Differences in the epigenetic and reprogramming properties of pluripotent and extra-embryonic stem cells implicate chromatin remodelling as an important early event in the developing mouse embryo
© Santos et al; licensee BioMed Central Ltd. 2010
Received: 1 July 2009
Accepted: 12 January 2010
Published: 12 January 2010
During early mouse development, two extra-embryonic lineages form alongside the future embryo: the trophectoderm (TE) and the primitive endoderm (PrE). Epigenetic changes known to take place during these early stages include changes in DNA methylation and modified histones, as well as dynamic changes in gene expression.
In order to understand the role and extent of chromatin-based changes for lineage commitment within the embryo, we examined the epigenetic profiles of mouse embryonic stem (ES), trophectoderm stem (TS) and extra-embryonic endoderm (XEN) stem cell lines that were derived from the inner cell mass (ICM), TE and PrE, respectively. As an initial indicator of the chromatin state, we assessed the replication timing of a cohort of genes in each cell type, based on data that expressed genes and acetylated chromatin domains, generally, replicate early in S-phase, whereas some silent genes, hypoacetylated or condensed chromatin tend to replicate later. We found that many lineage-specific genes replicate early in ES, TS and XEN cells, which was consistent with a broadly 'accessible' chromatin that was reported previously for multiple ES cell lines. Close inspection of these profiles revealed differences between ES, TS and XEN cells that were consistent with their differing lineage affiliations and developmental potential. A comparative analysis of modified histones at the promoters of individual genes showed that in TS and ES cells many lineage-specific regulator genes are co-marked with modifications associated with active (H4ac, H3K4me2, H3K9ac) and repressive (H3K27me3) chromatin. However, in XEN cells several of these genes were marked solely by repressive modifications (such as H3K27me3, H4K20me3). Consistent with TS and XEN having a restricted developmental potential, we show that these cells selectively reprogramme somatic cells to induce the de novo expression of genes associated with extraembryonic differentiation.
These data provide evidence that the diversification of defined embryonic and extra-embryonic lineages is accompanied by chromatin remodelling at specific loci. Stem cell lines from the ICM, TE and PrE can each dominantly reprogramme somatic cells but reset gene expression differently, reflecting their separate lineage identities and increasingly restricted developmental potentials.
After fertilization, the mouse embryo undergoes a series of sequential cleavage divisions producing an eight-cell embryo, where blastomeres maximize their contact with one another in order to generate a compact sphere of cells. Subsequently, apico-basal polarization and asymmetric divisions generate two distinct cell populations at the 16-cell stage: large peripheral polarized cells and small apolar central cells . The outer, polar cells of the late morula change morphology to form an epithelial monolayer of cells - the trophectoderm (TE), which mediates the implantation and initiation of placentation, while the inner apolar cells become the inner cell mass (ICM) and contain the founder cells of the embryo proper. By the early blastocyst stage (E3.5), these two tissues are morphologically distinct - the outer polarized epithelium, the TE, enclosing the ICM, which is itself heterogeneous . Around the time of implantation, cells within the ICM segregate spatially and morphologically into the epiblast (EPI) and PrE lineages, through the migration of PrE cells to the blastocoelic surface of the ICM. Lineage studies have shown that the cells of the EPI are pluripotent and give rise to all tissues of the fetus plus extra-embryonic mesoderm. TE cells are multipotent differentiating exclusively into the trophoblast lineages that form the majority of the fetal placenta, while the PrE give rise to the visceral and parietal endoderm layers that will later line the yolk sack. Besides providing growth support and protection within the uterus, the extra-embryonic TE and PrE are sources of signals to the embryonic lineages to promote correct patterning and differentiation .
While the molecular mechanisms underlying the generation of the ICM, TE and PrE lineages are not fully understood, several transcription factors that play a role in the development of these three different lineages have been described, including Oct4, Cdx2 and Gata6, which are critical for the development of the ICM, TE and PrE, respectively [4–6]. An appropriate segregation of the ICM and TE has, in addition, been shown to be dependent upon the establishment and maintenance of cell polarity, involving E-cadherin and the Par3/aPKC complex [7–9].
Studies from several laboratories have provided evidence of global epigenetic differences between these early lineages that may be important in defining their developmental fate. In particular, a recent study has suggested that at the four-cell-stage mouse embryo, blastomeres with higher levels of histone H3 arginine methylation are more likely to contribute to the pluripotent cells of the ICM . Moreover, while the TE (and also the PrE) are hypomethylated both at repetitive and structural gene sequences [11, 12] throughout development, a striking increase in both DNA and H3K9 methylation levels characterizes the ICM at the blastocyst stage [13, 14]. In addition, epigenetic asymmetry between embryonic and extra-embryonic tissues is evident during X-inactivation, which is random in embryonic but imprinted in the TE and PrE lineages [15, 16].
