RESEARCH ARTICLE 1541
Development 138, 1541-1550 (2011) doi:10.1242/dev.056812
© 2011. Published by The Company of Biologists Ltd
Disruption of a conserved region of Xist exon 1 impairs Xist
RNA localisation and X-linked gene silencing during random
and imprinted X chromosome inactivation
Claire E. Senner1,2,*, Tatyana B. Nesterova1, Sara Norton2, Hamlata Dewchand2, Jonathan Godwin1,
Winifred Mak2 and Neil Brockdorff1,†
SUMMARY
In XX female mammals a single X chromosome is inactivated early in embryonic development, a process that is required to
equalise X-linked gene dosage relative to XY males. X inactivation is regulated by a cis-acting master switch, the Xist locus, the
product of which is a large non-coding RNA that coats the chromosome from which it is transcribed, triggering recruitment of
chromatin modifying factors that establish and maintain gene silencing chromosome wide. Chromosome coating and Xist RNAmediated silencing remain poorly understood, both at the level of RNA sequence determinants and interacting factors. Here, we
describe analysis of a novel targeted mutation, XistINV, designed to test the function of a conserved region located in exon 1 of
Xist RNA during X inactivation in mouse. We show that XistINV is a strong hypomorphic allele that is appropriately regulated but
compromised in its ability to silence X-linked loci in cis. Inheritance of XistINV on the paternal X chromosome results in embryonic
lethality due to failure of imprinted X inactivation in extra-embryonic lineages. Female embryos inheriting XistINV on the maternal
X chromosome undergo extreme secondary non-random X inactivation, eliminating the majority of cells that express the XistINV
allele. Analysis of cells that express XistINV RNA demonstrates reduced association of the mutant RNA to the X chromosome,
suggesting that conserved sequences in the inverted region are important for Xist RNA localisation.
INTRODUCTION
X inactivation is the dosage compensation mechanism that has
evolved in mammals to ensure equal levels of X-linked gene
products in females (XX) and males (XY). The master regulator of
this process is a large non-coding RNA termed Xist (inactive X
specific transcripts) (Brown et al., 1991; Brockdorff et al., 1991;
Borsani et al., 1991). X inactivation is initiated by the upregulation
of Xist expression. This upregulation leads to accumulation of
Xist RNA that can be detected by RNA fluorescence in situ
hybridisation (FISH) as a large domain coating the entire X
chromosome from which it is expressed (Sheardown et al., 1997;
Panning et al., 1997). Chromosome-wide transcriptional silencing
rapidly ensues. The X chromosome becomes heterochromatic,
exhibiting marks of transcriptionally silent chromatin such as
H3K27me3 (Silva et al., 2003; Plath et al., 2003), H3K9me2
(Heard et al., 2001; Peters et al., 2002; Mermoud et al., 2002),
H4K20me1 (Kohlmaier et al., 2004), H2AK119u1 (de Napoles et
al., 2004; Fang et al., 2004), enrichment of the variant histone
macroH2A (Mermoud et al., 1999; Costanzi et al., 2000), and
promoter CpG island methylation (Norris et al., 1991). The inactive
X chromosome (Xi) also loses marks associated with active
1
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1
3QU, UK. 2MRC Clinical Sciences Centre, Imperial College, Hammersmith Campus,
Du Cane Road, London W12 ONN, UK.
*Present address: Laboratory of Developmental Genetics and Imprinting, The
Babraham Institute, Cambridge CB22 3AT, UK
†
Author for correspondence ([email protected])
Accepted 26 January 2011
transcription, such as H3K4 methylation (Heard et al., 2001) and
acetylation of the four major histones H2A, H2B, H3 and H4
(Jeppesen and Turner, 1993; Belyaev et al., 1996).
In mouse there are two waves of X inactivation: imprinted X
inactivation of the paternal X chromosome initiated in early
preimplantation embryos and persisting in extra-embryonic
trophectoderm and primitive endoderm lineages; and random X
inactivation that occurs in cells of the embryo proper at
embryonic day (E)5.5-6.5. Embryo progenitor cells in the inner
cell mass of the blastocyst reverse imprinted X inactivation in
order to prepare for subsequent random X inactivation (Mak et
al., 2004; Okamoto et al., 2004). Initial inactivation of the
paternal X chromosome occurs as a result of a repressive imprint
inherited by the maternal Xist allele, with the paternal allele
being set to a default on state (Kay et al., 1994; Goto and Takagi,
2000; Tada et al., 2000). At the onset of random X inactivation,
Xist is expressed only in females and from only one of the two
X chromosomes present. The mechanism regulating this is not
fully understood but involves precise integration of Xist
repressors (for a review, see Senner and Brockdorff, 2009) and
activators (Barakat et al., 2010), as well as trans-interactions of
the two Xist loci. Xist expression is absolutely required for X
inactivation to occur in cis. XX embryonic stem (ES) cells or
female mice carrying one mutant Xist allele always inactivate the
X chromosome carrying the wild-type Xist allele (Penny et al.,
1996; Marahrens et al., 1997). Further to this, Xist expression is
sufficient to trigger the inactivation process. Ectopic expression
of Xist cDNA transgenes leads to transcriptional silencing and
establishment of the epigenetic modifications associated with the
Xi chromosome (Wutz and Jaenisch, 2000; Kohlmaier et al.,
2004).
DEVELOPMENT
KEY WORDS: X inactivation, Xist, Epigenetics, Mouse
1542 RESEARCH ARTICLE
MATERIALS AND METHODS
Gene targeting
129Ola genomic sequences for the arms of homology were isolated from a
DASHII genomic library made in house. A NotI-XhoI 9.2 kb fragment
(+2189 nt to +11,382 nt relative to the Xist start site) was purified from
pMLC1 phage clone and cloned into pBluescript SK+. The plasmid was
digested with HpaI, which linearised the plasmid and separated the 3.8 kb
5⬘ homology region from the 5.4 kb 3⬘homology region. A blunt-ended
LoxP-PGKneo-LoxP cassette was then ligated into the dephosphorylated
HpaI site in the vector. Plasmid DNA was digested with XhoI to accept a
SalI/XhoI fragment of the diptheria-toxin A gene. The latter was used for
negative selection of random integrants (Yagi et al., 1990). The final
construct pNBXT1 was linearised with NaeI and electroporated into the
XistEx4del XY ES cell line as described elsewhere (Caparros et al., 2002).
