Lineage-specific regulation of imprinted X inactivation in extraembryonic endoderm stem cells
© Merzouk et al.; licensee BioMed Central Ltd. 2014
Received: 14 April 2014
Accepted: 2 June 2014
Published: 20 June 2014
Silencing of the paternal X chromosome (Xp), a phenomenon known as imprinted X-chromosome inactivation (I-XCI), characterises, amongst mouse extraembryonic lineages, the primitive endoderm and the extraembryonic endoderm (XEN) stem cells derived from it.
Using a combination of chromatin immunoprecipitation characterisation of histone modifications and single-cell expression studies, we show that whilst the Xp in XEN cells, like the inactive X chromosome in other cell types, globally accumulates the repressive histone mark H3K27me3, a large number of Xp genes locally lack H3K27me3 and escape from I-XCI. In most cases this escape is specific to the XEN cell lineage. Importantly, the degree of escape and the genes concerned remain unchanged upon XEN conversion into visceral endoderm, suggesting stringent control of I-XCI in XEN derivatives. Surprisingly, chemical inhibition of EZH2, a member of the Polycomb repressive complex 2 (PRC2), and subsequent loss of H3K27me3 on the Xp, do not drastically perturb the pattern of silencing of Xp genes in XEN cells.
The observations that we report here suggest that the maintenance of gene expression profiles of the inactive Xp in XEN cells involves a tissue-specific mechanism that acts partly independently of PRC2 catalytic activity.
KeywordsEpigenetic Single-cell analyses Stem cells X-inactivation
During embryonic preimplantation development of female mice, starting at the four-cell stage, the paternal X chromosome (Xp) undergoes global epigenetic silencing associated with the establishment of imprinted X chromosome inactivation (I-XCI) [1–5]. In the inner cell mass (ICM) of the implanting blastocyst, the Xp is then reactivated and a random XCI of the Xp or the maternal X chromosome (Xm) occurs de novo during the formation of the epiblast that will subsequently give rise to the adult tissues [1, 6]. In contrast, the extraembryonic lineages of the trophectoderm (TE) and the primitive endoderm (PrE) exhibit I-XCI of the Xp, which is maintained afterward in the derived lineages of the placenta and the yolk sac, respectively [7, 8].
Many studies have concentrated on the characterisation of random XCI using the ex vivo model of ICM-derived female embryonic stem (ES) cells, the differentiation of which is accompanied by the onset of X inactivation. In these cells, XCI initiates through the cis-accumulation of the Xist noncoding RNA (ncRNA) on the future inactive X (Xi), followed by recruitment of Polycomb repressive complexes PRC2 and PRC1 and other epigenetic modifications, which, together, result in the progressive establishment of an inactive state characterised by its extreme stability (for review, see [9–11] and references therein).
In contrast, I-XCI in extraembryonic tissues has been less extensively analysed. Studies of developing embryos or trophoblast stem (TS) cells derived from the TE  have revealed that, similarly to the randomly inactivated X, the inactive Xp in the TE lineage is associated with Xist ncRNA coating, depletion of active epigenetic marks and enrichment for the repressive H4K20me1 mark, the PRC2-dependent H3K27me3 mark and hypermethylation of CpG islands [3, 13–18]. Despite these cumulative regulatory locks ensuring the maintenance of Xp silencing, the inactive state in the TE seems to be less stable than that of adult somatic tissues because transient reactivation of some Xp-linked genes occurs spontaneously in both TS and TE cells . As a corollary, a large number of X-linked genes are expressed from both X chromosomes in female TS cells . Intriguingly, the magnitude and extent of this escape from I-XCI increase during TE differentiation into trophoblast giant cells, as revealed by an accrued frequency of reactivation of different Xp-linked transgenes and by reactivation of endogenous Xp loci [3, 16, 19–22]. This relaxed silencing is further exacerbated upon depletion of the PRC2 member EED, indicating that PRC2, possibly via its H3K27me3 catalytic activity, plays a role in the maintenance of I-XCI in the TE lineage [23, 24]. Collectively, these results suggest that I-XCI is more plastic than random XCI and indicate the interest in an in-depth analysis of the stability of I-XCI in the PrE and its derivatives.
The PrE originates from the ICM and gives rise, after differentiation, to the visceral endoderm (VE) and parietal endoderm (PE) that line the yolk sac, two tissues which maintain an inactive Xp . Extraembryonic endoderm (XEN) cells have been derived from the PrE and show many of the properties of PrE stem cells, including the ability to self-renew indefinitely ex vivo and to contribute in a lineage-appropriate manner in vivo, although undifferentiated XEN cells contribute predominantly to the PE and much less efficiently to the VE [25–27]. Whilst the inactive Xp in XEN cells is coated by the Xist ncRNA, it has been reported not to accumulate the PRC2 complex and associated H3K27me3 . EED-mutant embryos, however, show increased and frequent reactivation of an Xp-linked green fluorescent protein (GFP) transgene in cells of both the VE and the PE . X-linked GFP reexpression is also observed upon loss of Xist coating in the PE, suggesting that both Xist ncRNA and PRC2 activity are involved in the maintenance of Xp silencing in differentiated PrE cells .