ES (OS25), TS (B1) and XEN (IM8A1) cells selectively express genes characteristic of either the ICM, trophectoderm or primitive endoderm
Reverse transcription polymerase chain reaction (RT-PCR) was used to assess the relative abundance of different mRNA transcripts in ES (OS25, ), TS (B1, ) and XEN (IM8A1, ) cell lines. Consistent with previous reports, ES cells expressed Oct4, Nanog, Sox2, Fgf4, Rex1  and Esrrb , TS cells expressed Cdx2, Eomes, Esrrb and Hand1 , and XEN cells selectively expressed Gata4, Gata6, Foxa2 and Hnf4  (Figure 1B and Additional file 1). Rex1 transcripts were detected in all three cell types but were most abundant in ES cells; Sox2 transcripts were detected in both ES and TS cells; Eomes transcripts were detected in all three cell types but were most abundant in TS cells. These data show that each of the stem cell lines displays a different profile of gene expression, in line with previous studies [21, 22, 24] and with their different origins. At the level of specific genes, however, there is considerable overlap in expression between the cell lines.
ES, TS and XEN cell lines have similar but distinct replication timing profiles
Most of the genes analysed replicated in early (or middle-early) S-phase in all three stem cell lines (44, 44, 45 out of 58 genes in ES, TS and XEN cells, respectively, Figure 2A). These included a subset of ICM-associated genes (Oct4, Nanog and Fgf4) expressed by ES cells, as well as genes associated with TE (Cdx2 and Hand1) and PrE (Gata4 and Hnf4). In addition, many genes that are not thought to be expressed at significant levels in any of these cell types, for example Math1, Scl and Myog, replicate early in all three embryonic stem cell lines. These data suggests that in TS and XEN cells, many developmental regulator genes remain 'accessible' - as reflected by the prevalence of early replicating loci - similar to that reported previously for ES cells . Overall, the replication timing profiles of ES and TS cells were similar (41/58) or identical (32/58), while XEN cells showed a greater disparity. This is illustrated by a delayed replication of several pluripotency-associated genes in XEN cells (for example, Rex1 and Sox2) and the early replication of PrE-associated genes Gata6 and Foxa2 (Figure 2B) and is in keeping with the idea that some tissue-specific genes may replicate earlier when transcriptionally active [29, 30]. Similarly, Pem and Psx1, which encode factors required for extra-embryonic lineages, replicated later in ES cells as compared to TS and XEN cells and the replication of Pl1, a TE-specific factor, was selectively advanced in TS cells (Figure 2B). These results were confirmed by analysing additional independent TS and XEN cell lines (Additional file 1) that were derived from mice carrying floxed Dicer alleles . Comparing TSB1 and TSDicerfx/fx or XENIM8A1 and XENDicerfx/fx (Figure 2 and Additional file 1), as well as numerous different ES cell lines [19, 32], confirmed that the replication timing profiles of different embryonic and extra-embryonic cell lines were robustly preserved.
Interestingly, the neural-associated genes Sox1 and Neurod that are not expressed by any of the embryonic stem cell lines, showed clear differences in replication timing between ES, TS and XEN cells (Figure 2B, lower panel). Sox1 replication was advanced in ES cells while Neurod replicated early in XEN cells. Although unexpected, these results suggest underlying changes in the chromatin context of these genes in the stem cell lines. In the case of Neurod, although the transcription factor is known to function in neuronal development, it has also been shown to have an important role in the development of specialized cell types arising from the gut endoderm . Despite being derived from the EPI and not from the PrE, gut endoderm cells have morphological and functional similarities to visceral endoderm cells . The advanced replication of Neurod in XEN cells might therefore reflect changes in transcriptional competence at the locus that is associated with an affiliation to the 'endoderm' lineage.
Chromatin profiling of gene promoters in stem cell lines
The chromatin profile of important regulator genes was compared between embryo-derived stem cell lines using chromatin immunoprecipitation (ChIP) in order to evaluate the abundance of specific histone modifications that are associated with either active (H3K4me2, H4ac and H3K9ac) or repressed (H3K27me3 and H4K20me3) chromatin. For these analyses primers were designed to recognize the promoter region (up to 600 kb upstream the transcriptional start site) of each candidate gene; genes that are known to be abundantly expressed by each cell type were used as positive controls for 'active' chromatin marks. Pericentric heterochromatin (γ-satellite repeats) provided controls for H4K20me3 immuno-precipitations, H3K27me3 was validated by analysing known bivalent loci in ES cells , and the abundance of modified histones was calculated relative to histone H3.
