Human Molecular Genetics, 2001, Vol. 10, No. 6 581-589
© 2001 Oxford University Press
Primary non-random X inactivation associated with disruption of Xist promoter regulation
1X inactivation Group, MRC Clinical Sciences Centre, ICSM, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK and 2MRC Clinical Sciences Centre Transgenic Facility
Received 15 November 2000; Revised and Accepted 29 January 2001.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
In this report we demonstrate primary non-random X chromosome inactivation following targeted mutagenesis of a region immediately upstream of Xist promoter P1. In heterozygous animals there is a preferential inactivation of the targeted X chromosome in 80–90% of cells. The phenotype correlates with inappropriate activation of Xist in a proportion of the mutant XY embryonic stem cells. Strand-specific analysis revealed increased sense transcription initiating upstream of Xist promoter P1. There was, however, no discernible effect on transcription from the antisense Tsix gene. We demonstrate that the in vitro and in vivo phenotypes are specifically attributable to the presence of a PGKneo cassette at the targeted locus. These findings are discussed in the context of understanding mechanisms of Xist gene regulation in X inactivation.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
X chromosome inactivation (X inactivation) is the mechanism of sex chromosome dosage compensation in mammals. A single X chromosome, selected at random, is inactivated in all female (XX) cells early in development (1). After initiation of X inactivation the inactive state is clonally inherited through subsequent cell generations.
Although X inactivation is normally random there are examples where partial or complete non-random X inactivation occurs. These can be divided into primary and secondary mechanisms. Primary non-random X inactivation is caused by a bias in the initial choice of which X to inactivate, whilst secondary non-randomness arises through selection against cells having elected to inactivate a given X. Examples of the former are provided by imprinted inactivation of the paternally derived X (Xp) in extraembryonic lineages in mouse (2), and skewed X inactivation caused by heterozygosity at the Xce locus (3,4). Examples of the latter include many X:autosome translocations where there is selection against cells that inactivate the translocated X chromosome (see for example ref. 5).
Regulation of X inactivation is mediated by a single cis-acting master switch locus, classically referred to as the X inactivation centre (Xic) (6). The Xic is involved in the process whereby cells determine how many (if any) and which X chromosomes to inactivate (counting and choice respectively), and is also required for propagation of X inactivation in cis (reviewed in ref. 7). Prevailing models suggest that counting and choice are achieved by cells blocking a single Xic, thereby marking the active X chromosome (8). In recent years Xic functions have been mapped to the X inactive specific transcript (Xist) locus and surrounding region (9–15). Xist produces a large non-protein coding RNA that is expressed exclusively from the inactive X chromosome and which is thought to propagate inactivation by accumulating along the entire length of the chromosome in cis (11,13,16).
Developmental regulation of Xist gene expression has been studied extensively in mouse. In XX somatic cells Xist is transcribed from two promoters, P1 and P2, that appear to be functionally equivalent (11,17). Prior to initiation of random X inactivation in the epiblast and in embryonic stem (ES) cells Xist transcripts are unstable (18,19). The basis for this is presently unclear. We have proposed transcription of a distinct isoform produced from an upstream promoter P0 based on the detection of transcription upstream of promoters P1 and P2 in undifferentiated ES cells (17). Subsequent studies have shown however that an antisense RNA, Tsix, initiated 15 kb downstream of Xist accounts for much of the transcription observed in the upstream region, arguing against a promoter switch (20–22). The above findings led to the hypothesis that Xist RNA stability is developmentally regulated and that Tsix expression is required to destabilize Xist RNA on the active X chromosome-elect at the onset of X inactivation (23). However, the site of initiation of unstable sense transcripts remains uncertain and analysis to date has failed to detect endogenous P1/P2 activation in normal ES cells. In addition, stable Xist RNA is observed in early pre-implantation stage embryos (17,19), and also following induction of an Xist cDNA transgene in undifferentiated ES cells (24). To resolve these apparent contradictions it has been suggested that there is a threshold level for Xist transcripts over which stabilization and spreading along the X chromosome occur (24). Regulation of imprinted X inactivation occurs by a different mechanism, with pre-emptive accumulation of Xist RNA on the paternal allele apparently underlying preferential Xp inactivation in differentiating trophectoderm and primitive endoderm lineages (17,19). In a recent report the Tsix promoter region has been shown to be required for repression of the Xm Xist allele in imprinted X inactivation (25).
Gene targeting and transgenic analysis have demonstrated that the Xic functions of chromosome counting and propagation map to genetically separable regions of the Xist locus. A complex picture is emerging with multiple elements being implicated. Gene targeting in XX ES cells demonstrated that deletion of P1 and P2, together with 7 kb of transcribed sequence disrupts cis-propagation of X inactivation but not counting (15). Transgene analysis led to narrowing down of the sequences required for counting to a 35 kb region encompassing Xist (14,26). Elements located both upstream of P1/P2 and downstream of the 3' end of Xist have been implicated in subsequent experiments (27,28).