In order to compare the characteristics of I-XCI in the PrE to the X-inactivation process occurring in other lineages, we combined two different approaches: (1) profiling of active and repressive chromatin features along the X chromosomes using both chromatin immunoprecipitation followed by chip hybridisation (ChIP-chip) and high-resolution immunofluorescence followed by fluorescent in situ hybridisation (immuno-FISH) studies and (2) an analysis of X-linked transcriptional activities at the level of single XEN cells by FISH on RNA (RNA-FISH) and reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR). We observed that the Xp in XEN cells, in contrast to findings previously reported by other researchers, was globally enriched in H3K27me3 compared to the Xm. Intriguingly, we observed that the topological distribution of H3K27me3 on the inactive Xp territory did not coincide strictly with that of Xist ncRNA, suggesting that some Xp loci may harbour only low levels of H3K27me3. Indeed, similarly to what is observed in TS cells, a large number of X-linked genes (approximately 15%) lack H3K27me3 on both X chromosomes in XEN cells. These H3K27me3-depleted genes tend to be expressed from the inactive Xp in a significant number of cells, indicating that they escape from I-XCI. The frequency of escape, which appears to be extremely variable from gene to gene, was not significantly modified after depletion of H3K27 trimethylation using an EZH2-specific inhibitor. In addition, I-XCI profiles appear to be tightly maintained upon differentiation of XEN cells into VE cells. Our observations indicate that whilst widespread escape from I-XCI emerges as a common characteristic of extraembryonic stem cells, this escape from I-XCI is associated with a different subset of genes in each extraembryonic lineage.
Inactive X chromosome territory is enriched in H3K27me3 in XEN cells
In this study, we analysed one male cell line (GHP7/7) and two independently derived female XEN cell lines (GHP7/9 and GHP7/3) . All three cell lines harboured morphological features typical of XEN cells and expressed PrE-specific lineage markers (Additional file 1). The female cell lines carry an Xp of 129Sv.Pgk1a origin and an Xm of 129Sv origin, allowing the identification of allele origin through the extensive polymorphisms located in the large Pgk1a-derived region surrounding the Xist gene.
This observation prompted us to reassess the distribution of H3K27me3 in XEN female cells using three-dimensional immuno-RNA-FISH for Xist and H3K27me3, followed by a deconvolution step (Figure 1B). Xist coating of the Xi was associated with a coenrichment in H3K27me3 in the majority of XEN cells, similarly to what we observed in TS cells (Figure 1C and Additional file 3). Although the volumes of H3K27me3 enrichment and of Xist domains on the Xi were not significantly different (Figure 1D), H3K27me3 and Xist nuclear foci did not show strict superimposability in the majority of XEN nuclei (Figure 1B), indicating that certain regions of the Xi, coated by Xist, may lack H3K27me3.
These results suggest that XEN and TS cells have distinct epigenetic identities. Notably, XEN cells highly express the Ezh2 gene. Consistent with this, the Xi in XEN cells is globally enriched in H3K27me3.
A large number of genes on paternal inactive X lack H3K27me3 in XEN cells
In order to confirm these results and identify the genomic regions of the inactive Xp showing lower levels of H3K27me3, we performed ChIP-chip analysis for this histone mark on male and female XEN cells using a high-resolution microarray covering the X chromosome and a subpart of chromosome 17, which served as a ChIP-chip efficiency control (see the Methods section for data treatment and quality controls). Comparing male and female profiles allowed us to detect differences marking specifically the Xi.
We then assessed whether this lack of H3K27me3 on specific Xi genes in female XEN cells was associated with a gain of active histone mark H3K4me2 using a similar ChIP-chip approach. Lowered H3K4me2 levels in female XEN cells than in male XEN cells were observed at the majority of expressed genes, consistent with an absence of active histone marks on Xi alleles (Additional file 5A). In contrast, H3K27me3-low Xi genes appeared significantly enriched in H3K4me2 compared to H3K27me3-high Xi genes (P = 0.037 by Kolmogorov–Smirnov test) (Additional file 5B and C). We noted, however, that this effect was not observed at all H3K27me3-low genes and that some H3K27me3-high genes may also show high levels of H3K4me2 in female cells, indicating that the anticorrelation between H3K4me2 and H3K27me3 is not strict.
On the basis of these ChIP-chip analyses, we conclude that both the active and repressive histone marks H3K4me2 and H3K27me3 are distributed in a lineage-specific manner on Xi genes. Many of the genes lacking H3K27me3 on the Xi of XEN cells specifically showed a gain of H3K4me2 compared to Xi genes enriched in H3K27me3, indicating that some of these H3K27me3-low genes adopted the characteristics of active chromatin.