Sexing embryos
Embryos were added to embryo lysis buffer (50 mM Tris HCl pH 8, 1 mM
EDTA, 0.5% Tween 20, 200 g/ml proteinase K) and incubated overnight
at 55°C to lyse cells and digest proteins. The samples were then incubated
at 95°C for 10 minutes to lyse any remaining cells and inactivate the
proteinase K. The embryo lysate was then pipetted up and down rapidly to
dissociate any remaining clumps and then spun briefly to pellet debris. The
supernatant was used for the sexing PCR reaction. The sexing PCR
amplifies the UBEX gene on the X chromosome as well as the homologous
UBEY gene on the Y chromosome (F 5⬘-TGGTCTGGACCCAAACGCTGTCCACA-3⬘, R 5⬘- GGCAGCAGCCATCACATAATCCAGATG-3⬘).
The same primers yield a larger product from the X linked gene than from
the Y. Accordingly, XX females have one band and XY males have two
bands (Chuma and Nakatsuji, 2001). Reaction conditions were: 94°C for 4
minutes, 35 cycles of 94°C for 30 seconds, 64°C for 30 seconds, 72°C for
30 seconds, and a final 72°C for 5 minutes.
Derivation and culture of mouse embryonic fibroblasts (MEFs)
E14.5 embryos were obtained from crosses between males carrying an Xlinked GFP transgene and XistINV carrying females. The heads and organs
were removed and carcasses were transferred to 0.25% trypsin/0.04%
EDTA/2% chick serum and the tissues roughly dissociated by passing them
through an 18 gauge needle (BD Biosciences). The dissociated carcasses
were then incubated in the 0.25% trypsin/0.04% EDTA/2% CS for 10
minutes at 37°C. The trypsin was inactivated by adding an equal volume
of MEF culture medium (DMEM supplemented with 10% foetal calf
serum, 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM Lglutamine, 1⫻ NEAA and 50 M -mercaptoethanol). Clumps of tissue
were further dissociated by passing them through a 21 gauge needle (BD
Biosciences). The resulting cells were washed in fresh MEF culture
medium and transferred to a tissue culture vessel at a density of roughly
one embryo per 25 cm2. All tissue culture reagents were from Invitrogen.
GFP positive and negative MEFs were sorted by fluorescence activated cell
sorting (FACS) after two passages on the basis of their GFP expression
level using a FACS VANTAGE SE as described previously (de Napoles et
al., 2007).
RT-PCR and northern blot
RNA was isolated from MEFs using TRIzol reagent (Sigma) according to
the manufacturer’s instructions. RNA was routinely treated with Turbo
DNA-free reagent (Ambion) to exclude the possibility of DNA
contamination. cDNA synthesis was primed from random hexamers
(GE Healthcare) with Superscript III reverse transcriptase (Invitrogen).
PCR was then carried out for primers located in Xist exon 1 (a, 5⬘GTGCTGTGTGAGTGAACCTATGG-3⬘; h, 5⬘-TACACACCAGAACAAAGATTGGG-3⬘; TA 65°C), in Xist exons 3-5 (e, 5⬘-CAATCCCTATGTGAGACTCAAGGACT-3⬘; l, 5⬘-ATTATATGGCATGAGTAGGGTAGCAGTG-3⬘; TA 62°C), flanking LoxP in XistINV allele (b, 5⬘-AAGTGTTGGCATATCTGGTTCCT-3⬘; e, 5⬘-CAATCCCTATGTGAGACTCAAGGACT-3⬘; TA 60°C) and for primers for -actin (F 5⬘-GATATCGCTGCGCTGGTCGT-3⬘; R 5⬘-AGATCTTCTCCATGTCGTCC-3⬘;
TA 61°C). Reaction conditions were: 94°C for 4 minutes, 35 cycles of 94°C
for 30 seconds, specific annealing temperature (TA) for 30 seconds, 72°C
DEVELOPMENT
One approach to understanding how Xist carries out its silencing
function has been to investigate the structure of the Xist gene
(Brown et al., 1992; Brockdorff et al., 1992; Nesterova et al.,
2001). Although primary sequence conservation is poor within the
Xist gene, the overall gene structure is well conserved and, in
particular, the conservation of several blocks of tandem repeats,
termed A-F, between mammalian species suggests that they might
be involved in Xist function.
In order to establish which parts of the Xist RNA transcript are
important for its function, Wutz et al. (Wutz et al., 2002) generated
ES cell lines with inducible Xist cDNA transgenes carrying deletion
of different regions of the transcript. This study concluded that only
the 5⬘ region of the transcript containing the A-repeats was
necessary for gene silencing. Further to this, it appeared that
sequences 3⬘ of the A-repeats act cooperatively to localise the RNA
to the chromosome but they are redundant as no single sequence is
absolutely required. The function of the A-repeats was
subsequently investigated in vivo using a gene targeting approach.
Interestingly, deleting the A-repeats altered the regulation of the
mutant Xist allele such that it was never expressed. As a
consequence it was not possible to examine the functionality of an
A-repeat deficient Xist in vivo (Hoki et al., 2009).
Although transgene studies (Wutz et al., 2002) have yielded
valuable information on functionally important regions of Xist
RNA there are disadvantages to this approach. First, overexpression of Xist transgenes could mask subtle loss-of-function
phenotypes and second, partial silencing by mutant Xist RNAs
might be insufficient to cause functional nullisomy of the X
chromosome, the basis of the silencing assay used by Wutz et al.