X chromosome choosing also appears to involve multiple and distinct elements. A targeted Xist allele has been reported to result in complete primary non-random X inactivation of the normal X chromosome in heterozygous animals (29). Based on comparison with earlier targeting studies these results were interpreted as evidence for a positive choice element located close to the centre of the Xist transcribed locus. In classical experiments heterozygosity at the Xce locus has been shown to cause partially skewed X inactivation at the primary level (4). Recent genetic mapping demonstrates that Xce is tightly linked to Xist but at least 40 kb downstream of the 3' end of the gene (30). More recently, deletion of the Tsix promoter located ~15 kb downstream of Xist was shown to result in near complete primary non-random X inactivation of the targeted chromosome in heterozygotes (23,25).
In this report we describe extreme primary non-random X inactivation attributable to insertion of a PGKneo cassette immediately upstream of Xist promoter P1. The phenotype correlates with probabilistic activation of P1/P2 promoters in a proportion of undifferentiated targeted XY ES cells and increased sense transcription initiated upstream of P1. We discuss these findings in the context of understanding mechanisms of Xist regulation in X inactivation.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Deletion of hypersensitive sites 3 and 4
We designed a gene targeting strategy to test the function of two DNase1 hypersensitive sites (HS3 and HS4) located 1.4 and 3 kb upstream of the P1 promoter, respectively (Fig. 1). Both HS3 and HS4 are detectable in XY and XX ES cells and also in XY and XX somatic cells (31 and unpublished data). The targeting construct was designed to replace a 2.5 kb region flanking HS3 and 4 with the neomycin resistance gene (neor) driven by the human PGK-1 promoter (PGKneo). The PGKneo cassette is flanked with loxP sites so that Cre recombinase-mediated deletion can be used to excise the selectable marker in a subsequent step. The construct was transfected into the 129/1 XY ES cell line and a correctly targeted line, designated Xist
hs, was identified by Southern blot hybridization at both 3' and 5' ends (data not shown). Transient transfection of Xisths ES cells with pMC-Cre, a plasmid encoding Cre recombinase was used to derive sub-lines in which the PGKneo cassette was deleted. These lines were designated Xisthsneo.
|
Both Xist
hs and Xisthsneo cells were injected into blastocysts to produce chimaeric animals. Male chimaeras were then bred to determine whether targeted ES cells are transmitted through the germ line. Previously it has been shown that XY ES cells with an Xist null allele contribute to the male germ line of chimaeric animals but that resultant female progeny die in early embryogenesis because of failed imprinted X inactivation in extra-embryonic lineages (32). In contrast to this we found that chimaeras for both Xisths and Xisthsneo cell lines were transmitted and gave rise to approximately equal numbers of male and female progeny. As all female progeny must carry the targeted Xp chromosome, this result demonstrates that the upstream mutation has not affected propagation of X inactivation from the targeted allele. The fact that female progeny are fully viable also indicates that the counting function of the Xist locus has not been disrupted.
Extreme non-random X inactivation of the Xisths allele
To determine whether the upstream mutation affects the randomness of X inactivation we quantitated allelic levels of Xist and the X-linked Pgk-1 gene in Xisths or Xisthsneo female heterozygotes. Single Nucleotide Primer Extension (SNuPE) analysis was carried out using characterized polymorphisms between standard laboratory strain and PGK strain animals which carry a polymorphic X chromosome derived from a wild strain. Data from a number of independent experiments are summarized in Figure 2A. In control (129x PGK) F1 females we observed slightly higher levels of the 129 strain allele for Xist (59%) and lower levels for Pgk-1 (42%). This mild skewing is attributable to the Xce effect (the PGK strain carries a strong Xcec allele whereas Xisths and Xisthsneo are on a 129 strain X chromosome which carries a weak Xcea allele). Targeted (Xisths x PGK) F1 females, however, exhibited extreme non-random X inactivation. The 129 allele of Xist and Pgk-1 represented 92 and 14% of the total signal, respectively. This implies that in heterozygous animals, ~90% of cells inactivate the targeted 129 derived X chromosome. Similar results were obtained in reciprocal crosses demonstrating that parent of origin effects do not play a role (data not shown). Interestingly, Xisthsneo heterozygotes showed reversion to a wild-type phenotype (Fig. 2A), indicating that insertion of the PGKneo cassette rather than deletion of HS3 and HS4 underlies the non-random X inactivation.
|
We went on to test the extent of skewed X inactivation in the absence of Xce allelic differences. To do this we crossed Xist
hs males with a female line in which the polymorphic Pgk-1 allele had been transferred to an Xcea X chromosome (see Materials and Methods). Analysis of Pgk-1 expression in female progeny from this cross is shown in Figure 2B. A slight decrease in the degree of skewing from ~14% expression of the 129 allele to ~21% was observed. This result indicates that non-random X inactivation attributable to the Xisths allele occurs independently of Xce effects.