Genes lacking H3K27me3 in female XEN cells escape from imprinted XCI to various degrees
In agreement with our chromatin profile analysis, most H3K27me3-high Xi genes in each extraembryonic cell type were transcribed predominantly from the Xa (Xist-lacking X) and not from the Xi (Xist-coated X), indicating that these genes are subject to I-XCI. H3K27me3-low genes, on the other hand, appeared to be transcribed from the Xi in a significant percentage of nuclei in both cell types, indicating that they escaped from I-XCI (Figure 4). Two exceptions were observed. Hdac8, which we expected to escape from I-XCI in XEN cells based on its H3K27me3 profile, showed no significant expression from the Xi in these cells (Figure 4B). This may be related to its globally highly reduced expression level (low steady-state level of mature transcripts ), which may introduce some stochasticity into transcription initiation. Conversely, the Kdm6a gene is transcribed from the Xi in a significant percentage of TS nuclei, despite showing significant levels (about 47%) of H3K27me3 on the Xi in this cell line (Figure 4D). However, the proportion of nuclei showing this behaviour was significantly smaller than that seen in female XEN cells in which Kdm6a was poorly enriched (about 10%) in H3K27me3. Kdm6a is known to robustly escape from XCI in somatic tissues .
As previously described for TS cells  and other adult cell types , we observed that maternal allelic expression prevailed over that of the paternal allele in XEN cells for all the escapers examined, suggesting that, for most XCI escapers, the escape mechanism was not fully efficient in relieving the inactivation. Indeed, the degree of escape, as estimated both by the number of nuclei exhibiting transcriptional activity from the Xi detectable by RNA-FISH and by the number of cells showing significant paternal expression detectable by single-cell RT-qPCR, appeared to be extremely variable, not only between cell types (for example, Ftx showed different levels of escape in TS cells compared to XEN cells) but also from gene to gene. This supports the idea that expression from the Xi at loci escaping from I-XCI is a locally regulated phenomenon.
Level and extent of escape from imprinted XCI are not drastically modified upon inhibition of H3K27me3 in XEN cells
To assess the effect of GSK126-induced depletion of H3K27me3 on the expression of genes on the Xi, we analysed the expression of Xist, three genes subjected to I-XCI (Atp7a, Chic1 and Hmgn5) and three genes escaping from I-XCI (Ftx, Pbdc1 and Jpx) using allelic RT-qPCR on pools of XEN cells treated for 5 days with the drug. The expression levels of some of the X-linked genes, including Xist, were significantly increased after GSK126 treatment. Importantly, this increase affected exclusively alleles that were already active prior to any treatment—that is, the paternal Xist allele and maternal alleles of some other X-linked genes—suggesting that the inhibitor treatment leads to further derepression of active promoters (Figure 5B). In contrast, no significant change in expression levels of Xp alleles (except from Xist, as mentioned previously) was observed in GSK126-treated compared to untreated XEN cells, indicating that the marked reduction of H3K27me3 on the Xi of XEN cells did not significantly modify the efficiency of Xi gene silencing (Figure 5B). Similar results were obtained with two other H3K27me3-low genes (Kdm6a and Nkap) at the single-cell level using RNA-FISH following H3K27me3 immunostaining (Additional file 7D and E). Note that neither of the two analyses completely excludes the possibility that highly localised H3K27me3 may have remained associated with gene promoters, after GSK126 treatment, in some cells.
This analysis shows clearly that, although H3K27me3 enrichment allows the selective identification/segregation of genes subjected to I-XCI compared to genes escaping from I-XCI, this histone mark is probably not, by itself, determinant for X-linked gene expression control. This implies that other epigenetic marks or transcriptional mechanisms might be implicated in the regulation of Xi expression in XEN cells.
Escape from imprinted X-inactivation is not modified upon XEN cell conversion into visceral endoderm
Until now, it has been unclear whether differences in the silencing of genes on the Xi in the extraembryonic TE lineage, compared to tissues such as liver or embryonic kidney , are linked to the implementation of different mechanisms of X inactivation (that is, imprinted versus random) or whether they are associated with tissue specificity. In this study, we identified a large number of genes that escape from I-XCI solely in female XEN cells and not in female TS cells. Unlike the relaxation of I-XCI observed upon TS/TE cell differentiation (Additional file 8 and ), we observed that gene expression profiles from the inactive Xp in XEN cells appeared to be tightly maintained upon XEN conversion into VE cells in vitro. Hadjantonakis et al. made the same observation for yolk sac formation in vivo. These observations strongly support the existence of distinct regulation of Xp silencing in each extraembryonic lineage of the blastocyst. According to our single-cell RT-qPCR analysis, the expression of I-XCI escapers does not correlate or anticorrelate significantly with the expression of any of the lineage biomarkers we tested. This is true in both XEN and TS cells, suggesting either that other, as yet unidentified regulators must be involved and/or that tissue specificity is established after higher-level regulation of these factors (Additional file 2B). For instance, such tissue specificity could be mediated by differences in the topological distribution of some of these factors along the inactive Xp in the PrE compared to the TE. Specific clustering of I-XCI escapers along the X chromosome was not observed in XEN cells, which is suggestive of gene-centred regulation (Additional file 2C). Gene-specific, cis-acting regions, either promoting escape from XCI or preventing the spreading of active chromatin into adjacent silent domain, have been identified in cells subject to random XCI, suggesting that similar cis-acting sequences may be recruited in extraembryonic tissues [36–38]. Adjacent ncRNAs and/or CTCF binding (CCCTC-binding factor (zinc finger protein)) may participate in insulating XCI escapers from globally inactive chromatin [39–44].
