where paternally imprinted X inactivation occurs, the level of escape is similar to that seen in the embryo proper. Levels of escape are variable in adult tissues, ranging from 20-70% expression from the inactive allele. Through the analysis of adult clonally derived mouse haematopoietic stem cell colonies we have provided evidence that the partial repression of Smcx occurs at the level of individual cells.

Variable levels of partial escape

Comparison of levels of escape between 8.5-9.5 d.p.c. embryos and adult tissues suggest that escape from X inactivation for Smcx may increase progressively through ontogeny. This could indicate near complete inactivation of Smcx at the time of onset of X inactivation, followed by increased escape, possibly resulting from inefficient maintenance of the inactive chromatin state of Smcx. However, the increase observed is modest, and because we have assayed only a limited selection of adult tissues it is possible that our results reflect stochastic variations. Consistent with this, no significant increase in escape was observed by Carrel et al. (see accompanying paper and below) (41 ), although the earliest embryonic stage analysed in their study was 10.5 d.p.c. If there is indeed a significant increase in escape through ontogeny then we would expect even lower levels of escape immediately following the onset of X inactivation in 5.5-6 d.p.c. embryos. Unfortunately it is not possible to address this in T16H embryos as selection against cells having inactivated the 16X chromosome is ongoing (27 ,28 ). An alternative approach would be to assay Smcx for skewed X inactivation in embryos heterozygous at the Xce locus, although it is unlikely that this approach would distinguish between complete inactivation and low levels of escape, for example 25-30%. Related to this point, the Xce effect that we observed for Smcx in differentiated XX ES cells (4 ) may reflect a low level of escape rather than complete inactivation upon initiation of X inactivation in vitro.

Table 1 Relative Smcx RNA level from the inactive X chromosome allele of individual T16H * PGK female embryos, extraembryonic endoderm samples (samples x1.4 and x2.2 were from non-translocation bearing embryos) and adult tissues
Stage/tissue

 Sample no.

 Escape (%)
8.5 d.p.c. embryo

e10.2

 27.9
 

 e10.5

 24.9
 

 e11.3

 32.3
9.5 d.p.c. embryo

 e12.2

 32.5
12.5 d.p.c. embryo

 e2.1

 45
 

 e7.1

 26.4
14.5 d.p.c. embryo

 e3.2

36
 

 e3.3

 32.4
12.5 d.p.c. endoderm

 x1.4

 30
 

 x2.2

 28
14.5 d.p.c. endoderm

 x3.2

 21.7
 

 x3.3

 19.3
Adult liver

 m4.1

66.3
 

 m5.1

50.5
 

 m5.2

55.3
 

 m5.3

73
 

 m5.4

42.4
 

 m6.1

 56
Adult spleen

 m4.1

41.2
 

 m5.1

44.5
 

 m5.2

34.6
 

 m5.3

39.4
 

 m5.4

42
Adult lung

 m4.1

44.5
 

 m5.1

38.5
 

 m5.2

59
 

 m5.3

41.1
 

 m5.4

42
Adult kidney

 m4.1

21.6
 

 m5.1

36.5
 

 m5.2

32
 

 m5.3

38
 

 m5.4

38
 

 m6.1

 49.6
Results show RNA levels for the inactive X Smcx allele as a percentage of the active X allele.

Tissue specific variation in the level of escape was observed both in this study and in that of Carrel et al. (see accompanying paper) (41 ). The results obtained were broadly consistent, although Carrel et al. (41 ) observed significantly higher levels of escape in adult kidney but not in liver. This difference may be attributable to variable levels between different animals, or alternatively, to the different assay system or mouse strain used in the two studies. Tissue specific variation in levels of escape has also been observed for a number of X-linked genes in marsupials (19 ), and progressive escape from X inactivation through ontogeny, together with tissue specific variability, has recently been demonstrated for the X-linked Gpd gene in the Virginia oppossum (31 ).

The variable level of partial escape for Smcx demonstrates that conjectures concerning toleration of dosage imbalance for individual genes need to be carefully considered. In the case of Smcx, the near complete inactivation in early embryos means that dosage imbalance between males and females is minimal. Paradoxically Smcx has been characterised as an example of a gene tolerating escape from X inactivation due to the presence of a functional homologue on the Y chromosome (24 ,25 ). If Smcy is indeed functionally equivalent to Smcx, and if it is expressed at a similar level, it is males which will have the higher dose of gene product, most notably in early to mid-gestation embryos.