Non-random X inactivation occurs at the primary level
Non-random X inactivation can occur as a result of a bias in the initial choice of which X is inactivated (primary) or through cell selection against cells that keep a given X chromosome active (secondary). Given the extreme degree of skewing in heterozygotes, secondary cell selection events may be predicted to have a discernible effect on the development of Xisths female homozygotes. In fact we observed no such effects. Crosses between Xisths/+ heterozygous females and Xisths/Y males produced homozygous Xisths/Xisths females at the expected frequency (Table 1). This was also the case in equivalent crosses with Xisthsneo animals (Table 1). Xisths homozygous females and hemizygous males displayed no overt defects and showed no size differences relative to wild-type littermates.
|
We went on to address the issue of primary versus secondary mechanisms directly by assaying Xist and Pgk-1 alleles in early post-implantation embryos from a cross between Xist
hs heterozygous females and PGK strain males. X inactivation initiates between E5.5 and E6 (33). It has been generally assumed that non-random inactivation due to secondary cell selection events would be detectable as a progressive skewing occurring over the first few days after initiation of X inactivation whereas primary non-random X inactivation would be apparent immediately (see for example ref. 29). Therefore we analysed skewing in embryos as early as possible after the onset of X inactivation (E6.5), and at stages immediately thereafter (E7.5 and E8.5). Results from SNuPE analysis for Xist and Pgk-1 RNA are summarized in Figure 3. At all stages analysed we observed extreme non-random X inactivation of the targeted allele relative to control wild-type littermates. At the E6.5 and E7.5 stages the extent of skewing was less than at E8.5, but in wild-type littermates the Xce effect was also reduced (or absent). This difference is probably attributable to the fact that the visceral endoderm is retained in dissected embryonic regions at E6.5 and E7.5. Because cells in this lineage undergo imprinted X inactivation they will express only the 129 strain Pgk-1 allele and never express the 129 strain Xist allele. Taking this into consideration our results indicate that non-random X inactivation in Xisths heterozygotes occurs at the primary rather than the secondary level.
|
Inappropriate activation of Xist promoters P1/P2 in targeted ES cells correlates with preferential X inactivation
To investigate the basis for extreme non-random X inactivation we analysed Xist expression in Xist
hs XY ES cells using RNA FISH. As reported previously (17), using a probe located in exon 1 in conjunction with an upstream probe, we observed a single colocalizing pinpoint signal in control XY ES cells, corresponding to unstable transcripts (Fig. 4A, left panel). The majority of Xisths XY ES cells also showed this pattern. However, a proportion of cells exhibited a larger Xist RNA signal that was detectable only with the downstream probe (Fig. 4A, central and right hand panels). This pattern is similar to that seen in differentiated XX cells (see for example ref. 17), although in Xisths XY ES cells the signal often appears more diffuse (Fig. 4A, right panel). Accumulation of Xist RNA was not seen in Xisthsneo XY ES cells (data not shown). Therefore, as is the case for skewed X inactivation in vivo, the in vitro phenotype also correlates with insertion of the PGKneo cassette rather than deletion of HS3 and 4.
|
To determine the proportion of Xist
hs cells with accumulated transcripts, and to ensure that these cells had only a single X chromosome, we used the exon 1 probe for Xist RNA FISH in conjunction with a probe specific for X chromosome DNA. In a blind scoring, 12% of Xisths cells showed high level Xist expression compared with a frequency of 0.3% in control XY ES cells (Fig. 4B). Apparent signal in control cells is presumably attributable to non-specific background. No X chromosome aneuploidy was detected.
FISH analysis indicated that cells with accumulated transcripts had inappropriately activated the P1/P2 Xist promoters. We tested this at the molecular level using nuclease protection. As shown in Figure 4C, transcripts initiated from P2, the major promoter (17), were undetectable in both control XY ES cells and Xisthsneo cells. In Xisths cells, however, P2 transcripts were clearly detectable, albeit at a lower level than in XX somatic cells. In addition we observed full-length protected product characteristic of P1 transcripts (17). As P1/P2 bands are never detected in control XY ES cells this result points to inappropriate promoter activation in Xisths XY ES cells.
Accumulation of Xist transcripts in ~10% of targeted ES cells should result in steady-state levels approaching 10% of that seen in XX somatic cell lines. To test this we used quantitative RNA slot blot analysis as described previously (17). Combined results from several independent experiments are illustrated in Figure 4D. Using the w7d probe located in exon 1 we observed ~1.9 ± 0.4% (n = 6) of somatic cell RNA levels in control XY ES cells, consistent with previous results (19), but an elevated level of 5.2 ± 1.1% (n = 6; P < 0.0001) in targeted Xisths XY ES cells. This level is not as high as predicted, presumably reflecting less accumulation relative to XX somatic cells. No difference was observed using the mx3 probe located immediately upstream of P1/P2, consistent with elevated levels being attributable to inappropriate P1/P2 transcription (Fig. 4D). High levels of accumulated P1/P2 transcripts were not seen following differentiation of Xisths ES cells in vitro (data not shown). Presumably this reflects selection as a result of inactivation of the single X chromosome.