Another important observation concerns the variation in the degree of escape from gene to gene and, for certain genes, between XEN cell lines. Such variability has been reported, albeit to a lesser extent, in other tissues and organisms [31, 45–48]. This characteristic may be symptomatic of an accrued lability in the silencing of discrete genomic locations and/or a manifestation of dynamic transitions between on/off states at paternal alleles, as observed previously in TS cells . Ontology analyses of XEN-specific escapers did not reveal any striking overrepresentation of particular classes of biological function compared to that of genes stably subjected to I-XCI. Neither were we able to identify genes known to be involved specifically in yolk sac metabolism or development amongst the XEN-specific escapers. This could support the hypothesis that genes that are not crucial to XEN cell homeostasis are able to tolerate low levels of biallelic expression, whereas genes critical for XEN function might require tight control of their expression level. More detailed gene-by-gene functional studies are needed to definitively address this point and the role of such large-scale escape in PrE lineage development.
Our results show that the repressive histone mark H3K27me3 accumulates on the Xi in XEN cells in a manner similar to that observed in other cellular contexts, notably in its preferential targeting to the body of inactivated genes and in its exclusion from XCI escapers [17, 29, 31, 49, 50]. Strikingly, catalytic inactivation of EZH2 does not significantly affect the silencing of Xp genes in XEN cells, suggesting that H3K27me3 is not—at least not on its own—responsible for the maintenance of Xp gene silencing in XEN cells. Rather, the low levels of H3K27me3 at I-XCI escapers may be a by-product of the initial definition of loci escaping from XCI that is not critical in itself, but could reinforce the boundaries between active and silent chromatin along the inactive Xp. Because the disruption of PRC2 through the mutation of EED is associated with instability of I-XCI in extraembryonic derivatives [23, 24], this result also implies that a function of the complex distinct from its H3K27me3 activity could be responsible for the maintenance of Xp-linked silencing. Other PRC2 members or cofactors, such as JARID2, which has recently been shown to bridge PRC2 to Xist RNAs, may be involved [51, 52]. PRC1-mediated repressive marks, DNA methylation or macroH2A1 are other obvious candidates for serving this function [11, 53]. Alternatively, active histone marks such as H3K4me2 or associated histone acetylation may positively target XEN escapers.
Taken together, our results show that I-XCI in extraembryonic endoderm stem cells does not possess characteristics identical to X inactivation in other extraembryonic lineages or adult somatic tissues. Unlike the situation in TE cells, I-XCI in XEN cells is stably maintained in fully differentiated cells. We identified a large number of XEN-specific X inactivation escapers, which indicates that a mechanism dedicated to the definition of loci resistant to the global Xp silencing is at play in XEN cells. Importantly, this mechanism does not, at least not completely, rely on a XEN-specific distribution of H3K27me3. The next goal will be to unravel the nature of this mechanism and how it is recruited during initial cell commitment to the extraembryonic endoderm lineage.
Cell culture, drug treatment and ex vivo differentiation
Male GHP7/7 and female GHP7/9 and GHP7/3 XEN cell lines used in this study and have been described previously . GHP7/9 and GHP7/3 cell lines carry an Xp from a 129.Pgk1a origin and an Xm from a 129/Sv origin, providing polymorphisms allowing for allelic distinction. XEN cells were grown on gelatin-coated culture dishes and routinely cultured at 37°C in 8% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Carlsbad, CA, USA) supplemented with 15% foetal bovine serum (Life Technologies) and 100 mM β-mercaptoethanol . XEN cell conversion into visceral endoderm was induced upon addition of 25 ng/ml BMP4 (R&D Systems, Minneapolis, MN, USA) in serum containing DMEM . XEN cell treatment with GSK126 was carried out as follows: three concentrations of inhibitor were tested during a 5-day treatment period (0.5 μM, 2 μM and 3 μM GSK126). Male F2 and female F3 TS cell lines and culture conditions have been described previously [12, 18].
ChIP-chip procedure and data analysis
ChIP assays were performed as previously described  using an H3K27me3 antibody (07–449) and an H3K4me2 antibody (07–030; both from EMD Millipore, Billerica, MA, USA) and analysed as described previously  (see also Additional file 9 for the detailed protocol). ChIP-chip data have been deposited under Gene Expression Omnibus accession number [GSE:50587].
Raw data were analysed using the Tobias Straub protocol (http://www.epigenesys.eu/images/stories/protocols/pdf/20111025114444_p43.pdf) with the Bioconductor R interface. This protocol includes quality assessment, data normalization and transformation. Raw H3K27me3 data in male and female livers were taken from the Gene Expression Omnibus database [GSE:20617]  and subjected to the same statistical treatment as the TS and XEN data sets. The gene expression data of TS and XEN cells were extracted from [GSE:15519] , and gene expression data of liver cells were extracted from [GSE:13583] .