(Wutz et al., 2002), and might therefore lead to hypomorphic Xist
RNA mutations being scored as unaffected. With this in mind, it is
clearly important to complement transgene studies by generating
mutations of the endogenous Xist locus and analysing their
phenotype in vivo. The majority of knockout alleles produced to
date have deleted Xist promoter sequences and have therefore
ablated transcription of mutant alleles. These mutations have all
resulted in primary non-random X inactivation of the wild-type
allele in the epiblast of heterozygous female embryos. This is true
also for the specific deletion of the A-repeats, which is not
predicted to affect known Xist promoter sequences (Hoki et al.,
2009). The only exception is the targeted deletion of exon 4 that
includes a highly conserved sequence predicted to form a stable
stem loop structure (Caparros et al., 2002). Surprisingly, deleting
exon 4 in mice did not cause detectable effects on X inactivation,
as evidenced by viability and complete absence of skewing of
random X inactivation in heterozygous females. Mutant alleles
produced lower levels of Xist RNA but this did not impact Xist
function (Caparros et al., 2002).
To extend analysis of functional elements within Xist RNA by
gene targeting, we have used the Xistex4 allele to generate a targeted
inversion, XistINV, disrupting conserved sequences beginning 6 kb
into exon 1 and extending to intron 5. Female mice carrying a
maternally inherited XistINV allele exhibited secondary non-random
X inactivation, demonstrating that the inversion affects Xistmediated silencing but not Xist gene regulation. Female embryos
inheriting XistINV on the paternal X chromosome are lost at ~E11.5
as a result of failed imprinted X inactivation. This phenotype is less
severe than that reported for Xist null alleles (Marahrens et al.,
1997), suggesting that XistINV is a hypomorphic allele. Consistent
with this interpretation, molecular analyses suggest that XistINV
RNA localises to the X chromosome in cis, but at reduced
efficiency, and that silencing of X-linked genes is limited.
Development 138 (8)
Xist RNA localisation
RESEARCH ARTICLE 1543
for 30 seconds, and a final 72°C for 5 minutes. The relative positions of
Xist primers are indicated in Fig. S1 in the supplementary material.
Northern blots were carried out as described by Sambrook et al. (Sambrook
et al., 1989).
Single nucleotide primer extension (SNuPE)
Immunofluorescence
E6.5 embryos were obtained from crosses between wild-type females and
males carrying an X-linked GFP transgene and the XistINV allele or wildtype Xist allele. Females were identified by GFP expression. Embryonic
and extra-embryonic parts were separated. For each litter, embryonic parts
were pooled together and extra-embryonic parts were pooled together.
These pools were washed in PBST and then added to trypsin. They were
incubated for 15 minutes at 37°C. An excess of MEF culture medium was
added and the embryos were broken up into single cells and small clumps
by vigorous pipetting. The cell suspension was then cytospun onto
Superfrost slides (VWR). Immunofluorescence was then carried out
as described previously (de Napoles et al., 2004). Scoring of
immunofluorescence experiments was carried out on at least 100 cells in
all experiments.
RNA FISH
Digoxygenin-11-dUTP (DIG)-labelled DNA Xist probes were generated by
nick-translation from a plasmid containing the entire Xist cDNA sequence,
using a DIG nick-translation mix (Roche) according to the manufacturer’s
instructions. Metaphase spreads were prepared as described previously
(Duthie et al., 1999) and RNA FISH was then carried out as described
previously (Nesterova et al., 2008). For whole-mount RNA FISH, embryos
were obtained as for immunofluorescence. RNA FISH was then carried out
as described previously (Nesterova et al., 2008). Images were acquired on
a Leica TCS SP5 confocal microscope using LAS AF software.
RESULTS
Generation of a targeted inversion in Xist exon 1
Comparison of Xist cDNA sequences from mouse and human at
high stringency reveals conservation in only a limited number of
short regions (Fig. 1A). In addition to exon 4 and the A-repeats,
both of which have been functionally analysed in previous studies
(Caparros et al., 2002; Wutz et al., 2002; Chaumeil et al., 2006;
Hoki et al., 2009), high sequence conservation occurs over a region
of ~3 kb beginning 6 kb downstream of the transcriptional start site
Fig. 1. Strategy for generating a targeted inversion of Xist exon
1 conserved sequences. (A)Dotplot analysis of mouse and human
Xist/XIST cDNA. Full length mouse and human Xist/XIST cDNA
sequences were compared by dotplot analysis using the EMBOSS
dotmatcher program (Rice et al., 2000). Two sets of parameters with
different stringencies were used to demonstrate overall homology
between two sequences (window size 50, threshold 45) or to highlight
longer stretches with high homology (window size 150, threshold 95).
Exon structure for each transcript is shown above (mouse) or alongside
(human) the dotplots. Mouse intron 7, which contributes to a
proportion of splice variant transcripts, is shown as a white box. Exon 4,
the most conserved region, is shown in blue on the schematic and
encircled in blue on the dotplot. The region of inversion is indicated
with red bracketed arrows and encircled in red on the dotplot. Note the
extended homology over the whole region of the inversion. (B)A
schematic representation of the previously targeted Xist allele with a
deletion of exon 4 (Xistex4) (Caparros et al., 2002) and the pNBXT1
targeting construct. Conserved repetitive elements A-F within exons 1
and 7 are shown as shaded boxes. LoxP sequences (grey triangles) flank
a Pgk promoter-driven neomycin (neo) selection cassette (pale grey
rectangle) between 5⬘ and 3⬘ arms of homology (dark grey rectangles).