Xist/Tsix expression in targeted XY ES cells
As both sense and antisense RNAs contribute to Xist transcripts in undifferentiated ES cells we carried out strand-specific RNA FISH analysis on Xisths ES cells using labelled strand-specific Xist exon 1 oligonucleotides in conjuction with a double-stranded exon 1 probe (Fig. 5A). In cells with punctate RNA signal we could detect no overt difference in antisense expression between control and targeted ES cells. Punctate sense transcript was detectable in only 12% of control cells. This low frequency is probably attributable to the fact that we used short oligonucleotide probes as opposed to the larger single-stranded cDNA probes used in some other studies (for example ref. 20). In targeted cells however, punctate sense transcript was seen at a significantly higher frequency (44%).
|
To analyse this in more detail we carried out strand-specific RT–PCR. Antisense transcription was readily detectable in the upstream region in control and targeted XY ES cells (amplicons 2 and 4, Fig. 5B). In Xist
hs cells amplicon 2 product probably represents readthrough transcription from Tsix or the PGKneo cassette (which is transcribed in the opposite direction to Xist). In control XY ES cells sense transcription was only detected with amplicon 6 located in exon 1 (Fig. 5B). In targeted cells, however, upstream sense transcripts were also readily detectable using amplicon 4. Cre-mediated deletion of the PGKneo cassette in Xisthsneo cells restored the pattern seen in control ES cells. This result was fully reproducible in independent experiments. 5' RACE was carried out to determine where the upstream sense transcripts in Xisths cells initiated. Southern analysis of RACE PCR products using oligonucleotide probe 4s revealed a major band which sequence analysis demonstrated corresponds to initiation ~550 bp upstream of P1 (Fig. 5C). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
This study demonstrates that the deletion of two DNase1 hypersensitive sites immediately upstream of Xist, together with insertion of a PGKneo selection cassette, causes extreme non-random X inactivation of the chromosome bearing the targeted allele. Non-random X inactivation occurs at the primary level and correlates with premature and inappropriate activation of the Xist P1/P2 promoters in a proportion of undifferentiated targeted XY ES cells. These cells exhibit high level accumulation of Xist RNA similar to that seen in XX somatic cells. Additionally, we observed increased sense transcription from an initiation site upstream of P1. Both in vitro and in vivo phenotypes were effectively reversed by Cre-mediated deletion of PGKneo.
Our data indicate that non-random X inactivation in vivo occurs at the primary level and therefore corresponds to a bias in the choice of which Xist allele is expressed in a given cell. Whilst the basis for this effect is not clear, premature activation of P1/P2 promoters in undifferentiated XY ES cells in vitro appears to provide a possible model. Specifically, in heterozygous XX embryos, cells that have prematurely activated P1/P2 on the targeted allele could have effectively predetermined which X to inactivate. This would be equivalent to the situation in pre-implantation mouse embryos where accumulation of P1/P2 Xist transcripts from the Xp allele occurs prior to cellular differentiation (17,19). In this situation early P1/P2 expression apparently pre-selects for complete non-random inactivation of Xp in all cells during differentiation of trophectoderm and primitive endoderm lineages.
Disturbance of interactions between distant regulatory elements following insertion of selectable markers has been reported to occur at a number of targeted loci including the ß-globin LCR (34) and the immunoglobulin heavy chain constant region locus (35). In the present study it is possible that the inserted PGK promoter acts as a decoy for a negative regulatory element that normally acts either directly or indirectly to repress P1/P2 activity in ES cells. P1/P2 activation and non-random X inactivation are not observed when PGKneo is targeted 6.5 kb upstream of P1 in conjunction with a deletion of the putative P0 promoter (C. Johnston, manuscript in preparation). Similarly, insertion of PGKneo in intron 3 of Xist has no effect (M.-L. Caparros, manuscript in preparation), indicating that any such decoy effect must be highly position dependent. In this context it is interesting to note that a mutation in the region of the human XIST promoter has been correlated with non-random X inactivation (36).
Interference with either sense or antisense transcripts could also play a causative role in non-random X inactivation, either alone or in combination with premature P1/P2 activation. Previously it has been shown that deletion of the major Tsix antisense promoter in ES cells results in extreme but not complete non-random X inactivation of the targeted allele (23). In Xisths cells, however, we detected no difference in the antisense Tsix transcript relative to control cells but interestingly we saw a similar X inactivation phenotype associated with increased sense transcription from an upstream site. Although this transcription correlates with insertion of the PGKneo casette, the PGK promoter is oriented in the antisense rather than sense direction (Fig. 1). We mapped the initiation site for these transcripts to a region ~550 bp upstream of P1, indicating that PGKneo insertion has either activated or hyperactivated an endogenous promoter sequence. It is unclear whether the elevated upstream sense transcription detected by RT–PCR occurs in all cells or whether it is confined to the proportion of Xisths ES cells exhibiting accumulation of Xist RNA. Our data argue against the latter possibility since accumulated Xist RNA was never detected using an upstream RNA FISH probe. Additionally, the enhanced RNA FISH detection of punctate sense transcript relative to untargeted control cells is consistent with an increase in the level of unstable upstream sense transcript. Theoretically, increased sense transcription could be associated with an altered chromatin structure that in turn could underlie the non-random X inactivation phenotype.