RNA-FISH and Immuno-RNA-FISH
RNA-FISH and immuno-RNA-FISH procedures were carried out as described at http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-406-3_18?verPrint=print (H3K27me3 antibody, 07–449; EMD Millipore). For details of our experimental procedures, see Additional file 9. For probe localisation, see Additional file 2D. Z-stacks were captured (step = 0.2 μm) on a Zeiss Axioplan 2 microscope (Carl Zeiss Microscopy, Thornwood, NY, USA) equipped with a Hamamatsu ORCA ER charge-coupled device camera (Hamamatsu City, Japan) and controlled using Volocity Acquisition software (PerkinElmer, Waltham, MA, USA). Z-stacks deconvolution consisted in 50 iterations per each channel using Huygens Professional software (Scientific Volume Imaging, Hilversum, the Netherlands). Volume measurements were performed using Volocity Professional software, and intensity plot profiles were obtained on an image analysis platform in ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Single-cell gene expression analysis
Single-cell gene expression analysis was performed as described previously [18, 57, 58] and as recommended by Fluidigm (http://www.fluidigm.com/single-cell-expression.html; South San Francisco, CA, USA). Briefly, TS and XEN cells were sorted by fluorescence-activated cell sorting using the MoFlo system (Beckman Coulter, Brea, CA, USA), and individual cells were distributed into wells or 96-well plates containing 5 μl of CellsDirect resuspension buffer (Invitrogen, Carlsbad, CA, USA). The preamplification step consisted of 20 cycles using a mix of universal primer pairs to preamplify each gene simultaneously. Preamplification was followed by exonuclease I treatment (New England Biolabs, Ipswich, MA, USA), and allelic qPCR was performed on a BioMark thermal cycler (Fluidigm). Raw efficiencies of each PCR assay and allelic specificity were measured on control DNA within each experiment. Transcript levels were extrapolated using the raw PCR efficiencies, thus allowing the direct comparison of different genes. Controls for allelic specificities of PCR assays are available upon request. See Additional file 2A for primer sequences.
Capture hybridisation analysis of RNA targets with deep sequencing
Chromatin immunoprecipitation followed by chip hybridisation
Fluorescence recovery after photobleaching
Inner cell mass
Imprinted X chromosome inactivation
RNA antisense purification
Fluorescence in situ hybridisation on RNA
Reverse transcription followed by quantitative polymerase chain reaction
- TS cell:
Trophoblast stem cell
- XEN cell:
Extraendoderm stem cell
We thank Michel Cohen-Tannoudji, Jérôme Artus and Edith Heard for critical reading of the manuscript and Jérôme Artus for advice on XEN cell culture and differentiation. This work was supported by a Marie Curie ERG grant to CM (224945), by recurrent funding from the Institut Pasteur and the French National Centre for Scientific Research (CNRS) and by grants from the French National Agency for Research (ANR), the Epigenome Network of Excellence, REVIVE Labex and the Louis D Foundation of the Institut de France (Louis D Prize awarded to PA). JLD was supported by a postdoctoral fellowship from the Association for Research against Cancer (ARC). SM was supported by a doctoral fellowship from the French Ministry for Scientific Research, and by grants from the ARC and from the REVIVE Labex. PN and AD are supported on a permanent basis by the Pasteur Institute. CM is supported on a permanent basis by the French National Institute for Scientific and Medical Research (INSERM).
- Okamoto I, Otte AP, Allis CD, Reinberg D, Heard E: Epigenetic dynamics of imprinted X inactivation during early mouse development. Science. 2004, 303: 644-649.View ArticlePubMedGoogle Scholar
- Okamoto I, Arnaud D, Le Baccon P, Otte AP, Disteche CM, Avner P, Heard E: Evidence for de novo imprinted X-chromosome inactivation independent of meiotic inactivation in mice. Nature. 2005, 438: 369-373.View ArticlePubMedGoogle Scholar
- Patrat C, Okamoto I, Diabangouaya P, Vialon V, Le Baccon P, Chow J, Heard E: Dynamic changes in paternal X-chromosome activity during imprinted X-chromosome inactivation in mice. Proc Natl Acad Sci U S A. 2009, 106: 5198-5203.PubMed CentralView ArticlePubMedGoogle Scholar
- Kay GF, Barton SC, Surani MA, Rastan S: Imprinting and X chromosome counting mechanisms determine Xist expression in early mouse development. Cell. 1994, 77: 639-650.View ArticlePubMedGoogle Scholar