Mouse TS, XEN and ES cell lines dominantly reprogram lymphocytes in interspecies heterokaryons but induce the expression of different lineage-associated genes
In this study we show that stem cell lines derived from the ICM, TE and PrE, display distinct epigenetic properties as defined by replication timing, chromatin profiling and reprogramming potential. However, our studies revealed that many genes that are important in determining cellular fate are retained in an 'accessible' chromatin state (acetylated and early replicating) in trophoblast-restricted stem cells (TS), being co-marked also by PRC2-mediated H3K27me3. This chromatin configuration, often referred to as 'bivalent', is shared with ES cells [19, 20] and results in non-productive gene expression [41, 42]. It is thought to be important for priming specific cohorts of genes for future developmental expression [19, 43, 44], and may therefore be important for restraining differentiation [41, 45, 46]. In keeping with this idea, our data show that few developmental regulator genes appear to be primed (bivalent) in XEN cells as compared with ES or TS cells, perhaps reflecting their narrower developmental potential. In addition, the delayed replication of several neuronal-associated genes in XEN cells relative to ES (for example Otx2, Sox1 and Sox2), infers a change in chromatin status and the loss of promoter acetylation. Similar delays have been reported in mature B and T-lymphocytes , which, like XEN cells, have a more restricted (non-neuronal) fate and also in the case of F9 embryonic carcinoma cells, which show a propensity to differentiate to endoderm lineages . Interestingly, Sox1, Sox2 and the neural crest marker Foxd3 also display high levels of H4K20me3 levels at their promoters in XEN cells, consistent with reduced transcriptional competence. The exception to this general trend is Math1, a gene that appears to be functionally primed in XEN cells being simultaneously enriched for 'active' histone marks (H3K4me2, H3K9ac and H4ac) as well as 'repressive' PRC2-mediated H3K27me3. As this gene is known to be involved in the generation of the secretory cell lineages in the intestine  that are derived from the definitive endoderm, it is conceivable that Math1 has a conserved role in the development of extra-embryonic endoderm lineages. Consistent with this idea, XEN cells exhibit a strong bias to form parietal endoderm in chimeras, a tissue which is highly specialized for the synthesis and secretion of extracellular matrix proteins [22, 49].
The similar histone modifications and replication timing profiles between ES and TS cells is consistent with mounting evidence indicating that only relatively few genes are uniquely restricted to the placenta, the vast majority of candidate TS-associated genes being involved in the development of other organs within the embryo proper . Some genes such as Oct4 and Nanog, which are downregulated in TS cells (but remain early-replicating), probably rely on alternative epigenetic mechanisms to suppress transcription in extra-embryonic lineages. For example, in TS cells Oct4 and Nanog regulatory domains are hyper (DNA) methylated and hypoacetylated, relative to ES cells [51, 52]. Despite the overall similarity between ES and TS, the use of a candidate-based replication timing assay allows loci that are subject to chromatin re-modelling events early in mammalian development to be readily identified. A number of studies have suggested that the generation of the ICM and TE requires the development of cell polarity in the outer cells of the morula, and the linked asymmetric divisions of blastomeres at the eight-cell-stage . The significance of this polarization event is reflected by the identification of loci involved in cell polarity and cytoskeleton dynamics among candidates that replicate earlier in TS cells than in ES cells (such as Epb4.1l3, Fez2 and Cdh5, data not shown) in addition to Dab2, which are likely to be functional relevant for the biology of the trophoblast lineage.
The reprogramming properties of extra-embryonic stem cells have, to our knowledge, received little attention. Here, experimental heterokaryons were generated to ask whether TS and XEN cells were capable of dominant reprogramming human somatic cells and, if so, whether they could impose different lineage-specific gene expression programmes. We demonstrate that TS and XEN cells reprogramme human B-lymphocytes in order to establish TE- or PrE-specific gene expression, respectively, albeit at low levels. As these fusions were performed using cells from different mammalian species, low expression levels may reflect inter-species differences, such as mismatches between mouse factors and cis acting elements within human genes . Despite this, fusions using ES, TS or XEN cells reprogrammed human lymphocytes differently, the outcome reflecting discrete lineage affiliations. Interestingly, the expression of human transcripts by reprogrammed B-cell nuclei was not identical to that produced by the mouse stem cell eliciting the dominant reprogramming. This observation mirrors previous reports that fusion with mouse ES cells, results in human B cells expressing a human ES-specific gene expression profile (hSSEA4, hFGF2 and hFGFR1), while hallmark factors of mouse ES cells, such as Lif receptor and Bmp4, are not activated . In this context, it seems likely that reprogrammed hB cells display features of human extra-embryonic-specific gene expression upon heterokaryon formation with mTS or mXEN cells, in agreement with published data . Since extra-embryonic derived human stem cell lines have not been fully characterized, the generation of heterokaryon and hybrid cells using this approach could provide an important tool for studying human extra-embryonic lineages.