Activation of P1/P2 transcription in Xisths ES cells correlates with cis-accumulation of high levels of Xist RNA. This provides a further example demonstrating that Xist RNA stability is not developmentally regulated, but does not allow us to distinguish between the RNA isoform model (17) and the transcript threshold model (24). Clonal analysis of Xisths XY ES cells indicated that P1/P2 activation is not maintained through mitosis (data not shown). This may reflect negative selection as a result of inactivating the single X chromosome or alternatively may indicate that P1/P2 activation is dynamic and reversible. Evidence to date has indicated that cis-accumulation of stable Xist transcripts in undifferentiated cell types does not result in X inactivation. This is based on observations on cells of early pre-implantation embryos (17,19) and on mouse ES cells expressing human Xist RNA (37). We observed expression of X-linked loci in Xisths ES cells with accumulated P1/P2 RNA (data not shown), although quantitation of the data indicated a reduction in X-linked gene expression relative to cells without P1/P2 transcripts. Thus, partial inactivation of the X chromosome in response to Xist RNA accumulation may occur in undifferentiated ES cells. This is consistent with a recent study which demonstrated that cis-accumulation of Xist RNA and long-range gene silencing can occur following induction of an Xist cDNA transgene in ES cells (24).
In summary this study provides evidence for a novel mechanism in which a heterologous promoter upstream of the Xist locus can act as a decoy, perturbing normal interactions of regulatory elements that govern X chromosome choice at the onset of random X inactivation.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Cell lines
Gene targeting was carried out using the 129/1 XY ES cell line isolated in house (38). Cells were maintained and cultured on STO feeders. The XX somatic cell line C127 was maintained and cultured as described previously (39).
Generation of Xisths and Xisthsneo alleles
The 5' and 3' homologous regions correspond to nucleotides –7484 to –3623 and –1106 to +2380 relative to promoter P1. The construct was based on a standard positive(neor)/negative(thymidine kinase) strategy. The positive selection cassette PGKneo was flanked with loxP sites. 107 ES cells were electroporated with 10 µg linearized vector (200 V, 960 µF), and grown for 10 days under geneticin (400 µg/ml) and gancyclovir (2 µM) selection. Individual colonies were picked into 96-well plates and screened by Southern analysis. The recombinant Xisths line was recovered from 251 +/– selected colonies. To remove the PGKneo cassette, 107 Xisths cells were electroporated with 25 µg supercoiled pMC-Cre plasmid (Gibco BRL) (200 V, 960 µF). After plating out at low density, 163 Cre-transfected clones were analysed and four recombinant Xisthsneo lines (Cre 1.7D, 2.3E, 2.5F and 2.12E) were recovered. Both Xisths and Cre 1.7D (Xisthsneo) were found to be karyotypically stable (data not shown), and were used in all subsequent experiments. Male chimaeras produced by blastocyst injection of targeted ES cells were mated to C57BL/6 females. Xisths and Xisthsneo colonies were established by breeding with (C57BL/6 x CBA) F1 animals. Mice were genotyped by PCR analysis of tail-tip DNA; the Xisths allele was detected by the primer pair TNX35-NeoF2, the Xisthsneo allele by TNX35-SX2 and the wild-type Xist allele by TNX35-HS22 (TNX35: –4109 to –4088; SX2: –475 to –496; HS22: –3078 to –3098; NeoF2: 5’-CCCGTGATATTGCTGAAGAGC-3’). PCR reactions were given 35 cycles of 94°C for 1 min; 53°C for 1 min and 72°C for 1 min. All Xist sequence positions are given relative to +1 of Xist cDNA.
Analysis of X inactivation in vivo
PGK strain animals were bred in house. Xcea animals with the polymorphic Pgk-1a allele were kindly provided by Bruce Cattanach, MRC Harwell.
DNA and RNA were isolated from fresh tissues as described previously (15). Quantitative Xist and Pgk-1 SNuPE analysis was performed using primers and conditions described previously (15,40). To account for variations in specific activity of nucleotides, analysis of genomic DNA from a (PGK x 129) F1 female animal was used to normalize the data.
For embryo analysis timed matings were set up between Xisths heterozygous females and PGK strain males. Embryos were dissected out and separated into embryonic and extra-embryonic components. DNA was prepared from the extra-embryonic component and used for genotyping by PCR. Samples were incubated overnight in 10 µl TE/Tween with 40 µg proteinase K under mineral oil, before being heated at 95°C for 10 min. A total of 2 µl was used for genotyping by the TNX35-NeoF2 PCR as described above. Female embryos were genotyped as carrying either the Xisths or wild-type maternal X chromosome together with the PGK strain paternal X chromosome. RNA prepared from the embryonic part was used for quantitative SNuPE analysis. RT–PCR analysis of Xist expression using a defined HindIII polymorphism between the PGK strain and standard laboratory strains (38) was used to sex individual embryos.