- Nesterova TB, Barton SC, Surani MA, Brockdorff N: Loss of Xist imprinting in diploid parthenogenetic preimplantation embryos. Dev Biol. 2001, 235: 343-350.View ArticlePubMedGoogle Scholar
- Mak W, Nesterova TB, de Napoles M, Appanah R, Yamanaka S, Otte AP, Brockdorff N: Reactivation of the paternal X chromosome in early mouse embryos. Science. 2004, 303: 666-669.View ArticlePubMedGoogle Scholar
- Takagi N, Sasaki M: Preferential inactivation of the paternally derived X chromosome in the extraembryonic membranes of the mouse. Nature. 1975, 256: 640-642.View ArticlePubMedGoogle Scholar
- West JD, Frels WI, Chapman VM, Papaioannou VE: Preferential expression of the maternally derived X chromosome in the mouse yolk sac. Cell. 1977, 12: 873-882.View ArticlePubMedGoogle Scholar
- Escamilla-Del-Arenal M, da Rocha ST, Heard E: Evolutionary diversity and developmental regulation of X-chromosome inactivation. Hum Genet. 2011, 130: 307-327.PubMed CentralView ArticlePubMedGoogle Scholar
- Wutz A: Gene silencing in X-chromosome inactivation: advances in understanding facultative heterochromatin formation. Nat Rev Genet. 2011, 12: 542-553.View ArticlePubMedGoogle Scholar
- Brockdorff N: Noncoding RNA and Polycomb recruitment. RNA. 2013, 19: 429-442.PubMed CentralView ArticlePubMedGoogle Scholar
- Tanaka S, Kunath T, Hadjantonakis AK, Nagy A, Rossant J: Promotion of trophoblast stem cell proliferation by FGF4. Science. 1998, 282: 2072-2075.View ArticlePubMedGoogle Scholar
- Calabrese JM, Sun W, Song L, Mugford JW, Williams L, Yee D, Starmer J, Mieczkowski P, Crawford GE, Magnuson T: Site-specific silencing of regulatory elements as a mechanism of X inactivation. Cell. 2012, 151: 951-963.PubMed CentralView ArticlePubMedGoogle Scholar
- Plath K, Fang J, Mlynarczyk-Evans SK, Cao R, Worringer KA, Wang H, de la Cruz CC, Otte AP, Panning B, Zhang Y: Role of histone H3 lysine 27 methylation in X inactivation. Science. 2003, 300: 131-135.View ArticlePubMedGoogle Scholar
- Mak W, Baxter J, Silva J, Newall AE, Otte AP, Brockdorff N: Mitotically stable association of Polycomb group proteins Eed and Enx1 with the inactive X chromosome in trophoblast stem cells. Curr Biol. 2002, 12: 1016-1020.View ArticlePubMedGoogle Scholar
- Corbel C, Diabangouaya P, Gendrel AV, Chow JC, Heard E: Unusual chromatin status and organization of the inactive X chromosome in murine trophoblast giant cells. Development. 2013, 140: 861-872.View ArticlePubMedGoogle Scholar
- Senner CE, Krueger F, Oxley D, Andrews S, Hemberger M: DNA methylation profiles define stem cell identity and reveal a tight embryonic–extraembryonic lineage boundary. Stem Cells. 2012, 30: 2732-2745.View ArticlePubMedGoogle Scholar
- Dubois A, Deuve JL, Navarro P, Merzouk S, Pichard S, Commere PH, Louise A, Arnaud D, Avner P, Morey C: Spontaneous reactivation of clusters of X-linked genes is associated with the plasticity of X-inactivation in mouse trophoblast stem cells. Stem Cells. 2014, 32: 377-390.View ArticlePubMedGoogle Scholar
- Hadjantonakis AK, Cox LL, Tam PP, Nagy A: An X-linked GFP transgene reveals unexpected paternal X-chromosome activity in trophoblastic giant cells of the mouse placenta. Genesis. 2001, 29: 133-140.View ArticlePubMedGoogle Scholar
- Hemberger M, Kurz H, Orth A, Otto S, Lüttges A, Elliott R, Nagy A, Tan SS, Tam P, Zechner U, Fundele RH: Genetic and developmental analysis of X-inactivation in interspecific hybrid mice suggests a role for the Y chromosome in placental dysplasia. Genetics. 2001, 157: 341-348.PubMed CentralPubMedGoogle Scholar
- Tam PP, Williams EA, Tan SS: Expression of an X-linked HMG-lacZ transgene in mouse embryos: implication of chromosomal imprinting and lineage-specific X-chromosome activity. Dev Genet. 1994, 15: 491-503.View ArticlePubMedGoogle Scholar
- Tan SS, Williams EA, Tam PP: X-chromosome inactivation occurs at different times in different tissues of the post-implantation mouse embryo. Nat Genet. 1993, 3: 170-174.View ArticlePubMedGoogle Scholar
- Kalantry S, Mills KC, Yee D, Otte AP, Panning B, Magnuson T: The Polycomb group protein Eed protects the inactive X-chromosome from differentiation-induced reactivation. Nat Cell Biol. 2006, 8: 195-202.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang J, Mager J, Chen Y, Schneider E, Cross JC, Nagy A, Magnuson T: Imprinted X inactivation maintained by a mouse Polycomb group gene. Nat Genet. 2001, 28: 371-375.View ArticlePubMedGoogle Scholar