The positions of the arms of homology are indicated with black lines
below the schematic. A diptheria-toxin A negative selection cassette
(white rectangle) was included in the targeting construct. (C)The
resulting homologous recombinant allele (Xistneo) carrying both a
deletion of Xist exon 4 (replaced by a LoxP sequence) and a floxed neo
cassette in reverse orientation. (D)Transient expression of Crerecombinase generates a deletion of the neo cassette (Xistneo) and/or
inversion (XistINV) of the region between exon 1 and intron 4 (5947 bp
to 13,670 bp downstream of the transcriptional start site).
in the large exon 1 (highlighted in red in Fig. 1A, right panel). To
determine the importance of this region in native Xist RNA
transcripts we used homologous recombination in ES cells to
engineer an inducible inversion of the conserved exon 1 sequence
through to intron 5 (Fig. 1B-D). A loxP site was targeted 6 kb
upstream of the transcriptional site in XY ES cells and in the
DEVELOPMENT
RNA was isolated from embryos and SNuPE was carried out for Pgk-1
(Pgk1 – Mouse Genome Informatics) and Smc1l1 (Smc1a – Mouse
Genome Informatics) as described previously (Nesterova et al., 2003; Mak
et al., 2004). A novel polymorphism present in PGK strain mice was
identified in Gla (A to G at position 725 nt in exon 3, transcript
ENSMUST00000033621 in Ensembl Mouse database based on the NCBI
m37 mouse assembly). Nested RT-PCR was performed in order to generate
enough PCR product for SNuPE analysis. Primers for the first round of RTPCR were Gal18e (5⬘-CAGATCCCCAACGCTTTCCTA-3⬘, exon 2) and
Gal19e (5⬘-AGCGACTTCAACAATCTCCTTCTG-3⬘, exon 5) nested
with Gal20e (5⬘-TAGGGATTTATGCAGATGTTGG-3⬘, exon 3) and
Gal22e (5⬘-AATGCTTCGGCCTGTCCTGTT-3⬘, exon 4). The conditions
for the first round and nested PCR were: 94°C for 30 seconds, 59°C for 30
seconds, 72°C for 30 seconds for 17 and 32 cycles, respectively. The
SNuPE primer was SGal4 (5⬘-GATGGTTGTCACTGTGACAGTGT-3⬘)
and SNuPE conditions were: 94°C for 1 minute, 60°C for 1 minute, 72°C
for 4 minutes. To account for differences in specific activities of
radionucleotides and compensate for the incorporation of two G
nucleotides into the PGK allele versus one A into 129 allele, SNuPE bands
were normalised to those generated from genomic PCR of 129⫻PGK F1
females (Gal16i, 5⬘-CTTCCTGGTTGGTTTCCTATTG-3⬘, intron 2;
Gal17i, 5⬘-GGTTGGAAAGAGCGAACAGAT-3⬘, intron 3; 94°C for 30
seconds, 59°C for 30 seconds, 72°C for 30 seconds for 32 cycles). Bands
were quantified on a Typhoon FLA 7000 Biomolecular Imager (GE
Healthcare).
1544 RESEARCH ARTICLE
Development 138 (8)
opposite orientation to a loxP site present in the parent cell line
XistEx4del in which Xist exon 4 had previously been deleted
(Caparros et al., 2002). Correctly targeted cells carrying this allele,
designated Xistneo, were then transiently transfected with Cre
recombinase to generate the Xistneo allele in which the neo cassette
was excised and the floxed region of Xist exon 1 was in the correct
orientation.
Xistneo cells were injected into blastocysts and germ line
transmission was achieved from chimeric offspring. Xistneo
hemizygous males, heterozygous and homozygous females were
normal and fertile, indicating that insertion of a loxP site in exon 1
did not affect Xist gene function. This was confirmed by analysing
allelic ratios of Xist and Pgk-1 in female progeny from Xistneo ⫻
PGK strain crosses (data not shown). To induce inversion of Xist
exon 1 we used transient expression of Cre-recombinase in
fertilised oocytes of Xistneo homozygous animals. Resultant
progeny were analysed to identify males and female heterozygotes
in which sequences between the two loxP sites were stably
inverted, designated XistINV.
Fig. 2. Heterozygous female embryos with a maternally
transmitted XistINV allele exhibit secondary non-random X
inactivation. (A)Allele-specific RT-PCR analysis of Xist from XmINVXp
adult female kidneys (females 1-5). Top panel (wt) shows amplification
with wild-type Xist allele specific primers (e and l) and the bottom panel
presents amplification with XistINV specific primers (b and e). Numbers
above the top panel show the number of cycles used. Note the six to
nine cycle difference in amplification of wild-type versus XistINV
fragments between different females. (B)Light and GFP microscopy
images of E8.5 and E11.5 embryos with a maternally inherited XistINV
allele and a paternally inherited GFP transgene. The diminishing
proportion of green cells indicates secondary non-random X
inactivation. (C)Examples of FACS histograms showing relative
proportion of GFP-negative (M1) and GFP-positive (M2) cells in XmXpGFP
and XmINVXpGFP primary fibroblast cell lines established from E14.5
females. The x-axis represents the logarithmic level of GFP fluorescence
and the y-axis shows the number of cells. 50,000 total events were
acquired for each sample. (D)Quantitative analysis of FACS data
obtained for two primary control fibroblast cultures (XmXpGFP) and for
four primary fibroblast cultures from mutant heterozygous E14.5
embryos (XmINVXpGFP). (E)RT-PCR analysis of the Xist transcripts
produced by fibroblast cell lines expressing wild-type Xist, Xistex4 or
XistINV. Top panel, primers amplifying a sequence upstream of the
inversion in exon 1 (a and h); second panel, primers situated in exon 3
and exon 5, respectively (e and m); third panel, primers flanking the
inversion break-point that only amplify from the XistINV allele (b and e);
bottom panel, -actin loading control. + and – indicate reactions set up
with and without reverse transcriptase, respectively. Positions of all Xist
primers used are shown in Fig. S1 in the supplementary material.
specific for the wild-type allele amplified products of the predicted
size from both the GFP negative MEFs and the Xistex4 fibroblasts
and, as expected, did not amplify a product from the XistINV allele.
Primers located in exon 1, upstream of the inversion, detected
transcripts in all samples. A full RT-PCR analysis of XistINV
transcripts using primers across the locus is shown in Fig. S1 in the
supplementary material.
DEVELOPMENT
Females with a maternally inherited XistINV allele
exhibit secondary non-random X inactivation
To determine whether the XistINV mutation affects X inactivation
we first assayed expression of wild-type and XistINV alleles in
tissues of adult XistINV heterozygous females. As illustrated in Fig.
2A, expression of the XistINV allele was detected but at a
significantly reduced level relative to the wild-type allele. This
observation suggested extreme non-random X inactivation of the
X chromosome carrying the wild-type Xist allele.