Mechanism of escape from X inactivation

Our observations on clonal colonies derived from haematopoietic stem cells suggest that partial escape occurs within individual cells rather than as a combined result of clones fully expressing or fully repressing Smcx. This is supported by observations on fibroblast clones by Carrel et al. (see accompanying paper) (41 ). These results could be attributable to flanking heterochromatin giving rise either to a reduced rate of transcription from the inactive X Smcx allele, or alternatively to a reduced frequency of association of upstream activating elements with the Smcx promoter. We favour the latter hypothesis based on recent findings demonstrating that repressive chromatin reduces the frequency at which enhancers associate with linked promoters, rather than the actual rate of transcription (32 ). Thus, an Smcx enhancer element could be in dynamic equilibrium between association with the Smcx promoter and sequestration into heterochromatin of the inactive X chromosome. Tissue and stage specific variation in expression levels could then be attributable to stochastic variation in the level of factors interacting with the Smcx enhancer in different tissues and developmental stages. It should be possible to test this hypothesis using fluorescent in situ hybridisation to nascent RNA (33 ) to determine whether Smcx transcripts are produced from both alleles in all individual cells or whether a proportion of cells transcribe from a single allele only.

In conclusion, our findings for escape from X inactivation for the Smcx gene have important implications, both in terms of understanding the biological significance, and also the mechanism of escape from X inactivation. Identification of other genes that escape X inactivation on the mouse X chromosome will help to determine whether these findings are a general feature of genes that escape X inactivation. It is of interest to note that conflicting results for escape from inactivation for the mouse pseudoautosomal Sts gene (based on assaying Sts enzyme activity), led to the suggestion that this locus may be subject to partial and variable levels of escape (34 ). The Sts gene has recently been cloned and shown to escape X inactivation (35 ). Quantitative data on levels of escape have not been obtained as yet.

MATERIALS AND METHODS

Mouse strains

T16H, PGK and 129ola animals were all bred in house. Timed matings were set up between T16H females and PGK strain males (T16H Pgk-1b Smcxb/ + Pgk-1b Smcxb x + Pgk-1a Smcxa /Y). Embryos were harvested from timed matings, and dissected out, according to standard methods (36 ). Embryo material was split into two parts and then snap frozen on liquid nitrogen prior to DNA or RNA isolation. The extraembryonic endoderm was isolated from yolk sacs following partial trypsin/pancreatin digestion (36 ,37 ). Adult T16H females were identified by phenotypic scoring for X-linked mutations (tabby or mottled) which were segregating in the crosses.

SNuPE analysis

RNA was prepared using RNAzol reagent (Biogenesis Ltd). The PGK12.1 ES cell line is described elsewhere (4 ). Primer sets and conditions for RT-PCR and SNuPE analysis of Pgk-1 and Smcx have been described in detail elsewhere (4 ,29 ). In both cases the T16H Mus musculus domesticus allele is identical to the allele described previously for 129ola animals. Mixing experiments were performed to verify that accurate quantitative data could be obtained for each of the polymorphisms (4 ,29 ). SNuPE reactions were electrophoresed on 12.5% denaturing polyacrylamide gels which were then exposed to X-ray film for 10-20 min. For quantitative purposes, duplicate samples were loaded, and gels were exposed to phosphor screens for 5 min. Quantitation was carried out on a Molecular Dynamics PhosphorImager. Because of batch variations in the specific activity of [32P]dNTPs, each experiment was normalised using a sample of known allelic ratio. Individual embryos were sexed as described previously using PCR analysis of genomic DNA with primers for the Y-linked gene Zfy (28 38). Male embryos were excluded from further analysis. Non-translocation bearing female embryos, which expressed both alleles of the Pgk-1 gene at a similar level, were also excluded from further analysis. Balanced T16H female embryos were identified as those expressing the Pgk-1b allele exclusively. Secondary non-random X inactivation in T16H has previously been shown to be complete between 7.5 and 10.5 d.p.c. (27 ,28 ). In this study we found that selection was complete by 8.5 d.p.c. Unbalanced T16H female embryos have occasionally been observed to survive to this stage (39 ); such embryos would be expected either to express the Pgk-1a allele exclusively or both alleles at a similar level.

Haematopoietic colony assays

Normal bone marrow cells were flushed from the femur of individual female (PGKx129)F1 animals (6-8 weeks age). Mononuclear cells were separated over Ficoll-Hypaque, washed twice in serum containing medium, and passed gently through a 25 gauge needle to ensure a single cell suspension. Between 104 * and 2 * 105 cells were plated in 35 mm dishes in IMDM media containing 0.27% agar, 20% FCS, and appropriate growth factors (40 ), and incubated in a humid 5% CO2 atmosphere at 37oC for 7-10 days. Isolated single colonies were identified, individually picked with ultrafine glass capillaries, and used to prepare RNA. All of the RNA from a single colony was used for RT-PCR. PCR for Smcx and Pgk-1 was as described previously (4 ), except 35 cycles (Pgk-1) and 40 cycles (Smcx) were used.

ACKNOWLEDGEMENTS

We would like to thank Vasso Episkopou and members of the Comparative Biology group for helpful discussions. This work was supported by the Medical Research Council of Great Britain.

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*To whom correspondence should be addressed

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