- Fowler KJ, Mitrangas K, Dziadek M: In vitro production of Reichert’s membrane by mouse embryo-derived parietal endoderm cell lines. Exp Cell Res. 1990, 191: 194-203.View ArticlePubMedGoogle Scholar
- Kunath T, Arnaud D, Uy GD, Okamoto I, Chureau C, Yamanaka Y, Heard E, Gardner RL, Avner P, Rossant J: Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts. Development. 2005, 132: 1649-1661.View ArticlePubMedGoogle Scholar
- Niakan KK, Schrode N, Cho LT, Hadjantonakis AK: Derivation of extraembryonic endoderm stem (XEN) cells from mouse embryos and embryonic stem cells. Nat Protoc. 2013, 8: 1028-1041.PubMed CentralView ArticlePubMedGoogle Scholar
- Ohhata T, Senner CE, Hemberger M, Wutz A: Lineage-specific function of the noncoding Tsix RNA for Xist repression and Xi reactivation in mice. Genes Dev. 2011, 25: 1702-1715.PubMed CentralView ArticlePubMedGoogle Scholar
- Rugg-Gunn PJ, Cox BJ, Ralston A, Rossant J: Distinct histone modifications in stem cell lines and tissue lineages from the early mouse embryo. Proc Natl Acad Sci U S A. 2010, 107: 10783-10790.PubMed CentralView ArticlePubMedGoogle Scholar
- Greenfield A, Carrel L, Pennisi D, Philippe C, Quaderi N, Siggers P, Steiner K, Tam PP, Monaco AP, Willard HF, Koopman P: The UTX gene escapes X inactivation in mice and humans. Hum Mol Genet. 1998, 7: 737-742.View ArticlePubMedGoogle Scholar
- Yang F, Babak T, Shendure J, Disteche CM: Global survey of escape from X inactivation by RNA-sequencing in mouse. Genome Res. 2010, 20: 614-622.PubMed CentralView ArticlePubMedGoogle Scholar
- McCabe MT, Ott HM, Ganji G, Korenchuk S, Thompson C, Van Aller GS, Liu Y, Graves AP, Della Pietra A, Diaz E, LaFrance LV, Mellinger M, Duquenne C, Tian X, Kruger RG, McHugh CF, Brandt M, Miller WH, Dhanak D, Verma SK, Tummino PJ, Creasy CL: EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature. 2012, 492: 108-112.View ArticlePubMedGoogle Scholar
- Sado T, Fenner MH, Tan SS, Tam P, Shioda T, Li E: X inactivation in the mouse embryo deficient for Dnmt1: distinct effect of hypomethylation on imprinted and random X inactivation. Dev Biol. 2000, 225: 294-303.View ArticlePubMedGoogle Scholar
- Artus J, Douvaras P, Piliszek A, Isern J, Baron MH, Hadjantonakis AK: BMP4 signaling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm identity. Dev Biol. 2012, 361: 245-262.PubMed CentralView ArticlePubMedGoogle Scholar
- Paca A, Séguin CA, Clements M, Ryczko M, Rossant J, Rodriguez TA, Kunath T: BMP signaling induces visceral endoderm differentiation of XEN cells and parietal endoderm. Dev Biol. 2012, 361: 90-102.View ArticlePubMedGoogle Scholar
- Yang C, McLeod AJ, Cotton AM, de Leeuw CN, Laprise S, Banks KG, Simpson EM, Brown CJ: Targeting of >1.5 Mb of human DNA into the mouse X chromosome reveals presence of cis-acting regulators of epigenetic silencing. Genetics. 2012, 192: 1281-1293.PubMed CentralView ArticlePubMedGoogle Scholar
- Li N, Carrel L: Escape from X chromosome inactivation is an intrinsic property of the Jarid1c locus. Proc Natl Acad Sci U S A. 2008, 105: 17055-17060.PubMed CentralView ArticlePubMedGoogle Scholar
- Horvath LM, Li N, Carrel L: Deletion of an X-inactivation boundary disrupts adjacent gene silencing. PLoS Genet. 2013, 9: e1003952.PubMed CentralView ArticlePubMedGoogle Scholar
- Filippova GN, Cheng MK, Moore JM, Truong JP, Hu YJ, Nguyen DK, Tsuchiya KD, Disteche CM: Boundaries between chromosomal domains of X inactivation and escape bind CTCF and lack CpG methylation during early development. Dev Cell. 2005, 8: 31-42.View ArticlePubMedGoogle Scholar
- Lopes AM, Arnold-Croop SE, Amorim A, Carrel L: Clustered transcripts that escape X inactivation at mouse XqD. Mamm Genome. 2011, 22: 572-582.View ArticlePubMedGoogle Scholar
- Tsuchiya KD, Greally JM, Yi Y, Noel KP, Truong JP, Disteche CM: Comparative sequence and X-inactivation analyses of a domain of escape in human Xp11.2 and the conserved segment in mouse. Genome Res. 2004, 14: 1275-1284.PubMed CentralView ArticlePubMedGoogle Scholar
- Carrel L, Park C, Tyekucheva S, Dunn J, Chiaromonte F, Makova KD: Genomic environment predicts expression patterns on the human inactive X chromosome. PLoS Genet. 2006, 2: e151.PubMed CentralView ArticlePubMedGoogle Scholar