Skewed XistINV expression could result from primary nonrandom X inactivation at the time X inactivation is initiated (~E5.56.5), or alternatively from secondary non-random X inactivation in
which cells expressing the XistINV allele are selected against
subsequent to the onset of random X inactivation. To address this,
we crossed heterozygous XistINV females with males carrying an Xlinked GFP transgene (XGFP) and a wild-type Xist allele. The GFP
transgene is located on the proximal part of the X chromosome and
has previously been shown to undergo silencing both in imprinted
X inactivation in extra-embryonic lineages and in random X
inactivation in the embryo (Hadjantonakis et al., 1998;
Hadjantonakis et al., 2001). Primary non-random X inactivation is
predicted to lead to absence of GFP in both extra-embryonic and
embryonic cells immediately following initiation of random X
inactivation (E5.5-6.5). Conversely, in secondary non-random X
inactivation loss of GFP expression is predicted to occur more
gradually in cells of the embryo proper, the rate of loss being
determined by the degree to which dosage compensation is
compromised.
XX embryos with a maternally inherited XistINV allele exhibited
progressive loss of GFP expressing cells between E6.5 and E14.5,
consistent with secondary non-random X inactivation. Figure 2B
shows representative embryos at E8.5 where significant GFP
expression is seen throughout the embryo and at E11.5 where GFP
expression is restricted to the heart.
FACS analysis of MEFs from E14.5 embryos identified a small
proportion (1-10%) of GFP positive cells (Fig. 2C,D). We carried
out RT-PCR on sorted populations using primer pairs to detect
expression of wild-type and XistINV RNAs. As a control we
analysed MEFs expressing the Xistex4 allele (Caparros et al.,
2002). Using primers flanking one of the break points in the XistINV
allele we confirmed that only the GFP positive MEFs express this
allele (Fig. 2E). Primers (located in exon 3 and exon 5) that are
Xist RNA localisation
RESEARCH ARTICLE 1545
Fig. 3. Embryo lethality in XX embryos
inheriting XistINV on the paternal X
chromosome. Embryos were dissected from
uteri at E6.5-10.5. Xm carries the wild-type
Xist allele and so male (XmY) embryos are wild
type. Xp carries the XistINV allele and so
females are heterozygous for the inversion.
The embryos shown are representatives from
three litters dissected at each gestational age.
Xm, maternally transmitted X chromosome;
Xp, paternally transmitted X chromosome.
Scale bars: 500m.
Embryonic lethality of a paternally inherited
XistINV allele
We observed that male animals carrying the XistINV allele are fertile
but fail to sire daughters, suggesting failure of imprinted X
inactivation in the extra-embryonic tissues where only the
paternally transmitted Xist allele is expressed. To investigate this
further, litters fathered by XistINV carrying males were dissected at
various gestational timepoints. The morphology and size of all
embryos in a litter was noted and then PCR was carried out to
determine the sex of each embryo. Examples are shown in Fig. 3.
XistINV female embryos were indistinguishable from their wild-type
male littermates at E6.5 but appeared growth retarded as early as
E7.5 (Fig. 3). At E8.5 XistINV female embryos appeared markedly
smaller. At E9.5 XistINV female embryos were much smaller and
were often developmentally delayed. For example, at E9.5 neural
tube closure is usually complete but some XistINV female embryos
had not completed caudal fusion at this stage. Furthermore, E9.5
XistINV female embryos showed no signs of developing a placenta
whereas placental development of male wild-type littermates
appeared normal. By E10.5 XistINV female embryos were
disintegrating.
Loss of female embryos with a paternally inherited XistINV allele
during the first half of gestation and in particular the failure to
develop a placenta is likely to be due to a failure of imprinted X
inactivation in the extra-embryonic tissues where only the
paternally inherited XistINV allele is expressed. However, the
embryonic phenotype observed was not as severe as that noted
previously in studies analysing a null allele, dXist (Marahrens et
al., 1997).
Silencing of X-linked genes is compromised in
cells expressing XistINV RNA
To assess expression of X-linked genes in cells expressing XistINV
RNA, we first crossed wild-type females with male mice carrying
both XGFP and the XistINV allele on their single X chromosome
and analysed GFP expression in tissues that undergo imprinted X
inactivation. As shown in Fig. 4A, E3.5 blastocyst stage embryos,
in which all cells have undergone imprinted X inactivation, have
high levels of GFP expression relative to wild-type controls. There
was, however, some variability between embryos and between
patches of cells within each individual embryo and overall GFP
expression levels appeared lower than in E3.5 embryos, in which
XGFP is located on the active maternally derived X chromosome
(Fig. 4A; arrowhead). We also analysed E6.5 embryos (Fig. 4B).
In wild-type controls GFP expression was undetectable in extraembryonic ectoderm, consistent with paternally imprinted X
inactivation, whereas in XistINV females we observed low-level
GFP expression throughout with some patches of stronger GFP
expression. In embryonic ectoderm there was strong GFP
expression both in wild-type and XistINV embryos, consistent with
random X inactivation. These results suggest that cells expressing
the XistINV allele fail to initiate or maintain silencing of the XGFP
transgene appropriately.
Next, we analysed the expression of endogenous X-linked genes.
E3.5 embryos were obtained from crosses between either wild-type
or XistINV males carrying the XGFP transgene and PGK strain
females. Polymorphisms arising between the PGK strain and the
targeted 129 strain allowed expression from the maternally derived
(PGK) and paternally derived (129) alleles to be distinguished by
single nucleotide primer extension (SNuPE). Female embryos were
identified by GFP expression as above and then pooled together for
analysis. We assessed expression of three X-linked genes, Pgk-1,
DEVELOPMENT
Northern blot analysis of GFP-positive MEFs revealed that the
XistINV allele transcribes two major RNA species, approximately 12
and 20 kb in length (see Fig. S2A in the supplementary material).
The 20 kb species corresponds to the maximum length transcript
predicted from the sequence of the genomic locus and RT-PCR
analysis (see Fig. S1 and Fig. S2B in the supplementary material).
The 12 kb species probably results from premature termination.