Analysis of Xist expression in ES cells in vitro
For FISH analysis preparation of slides, labelling of probes and RNA FISH were carried out as described previously (19). Probes used were GPT16 (Xist exon 1) and mx8 (Xist upstream region) (17). RNA/DNA FISH was performed as described previously (41). The BAC141 probe is a Bac genomic DNA clone isolated by screening with the Smh141 probe corresponding to an X-specific repeat sequence (42). Strand-specific RNA FISH was performed using exon 1 oligonucleotide probes as described previously (21). Probes were kindly provided by E. Heard.
Steady-state RNA analysis was performed as described previously (17,19), using the upstream probe mx3 and the exon 1 probe w7d. Samples of total RNA (10 µg) were loaded in duplicate slots and 2 ng Xist cosmid was loaded as a control. Duplicate samples were subtracted for background, and then normalized to 28S rRNA. Hybridization to Xist cosmid was used to normalize the data. Quantitation was performed using a PhosphorImager (Molecular Dynamics; ImageQuant).
Nuclease protection analysis was performed on 10 µg total RNA using the antisense probe RP2 as described previously (17).
Strand-specific RT–PCR was performed essentially as described previously (20). RNA was prepared from ~50 mg freshly dissected tissue or from 106 to 107 cultured cells, using RNAzol B (Biogenesis). Total cellular RNA (10 µg) was treated with 1 U RNase-free DNase I (Ambion) for 10 min at 37°C. The RNA was then re-extracted and resuspended in a volume of 50 µl RNase-free water. First strand cDNA synthesis was performed using 6 pmol each of the strand-specific Xist primers 2S, 2AS, 4S, 4AS, 6S and 6AS described previously (20). Reactions were incubated at 50°C for 1 h and terminated by heating at 80°C for 10 min. Water (10 µl) was added to each tube, and 1 µl cDNA used for PCR amplification with the appropriate sense and antisense primer pairs as described previously (20) with the exception that amplicon 2 was amplified using the pair 2S and TNX6 (5’-GCA AGA TCT TAG CCA AAC GAG-3’), since in our hands this combination gave a cleaner product. Amplicons 2 and 4 were amplified using an annealing temperature of 51°C and were given 35 cycles, amplicon 6 was annealed at 54°C and given 32 cycles.
5' RACE used the Marathon cDNA amplification kit (Clontech). Double-stranded cDNA was synthesized from 10 µl 4AS-primed first strand cDNA (see above). Following adaptor ligation, first round PCR product was generated between XRACE1 (located –386 to –359 upstream of Xist promoter P1) and the adaptor primer AP1. Second round product was then generated between XRACE2 (–415 to –385) and adaptor primer AP2.
|
ACKNOWLEDGEMENTS |
|---|
We would like to thank members of the X inactivation group for critical reading of this manuscript and Graham Reed and Richard Newton for help with preparing figures. This work was supported by the Medical Research Council, UK. T.N. is supported by an international development award from the Wellcome Trust.
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +44 208 383 8278; Fax: +44 208 383 8303; Email: nbrockdo@hgmp.mrc.ac.uk
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Lyon, M.F. (1961) Gene action in the X chromosome of the mouse (Mus musculus L.). Nature, 190, 372–373.[Medline]
2 Takagi, N. and Sasaki, M. (1975) Preferential inactivation of the paternally derived X chromosome in the extraembryonic membranes of the mouse. Nature, 256, 640–642.[Medline]
3 Cattanach, B.M., Pollard, C.E. and Perez, J.N. (1969) Controlling elements in the mouse X-chromosome. I. Interaction with the X-linked genes. Genet. Res., 14, 223–235.[Web of Science][Medline]
4 Rastan, S. (1982) Primary non-random X-inactivation caused by controlling elements in the mouse demonstrated at the cellular level. Genet. Res., 40, 139–147.[Web of Science][Medline]
5 McMahon, A. and Monk, M. (1983) X-chromosome activity in female mouse embryos heterozygous for Pgk-1 and Searle’s translocation, T(X; 16) 16H. Genet. Res., 41, 69–83.[Web of Science][Medline]
6 Russell, L.B. and Montgomery, C.S. (1965) The use of X-autosome translocations in locating the X-chromosome inactivation centre. Genetics, 52, 470–471.
7 Rastan, S. and Brown, S.D. (1990) The search for the mouse X-chromosome inactivation centre. Genet. Res., 56, 99–106.[Web of Science][Medline]
8 Rastan, S. (1983) Non-random X-chromosome inactivation in mouse X-autosome translocation embryos—location of the inactivation centre. J. Embryol. Exp. Morphol., 78, 1–22.