- Reinius B, Shi C, Hengshuo L, Sandhu KS, Radomska KJ, Rosen GD, Lu L, Kullander K, Williams RW, Jazin E: Female-biased expression of long non-coding RNAs in domains that escape X-inactivation in mouse. BMC Genomics. 2010, 11: 614.PubMed CentralView ArticlePubMedGoogle Scholar
- Ciavatta D, Kalantry S, Magnuson T, Smithies O: A DNA insulator prevents repression of a targeted X-linked transgene but not its random or imprinted X inactivation. Proc Natl Acad Sci U S A. 2006, 103: 9958-9963.PubMed CentralView ArticlePubMedGoogle Scholar
- Berletch JB, Yang F, Xu J, Carrel L, Disteche CM: Genes that escape from X inactivation. Hum Genet. 2011, 130: 237-245.PubMed CentralView ArticlePubMedGoogle Scholar
- Carrel L, Willard HF: X-inactivation profile reveals extensive variability in X-linked gene expression in females. Nature. 2005, 434: 400-404.View ArticlePubMedGoogle Scholar
- Lingenfelter PA, Adler DA, Poslinski D, Thomas S, Elliott RW, Chapman VM, Disteche CM: Escape from X inactivation of Smcx is preceded by silencing during mouse development. Nat Genet. 1998, 18: 212-213.View ArticlePubMedGoogle Scholar
- Prothero KE, Stahl JM, Carrel L: Dosage compensation and gene expression on the mammalian X chromosome: one plus one does not always equal two. Chromosome Res. 2009, 17: 637-648.View ArticlePubMedGoogle Scholar
- Marks H, Chow JC, Denissov S, Françoijs KJ, Brockdorff N, Heard E, Stunnenberg HG: High-resolution analysis of epigenetic changes associated with X inactivation. Genome Res. 2009, 19: 1361-1373.PubMed CentralView ArticlePubMedGoogle Scholar
- Cerase A, Smeets D, Tang YA, Gdula M, Kraus F, Spivakov M, Moindrot B, Leleu M, Tattermusch A, Demmerle J, Nesterova TB, Green C, Otte AP, Schermelleh L, Brockdorff N: Spatial separation of Xist RNA and polycomb proteins revealed by superresolution microscopy. Proc Natl Acad Sci U S A. 2014, 111: 2235-2240.PubMed CentralView ArticlePubMedGoogle Scholar
- Casanova M, Preissner T, Cerase A, Poot R, Yamada D, Li X, Appanah R, Bezstarosti K, Demmers J, Koseki H, Brockdorff N: Polycomblike 2 facilitates the recruitment of PRC2 Polycomb group complexes to the inactive X chromosome and to target loci in embryonic stem cells. Development. 2011, 138: 1471-1482.PubMed CentralView ArticlePubMedGoogle Scholar
- da Rocha ST, Boeva V, Escamilla-Del-Arenal M, Ancelin K, Granier C, Matias NR, Sanulli S, Chow J, Schulz E, Picard C, Kaneko S, Helin K, Reinberg D, Stewart AF, Wutz A, Margueron R, Heard E: jarid2 is implicated in the initial Xist-induced targeting of PRC2 to the inactive X chromosome. Mol Cell. 2014, 53: 301-316.View ArticlePubMedGoogle Scholar
- Changolkar LN, Singh G, Cui K, Berletch JB, Zhao K, Disteche CM, Pehrson JR: Genome-wide distribution of macroH2A1 histone variants in mouse liver chromatin. Mol Cell Biol. 2010, 30: 5473-5483.PubMed CentralView ArticlePubMedGoogle Scholar
- Navarro P, Pichard S, Ciaudo C, Avner P, Rougeulle C: Tsix transcription across the Xist gene alters chromatin conformation without affecting Xist transcription: implications for X-chromosome inactivation. Genes Dev. 2005, 19: 1474-1484.PubMed CentralView ArticlePubMedGoogle Scholar
- Fujimoto T, Miyasaka K, Koyanagi M, Tsunoda T, Baba I, Doi K, Ohta M, Kato N, Sasazuki T, Shirasawa S: Altered energy homeostasis and resistance to diet-induced obesity in KRAP-deficient mice. PLoS One. 2009, 4: e4240.PubMed CentralView ArticlePubMedGoogle Scholar
- Chaumeil J, Okamoto I, Heard E: X-chromosome inactivation in mouse embryonic stem cells: analysis of histone modifications and transcriptional activity using immunofluorescence and FISH. Methods Enzymol. 2004, 376: 405-419.View ArticlePubMedGoogle Scholar
- Guo G, Huss M, Tong GQ, Wang C, Sun LL, Clarke ND, Robson P: Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. Dev Cell. 2010, 18: 675-685.View ArticlePubMedGoogle Scholar
- Rugg-Gunn PJ, Cox BJ, Lanner F, Sharma P, Ignatchenko V, McDonald AC, Garner J, Gramolini AO, Rossant J, Kislinger T: Cell-surface proteomics identifies lineage-specific markers of embryo-derived stem cells. Dev Cell. 2012, 22: 887-901.PubMed CentralView ArticlePubMedGoogle Scholar
- Silva J, Barrandon O, Nichols J, Kawaguchi J, Theunissen TW, Smith A: Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol. 2008, 6: e253.PubMed CentralView ArticlePubMedGoogle Scholar
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