Consistent with this, there were potential polyadenylation signals
clustered ~12 kb downstream of the promoter within inverted exon
1 sequence (see Fig. S2B in the supplementary material). The
northern blot analysis also indicated that the level of XistINV RNA is
similar to that seen with the Xistex4 allele, both of which are lower
than Xist RNA levels in wild-type cells (see Fig. S2A in the
supplementary material). It should be noted that because the Xistex4
mutation does not affect X inactivation (Caparros et al., 2002), the
phenotype of the XistINV mutation is unlikely to be attributable to
reduced RNA levels. In summary, the XistINV allele produces a
contiguous transcript (including inverted sequences) up to Xist exon
5, subsequent to which processing is identical to wild-type Xist
RNA. A proportion of XistINV transcripts truncate prematurely at
polyadenylation signals 12Kb downstream of the promoter.
Taken together, these results demonstrate that random X
inactivation occurs in female embryos with a maternally inherited
XistINV allele and that subsequently there is a progressive selection
against cells that express the XistINV allele. A small proportion of
cells expressing XistINV RNA persist through ontogeny and in adult
animals.
1546 RESEARCH ARTICLE
Development 138 (8)
blastocyst have reversed imprinted X inactivation patterns and then
proliferated under selection, we consider this to be unlikely both
because failure to develop a placenta argues against significant
rescue and because reversion of imprinted X inactivation under
selective pressure was not observed in previous studies using null
Xist alleles (Marahrens et al., 1997). In wild-type embryos it
appeared, as previously reported, that Smc1l1 is only partially
inactivated at this stage (Mak et al., 2004). However, XistINV
carrying embryos still had significantly higher expression levels
from the paternally derived allele compared with wild-type
embryos (Student’s t-test, P<0.05).
Gla and Smc1l1, for which there are known polymorphisms (Fig.
4C,D). As expected, in wild-type embryos expression of Pgk-1 and
Gla was predominantly from the maternally derived allele. Only
very low levels of paternally derived transcripts were detected,
reflecting the inactive state of the paternally inherited X
chromosome. In XistINV carrying embryos, however, significantly
more expression was detected from the paternally derived allele
(Student’s t-test, P<0.005 for Pgk-1 and Gla) indicating that the
process of imprinted X inactivation is indeed impaired. Although
we cannot rule out that a proportion of cells in the XistINV
Localisation of XistINV RNA to the X chromosome
is impaired
Next, we analysed localisation of XistINV RNA to the Xi
chromosome by RNA FISH on whole-mount E6.5 female embryos
(Fig. 6). Again, these embryos had a paternally inherited XistINV
allele and so extra-embryonic tissues expressed only the mutant
allele. When compared with wild-type embryonic and extraembryonic and XistINV embryonic cells, the size and intensity of
Xist domains in the XistINV extra-embryonic cells were reduced.
The apparent reduction in XistINV RNA domains could underlie
the compromised silencing of X-linked genes, either because
overall transcript levels are insufficient for chromosome wide
silencing or because of compromised localisation of XistINV RNA
to Xi in cis. Consistent with the former possibility, XistINV
DEVELOPMENT
Fig. 4. Failure of X-linked gene silencing in female embryos
carrying a paternally inherited XistINV allele. (A,B)Light and GFP
microscopy images of E3.5 (A) and E6.5 (B) female embryos with a
paternally inherited XistINV allele and XGFP transgene. GFP expression in
blastocysts (E3.5) and extra-embryonic tissue at E6.5 indicates that
XistINV cannot establish or maintain appropriate levels of X-linked gene
silencing. The arrowhead marks a single blastocyst with a maternally
inherited GFP transgene to show the intensity of full activation of the
transgene. Scale bars: 100m in A, 500m in B. emb, embryonic part;
ex, extra-embryonic part; wt, wild type. (C)Allele-specific gene
expression analysis of three X-linked genes: Pgk-1, Gla and Smc1l1.
E3.5 embryos were obtained from crosses between either wild-type or
XistINV-carrying males and PGK females. Polymorphisms arising between
the PGK strain and the targeted 129 strain allowed expression from Xm
(PGK) and Xp (129) alleles to be distinguished by single nucleotide
primer extension (SNuPE). Female embryos were identified by GFP
expression and pooled together for analysis. At least six embryos were
included in each pool and at least four pools were analysed. Examples
show duplicate loading of RT-PCR reactions detecting the presence of
expression from Xp (129) and Xm (PGK) alleles. PGK ⫻ 129 F1 genomic
DNA was also included in the analysis as a control. (D)The percentage
of total expression coming from the Xp allele in wild-type and XistINVcarrying embryos. PGK ⫻ 129 F1 genomic PCR fragments were used to
normalise the data. Xp, paternally inherited X chromosome; Xm,
maternally inherited X chromosome. Error bars indicate s.d.
Reduced H3K27me3 and H2AK119u1 domains in
cells expressing XistINV RNA
To determine whether cells expressing XistINV RNA exhibit
chromosomal features of X inactivation we analysed H3K27me3
and H2AK119u1, histone modifications that are enriched on Xi
as a consequence of Xist-dependent recruitment of polycomb
repressor complexes PRC2 and PRC1, respectively (Silva et al.,
2003; Plath et al., 2003; de Napoles et al., 2004; Fang et al.,
2004). E6.5 XX embryos carrying either a wild-type or XistINV
allele on the paternal X chromosome were analysed by
immunofluorescence using antibodies specific for H3K27me3
and H2AK119u1 (Fig. 5).
Similar numbers and sizes of domains of colocalising
H3K27me3 and H2AK119u1 were observed in wild-type
embryonic and extra-embryonic cells. A significant increase in the
number of cells with no domains was observed in XistINV
embryonic cells (Student’s t-test, P<0.05). However, large bright
domains similar to those seen in the wild-type embryos were
observed. Significantly fewer domains were seen in XistINV extraembryonic tissues (Student’s t-test, P<0.05) and those domains that
were seen were smaller and of reduced intensity (Fig. 5). More
frequent bright domains seen in embryonic cells probably represent
cells that inactivated the wild-type X chromosome. As a control,
NB18 TS cells carrying the Xistex4 allele were analysed and the
domains were found to be of a comparable size and intensity to
those observed in the wild-type embryonic and extra-embryonic
cells (see Fig. S3 in the supplementary material), indicating that the
reduction in these modifications is attributable to the inversion of
Xist RNA sequences and not to the deletion of exon 4. The reduced
H2AK119u1 domains observed in extra-embryonic cells of
mutant embryos were associated with underlying histone H4
hypoacetylation in many, but not all, instances and domains were
again relatively small (see Fig. S4 in the supplementary material).