9 Borsani, G., Tonlorenzi, R., Simmler, M.C., Dandolo, L., Arnaud, D., Capra, V., Grompe, M., Pizzuti, A., Muzny, D., Lawrence, C. et al. (1991) Characterization of a murine gene expressed from the inactive X chromosome. Nature, 351, 325–329.[Medline]
10 Brockdorff, N., Ashworth, A., Kay, G.F., Cooper, P., Smith, S., McCabe, V.M., Norris, D.P., Penny, G.D., Patel, D. and Rastan, S. (1991) Conservation of position and exclusive expression of mouse Xist from the inactive X chromosome. Nature, 351, 329–331.[Medline]
11 Brockdorff, N., Ashworth, A., Kay, G.F., McCabe, V.M., Norris, D.P., Cooper, P.J., Swift, S. and Rastan, S. (1992) The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus. Cell, 71, 515–526.[Web of Science][Medline]
12 Brown, C.J., Ballabio, A., Rupert, J.L., Lafreniere, R.G., Grompe, M., Tonlorenzi, R. and Willard, H.F. (1991) A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature, 349, 38–44.[Medline]
13 Brown, C.J., Hendrich, B.D., Rupert, J.L., Lafreniere, R.G., Xing, Y., Lawrence, J. and Willard, H.F. (1992) The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell, 71, 527–542.[Web of Science][Medline]
14 Lee, J.T., Strauss, W.M., Dausman, J.A. and Jaenisch, R. (1996) A 450 kb transgene displays properties of the mammalian X-inactivation center. Cell, 86, 83–94.[Web of Science][Medline]
15 Penny, G.D., Kay, G.F., Sheardown, S.A., Rastan, S. and Brockdorff, N. (1996) Requirement for Xist in X chromosome inactivation. Nature, 379, 131–137.[Medline]
16 Clemson, C.M., McNeil, J.A., Willard, H.F. and Lawrence, J.B. (1996) XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J. Cell Biol., 132, 259–275.
17 Johnston, C.M., Nesterova, T.B., Formstone, E.J., Newall, A.E., Duthie, S.M., Sheardown, S.A. and Brockdorff, N. (1998) Developmentally regulated Xist promoter switch mediates initiation of X inactivation. Cell, 94, 809–817.[Web of Science][Medline]
18 Panning, B., Dausman, J. and Jaenisch, R. (1997) X chromosome inactivation is mediated by Xist RNA stabilization. Cell, 90, 907–916.[Web of Science][Medline]
19 Sheardown, S.A., Duthie, S.M., Johnston, C.M., Newall, A.E., Formstone, E.J., Arkell, R.M., Nesterova, T.B., Alghisi, G.C., Rastan, S. and Brockdorff, N. (1997) Stabilization of Xist RNA mediates initiation of X chromosome. Cell, 91, 99–107.[Web of Science][Medline]
20 Lee, J.T., Davidow, L.S. and Warshawsky, D. (1999) Tsix, a gene antisense to Xist at the X-inactivation centre. Nature Genet., 21, 400–404.[Web of Science][Medline]
21 Debrand, E., Chureau, C., Arnaud, D., Avner, P. and Heard, E. (1999) Functional analysis of the DXPas34 locus, a 3' regulator of Xist expression. Mol. Cell. Biol., 19, 8513–8525.
22 Warshawsky, D., Stavropoulos, N. and Lee, J.T. (1999) Further examination of the Xist promoter-switch hypothesis in X inactivation: Evidence against the existence and function of a P(0) promoter. Proc. Natl Acad. Sci. USA, 96, 14424–14429.
23 Lee, J.T. and Lu, N. (1999) Targeted mutagenesis of Tsix leads to nonrandom X inactivation. Cell, 99, 47–57.[Web of Science][Medline]
24 Wutz, A. and Jaenisch, R. (2000) A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation. Mol. Cell, 5, 695–705.[Web of Science][Medline]
25 Lee, J.T. (2000) Disruption of imprinted X inactivation by parent-of-origin effects at Tsix. Cell, 103, 17–27.[Web of Science][Medline]
26 Herzing, L.B., Romer, J.T., Horn, J.M. and Ashworth, A. (1997) Xist has properties of the X-chromosome inactivation centre. Nature, 386, 272–275.[Medline]
27 Clerc, P. and Avner, P. (1998) Role of the region 3' to Xist exon 6 in the counting process of X- chromosome inactivation. Nature Genet., 19, 249–253.[Web of Science][Medline]
28 Lee, J.T., Lu, N. and Han, Y. (1999) Genetic analysis of the mouse X inactivation center defines an 80-kb multifunction domain. Proc. Natl Acad. Sci. USA, 96, 3836–3841.
29 Marahrens, Y., Loring, J. and Jaenisch, R. (1998) Role of the Xist gene in X chromosome choosing. Cell, 92, 657–664.[Web of Science][Medline]
30 Simmler, M.C., Cattanach, B.M., Rasberry, C., Rougeulle, C. and Avner, P. (1993) Mapping the murine Xce locus with (CA)n repeats. Mamm. Genome, 4, 523–530.[Web of Science][Medline]
31 Sheardown, S.A., Newall, A.E., Norris, D.P., Rastan, S. and Brockdorff, N. (1997) Regulatory elements in the minimal promoter region of the mouse Xist gene. Gene, 203, 159–68.[Web of Science][Medline]
32 Marahrens, Y., Panning, B., Dausman, J., Strauss, W. and Jaenisch, R. (1997) Xist-deficient mice are defective in dosage compensation but not spermatogenesis. Genes Dev., 11, 156–166.
33 Rastan, S. (1982) Timing of X-chromosome inactivation in postimplantation mouse embryos. J. Embryol. Exp. Morphol., 71, 11–24.[Web of Science][Medline]
34 Fiering, S., Epner, E., Robinson, K., Zhuang, Y., Telling, A., Hu, M., Martin, D.I., Enver, T., Ley, T.J. and Groudine, M. (1995) Targeted deletion of 5'HS2 of the murine beta-globin LCR reveals that it is not essential for proper regulation of the beta-globin locus. Genes Dev., 9, 2203–2213.
35 Seidl, K.J., Manis, J.P., Bottaro, A., Zhang, J., Davidson, L., Kisselgof, A., Oettgen, H. and Alt, F.W. (1999) Position-dependent inhibition of class-switch recombination by PGK-neor cassettes inserted into the immunoglobulin heavy chain constant region locus. Proc. Natl Acad. Sci. USA, 96, 3000–3005.
36 Plenge, R.M., Hendrich, B.D., Schwartz, C., Arena, J.F., Naumova, A., Sapienza, C., Winter, R.M. and Willard, H.F. (1997) A promoter mutation in the XIST gene in two unrelated families with skewed X-chromosome inactivation. Nature Genet., 17, 353–356.[Web of Science][Medline]
37 Heard, E., Mongelard, F., Arnaud, D., Chureau, C., Vourc'h, C. and Avner, P. (1999) Human XIST yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells. Proc. Natl Acad. Sci. USA, 96, 6841–6846.
38 Kay, G.F., Penny, G.D., Patel, D., Ashworth, A., Brockdorff, N. and Rastan, S. (1993) Expression of Xist during mouse development suggests a role in the initiation of X chromosome inactivation. Cell, 72, 171–182.[Web of Science][Medline]
39 Jeppesen, P. and Turner, B.M. (1993) The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression. Cell, 74, 281–289.[Web of Science][Medline]
40 Singer-Sam, J., LeBon, J.M., Dai, A. and Riggs, A.D. (1992) A sensitive, quantitative assay for measurement of allele-specific transcripts differing by a single nucleotide. PCR Methods Appl., 1, 160–163.[Medline]
41 Duthie, S.M., Nesterova, T.B., Formstone, E.J., Keohane, A.M., Turner, B.M., Zakian, S.M. and Brockdorff, N. (1999) Xist RNA exhibits a banded localization on the inactive X chromosome and is excluded from autosomal material in cis. Hum. Mol. Genet., 8, 195–204.
42 Nasir, J., Fisher, E.M., Brockdorff, N., Disteche, C.M., Lyon, M.F. and Brown, S.D. (1990) Unusual molecular characteristics of a repeat sequence island within a Giemsa-positive band on the mouse X chromosome. Proc. Natl Acad. Sci. USA, 87, 399–403. [Erratum (1990) Proc. Natl Acad. Sci. USA, 87, 9508.]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  K. V. Prasanth and D. L. Spector Eukaryotic regulatory RNAs: an answer to the 'genome complexity' conundrum Genes & Dev., January 1, 2007; 21(1): 11 - 42. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Tufarelli The silence RNA keeps: cis mechanisms of RNA mediated epigenetic silencing in mammals Phil Trans R Soc B, January 29, 2006; 361(1465): 67 - 79. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Gribnau, S. Luikenhuis, K. Hochedlinger, K. Monkhorst, and R. Jaenisch X chromosome choice occurs independently of asynchronous replication timing J. Cell Biol., January 31, 2005; 168(3): 365 - 373. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Thakur, V. K. Tiwari, H. Thomassin, R. R. Pandey, M. Kanduri, A. Gondor, T. Grange, R. Ohlsson, and C. Kanduri An Antisense RNA Regulates the Bidirectional Silencing Property of the Kcnq1 Imprinting Control Region Mol. Cell. Biol., September 15, 2004; 24(18): 7855 - 7862. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. B. Nesterova, C. M. Johnston, R. Appanah, A. E.T. Newall, J. Godwin, M. Alexiou, and N. Brockdorff Skewing X chromosome choice by modulating sense transcription across the Xist locus Genes & Dev., September 1, 2003; 17(17): 2177 - 2190. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||