These observations are consistent with compromised gene silencing
by XistINV RNA.
Xist RNA localisation
RESEARCH ARTICLE 1547
illustrated in Fig. 7, XistINV RNA localised along the entire
chromosome and there was no detectable concentration of
transcripts within a smaller region of the chromosome. However,
overall levels were substantially lower than those seen for wildtype and Xistex4 controls. These results suggest that XistINV RNA
does associate with the X chromosome, but with reduced affinity,
and that this underlies defective silencing of X-linked genes and
female embryo lethality.
DISCUSSION
Here we describe analysis of a novel targeted mutation, XistINV,
designed to test the function of a conserved region located in exon
1 of Xist RNA during X inactivation. We show that XistINV is
regulated appropriately but is compromised in its ability to silence
X-linked genes in cis. Inheritance of XistINV on the paternal X
chromosome results in embryonic lethality due to failure of
imprinted X inactivation in extra-embryonic lineages. Female
embryos inheriting XistINV on the maternal X chromosome undergo
extreme secondary non-random X inactivation, eliminating the
majority of cells that express the XistINV allele. Analysis of cells
that express XistINV demonstrates that the mutant RNA shows
reduced association with the X chromosome, suggesting that
conserved sequences in the inverted region are important for Xist
RNA localisation.
transcript levels are lower than wild-type Xist RNA (see Fig. S2A
in the supplementary material). However, levels are equivalent to
the Xistex4 allele that was shown previously to function normally
(Caparros et al., 2002). RNA FISH analysis of interphase nuclei in
MEF cell lines, however, indicated that XistINV RNA domains are
reduced in size compared with both wild type and Xistex4,
suggesting that in cis spreading of XistINV RNA might occur over
only a limited region of the chromosome. To test this, we analysed
in cis localisation of XistINV RNA on metaphase chromosomes. As
Fig. 6. Reduced Xist domains in extra-embryonic tissue of
female embryos with a paternally inherited XistINV allele.
RNA FISH on female embryos collected at E6.5 reveals Xist RNA
(green) coating of the inactive X chromosome throughout the
whole of the wild-type female embryo (left panel) and in the
increased magnifications of the epiblast (epi) and ectoplacental
cone (epc) (middle right panels). Xist RNA coating can be seen
at levels comparable to wild type in the XistINV epiblast only
(middle left and bottom right panels). In the extra-embryonic
part of the embryo the domains appear smaller and weaker
(middle left and top right panels).
DEVELOPMENT
Fig. 5. Reduced H3K27me3 and H2AK119u1 on the Xi in female
embryos with a paternally inherited XistINV allele.
(A)Immunofluorescence images taken of cells from wild-type and
XistINV female embryonic and extra-embryonic parts at E6.5 stained for
H3K27me3 (red) and H2AK119u1 (green). DNA was counterstained
with DAPI (blue). (B)Scoring data showing the numbers of H3K27me3
and H2AK119u1 domains detected by immunofluorescence in cells
from wild-type and XistINV female embryonic and extra-embryonic parts
at E6.5. Error bars indicate s.d.
XistINV compromises X chromosome silencing
Paternally inherited XistINV was appropriately expressed in extraembryonic ectoderm of E6.5 embryos, indicating that the
mutation does not affect the regulation of Xist imprinting. There
was, nevertheless, a failure of imprinted X inactivation
demonstrated by inefficient silencing of an X-linked GFP
transgene both at the blastocyst stage (E3.5) and at E6.5.
Endogenous X-linked genes Pgk-1, Gla and Smc1l1 also showed
impaired silencing at the blastocyst stage. This impairment
contrasts with a recently published study in which female
embryos with a paternally inherited Xist knockout allele were
suggested to initiate X inactivation normally (Kalantry et al.,
2009). In these experiments, blastocysts with an identical
paternally inherited GFP transgene silenced the transgene to the
same degree in mutant and wild-type female embryos. It was only
later that the GFP was reactivated. Based on these results, it was
suggested that initiation of imprinted X inactivation is
independent of Xist. Here, GFP silencing was clearly not
observed at the blastocyst stage to the same extent as in wild-type
embryos with a paternally inherited XistINV allele, suggesting that
initiation of silencing is dependent on Xist. Further studies will
be required to resolve this apparent paradox.
1548 RESEARCH ARTICLE
Development 138 (8)
Fig. 7. XistINV RNA does not localise
efficiently to the X chromosome.
Representative examples illustrating
Xist RNA localisation (green)
determined by RNA FISH on (A)
interphase and (B) metaphase
chromosomes from fibroblast cell lines
expressing wild-type Xist, Xistex4 or
XistINV. DNA was counterstained with
DAPI (blue).
Impaired localisation of XistINV transcripts
RNA FISH on whole-mount embryos and metaphase chromosomes
revealed that XistINV RNA is not localising efficiently to the X
chromosome from which it is transcribed. This does not appear to
relate to the low abundance of XistINV RNA, as RT-PCR analysis
of the transcript showed equivalent levels compared with Xistex4
RNA, which localises and executes X inactivation normally.
Moreover, Xist RNA levels in some wild-derived mouse strains are
significantly lower than in laboratory mice (Brockdorff et al., 1991;
Buzin et al., 1994) but X inactivation appears to be unaffected.
There does not appear to be a problem with spreading of XistINV
transcripts as chromosome coating is detected all along the
chromosome. However, there are clearly lower levels of XistINV
RNA associated with the X chromosome at metaphase, suggesting
that the mutant transcript adheres with reduced affinity.
Previously, the entire XistINV region was deleted in an Xist cDNA
transgene and, upon ectopic expression in ES cells, silencing and
localisation appeared approximately the same as observed with the
full length transgene (Wutz et al., 2002). This suggests that the
sequence of the inverted region is not directly involved in
localisation. It is possible that expression of the transgene was
greater than endogenous Xist and that this partially overrides
reduced localisation efficiency. Alternatively, inclusion of
additional reversed exons and introns between the LoxP sites in the
XistINV transcript might have changed the structure of the molecule
in such a way as to inhibit interactions required for localisation.
Wutz et al. (Wutz et al., 2002) previously reported that redundant
sequences throughout the 3⬘ end of the molecule mediate
localisation, including a region in exon 1 immediately adjacent to
the inverted region described here. It is possible that our targeted
inversion impairs the interactions that this adjacent region is
involved in.
Levels of enrichment of the repressive histone modifications
H2AK119u1 and H3K27me3 catalysed by recruitment of PRC1
and PRC2 polycomb complexes to Xi are strongly reduced in cells
of E6.5 embryos expressing XistINV RNA. This is consistent with
previous data analysing blastocysts expressing XistINV RNA from
the paternal X chromosome (Silva et al., 2003). In both cases,
reduced polycomb complex recruitment is likely to be a direct
reflection of reduced Xist RNA domains. However, we cannot rule
out that XistINV RNA recruits PRC1 and PRC2 less efficiently.
Previous work has demonstrated that sequences througout Xist
RNA have a role in recruiting PRC1 and PRC2 and, moreover, that
the complexes are recruited by a silencing deficient Xist RNA
transgene in which the A-repeats have been deleted (Kohlmaier et
al., 2004; Chaumeil et al., 2006).
XistINV is a hypomorphic allele
Female mice carrying a paternally inherited XistINV allele are lost
during embryogenesis. However, the phenotype of these mice is
less severe than female embryos carrying a paternally inherited Xist
knockout allele. Female embryos with a paternally inherited XistINV
allele are indistinguishable from male littermates at E6.5 and
appear smaller at E8.5 but have no developmental phenotype at that
stage. By contrast, female embryos with a paternally inherited Xist
null allele are reduced in size as early as E6.5 and are severely
dysmorphic by E8.5 (Marahrens et al., 1997). This disparity raises
the possibility that XistINV is a hypomorphic allele. XistINV is clearly
not capable of efficiently silencing the X chromosome. However,
in blastocysts with a paternally inherited XistINV on the same
DEVELOPMENT
The phenotype of XistINV is not limited to imprinted X
inactivation. During random X inactivation female embryos
exhibited progressive loss of cells in which XistINV RNA was
expressed. Thus, like in imprinted X inactivation, appropriate
regulation of Xist expression is unaffected by the presence of the
XistINV allele but downstream silencing by XistINV RNA is
compromised. This contrasts with previously studied Xist
mutants that in most cases trigger primary non-random X
inactivation of the non-mutant allele or, in the case of deletion
of Xist exon 4, show no discernible phenotype. XistINV is the first
Xist mutant allele, to our knowledge, shown to cause secondary
non-random X inactivation. It is interesting to note that a small
number of XistINV RNA-expressing cells are viable both in MEF
cultures and in adult tissues. These could represent cells in which
failure of dosage compensation has been tolerated and/or cells in
which silencing by XistINV RNA is more robust than is generally
the case.
chromosome as a GFP transgene there was evidence for some
degree of silencing of the GFP transgene. In some places the GFP
expression was in the same range as that of the maternally inherited
GFP but in others it was almost as low as in the wild-type embryos.
Although endogenous genes also showed increased expression
from the paternally inherited X chromosome, expression levels did
not reach the levels detected from the maternally inherited X
chromosome. This observation indicates partial silencing of the
paternally inherited X chromosome and again supports the idea that
XistINV is a hypomorphic allele. In further support of this is the
observation that enrichment of the XistINV extra-embryonic tissues
at E6.5 with H3K27me3 and H2AK119u1 was substantially
reduced but not completely abolished. Underlying H4
hypoacetylation was found coincident with H2AK119u1 foci at
interphase, but this was only in a proportion of cells and did not
extend beyond the relatively small H2AK119u1 foci observed in
XistINV extra-embryonic tissues. This is suggestive of reduced
efficiency of silencing in response to XistINV RNA.
A small proportion of genes might be silenced in each cell and
this silencing maintained, for example, through the recruitment
of polycomb complexes. Partial silencing would not be sufficient
for normal embryonic development but it could perhaps carry the
embryos slightly further along embryonic development than a
full Xist knockout. The low level of XistINV RNA localisation
observed could constitute a smaller repressive compartment into
which some genes could be recruited. Given the variability of the
GFP expression levels throughout each embryo, different genes
might be silenced in different cells in a stochastic manner.
Analysis of endogenous genes in pooled blastocysts, however,
suggests that individual loci are subject to different degrees of
silencing and that this might relate to distance from the Xist
locus. Future studies, for example using global analysis of gene
expression in XistINV mutants, might discriminate weak silencing
of all or most genes from relatively efficient silencing of only a
subset of genes.
In summary, this study suggests that conserved sequences
located in the distal half of Xist exon 1 are important for in cis
localisation of Xist RNA. A recent study has demonstrated that
scaffold attachment factor A (SAF-A; HNRNPU – Human Gene
Nomenclature Database) mediates Xist RNA localisation,
interacting with Xist RNA through an RRM RNA binding domain,
and immunoprecipitation of UV cross-linked SAF-A preferentially
enriched sequences at the distal end of Xist exon 1 (Hasegawa et
al., 2010). Conserved exon 1 sequences included in the XistINV
region therefore provide good candidates for further analysis to
determine the precise RNA sequence/structure required for SAFA
binding which in turn might provide significant insight into the
mechanism of in cis localisation of Xist RNA.
Acknowledgements
We thank members of the lab for helpful discussion and Vasso Episkopou for
assistance with embryo studies. This work was funded by the Medical Research
Council, UK, and the Wellcome Trust. Deposited in PMC for release after 6
months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.056812/-/DC1
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