This report provides a preliminary epigenetic characterization of mouse TE and PrE extra-embryonic lineages using stem cell lines as a model. We provide evidence of qualitative differences in the chromatin profiles between embryo-derived stem cell lines that accurately reflect their different transcriptional, lineage commitment and developmental potentials. These data support previous in vivo studies of pre-implantation stage embryos [13, 14], showing that dynamic changes in chromatin occur at the earliest stages of mammalian development and are likely to be important for refining cellular potential.
Cell lines and cell culture
ES cells (OS25) were maintained in an undifferentiated state on 0.1% gelatin (StemCell Technologies, Vancouver, Canada)-coated flasks (Fisher Scientific UK Ltd, Leicestershire, UK) in G-MEM-BHK 21 medium (Invitrogen Ltd, Paisley, UK) supplemented with 10% fetal calf serum (FCS; PAA Laboratories, Gmbh, Pasching, Austria), non-essential amino acids, sodium pyruvate, sodium bicarbonate, antibiotics, L-glutamine, β-mercaptoethanol (Sigma-Aldrich Co Ltd, Gillingham, UK) and ESGRO-LIF (1000 U/ml) (Chemicon/Millipore, Billerica, USA). TS cell lines (B1 and Dicerfx/fx) were cultured in the presence of 70% mitotically inactivated mouse embryo fibroblast cells-conditioned medium and 30% TS medium to which human recombinant Fgf4 (25 ng/ml) (Sigma-Aldrich) and heparin (1 μg/ml) (Sigma-Aldrich) were added. The TS cell medium was RPMI 1640 supplemented with 20% FCS (GlobePharm, Cork, Ireland), sodium pyruvate, β-mercaptoethanol, L-glutamine and antibiotics. XEN cell lines (IM8A1 and Dicerfx/fx) were maintained on 0.1% gelatin-coated flasks in RPMI 1640 supplemented with 20% FCS (GlobePharm), sodium pyruvate, L-glutamine, antibiotics and β-mercaptoethanol. EBV-transformed human B-lymphocyte clones were maintained in RPMI medium supplemented with 10% FCS (GlobePharm), L-glutamine and antibiotics. All cell lines used in this study were subjected to karyotypic analysis to check chromosome number. XEN cell lines routinely contained 40-46 chromosomes consistent with their previously reported aneuploid status  while ES and TS cell lines appeared normal.
RNA extraction from ES, TS, XEN cells and heterokaryons was performed using RNeasy protect mini kit (Qiagen, USA) and RNase-free DNase set (Qiagen) for digestion of residual DNA. Total RNA (2.5 μg) was then reverse transcribed using the Superscript first-strand synthesis system (Invitrogen) and cDNA of interest amplified in a total reaction volume of 50 μL using 500 nM primers, and 1.25 U of HotStarTaq (Qiagen). The PCR cycling conditions were as follows: 95°C for 2 min, 30 cycles 95°C for 30 s, annealing at 60°C or 65°C for 30 s and elongation at 72°C for 2 min, finishing with a step at 72°C for 10 min.
Replication timing assay
BrdU-labelling, ethanol fixation, cell cycle fractionation by flow cytometry and isolation of BrdU-labelled DNA by immunoprecipitation were carried out as previously described  with the same BrdU-pulse labelling time for all three stem cell populations (30 min). The abundance of newly replicated DNA in each cell-cycle fraction was determined by real-time PCR amplification.
Real-Time PCR analysis
Real-Time PCR analysis was carried out on a Opticon™ DNA engine (MJ Research, Inc, MA, USA) under the following cycling conditions: 95°C for 15 min, 40 cycles at 94°C for 15 s, 60°C for 30 s, 72°C for 30 s followed by plate read. PCR reactions were performed in a 30 μL reaction volume containing 2× SYBR Green (Qiagen), 1.5 μL of template and 300 nM primers. Each measurement was performed in duplicate. For heterokaryon analysis data were normalised to human GAPDH expression.
Chromatin immunoprecipitation analysis
Exponentially growing ES, TS and XEN cells were processed for ChIP analysis as described previously . 140 μg chromatin was subjected to immunoprecipitation with 5 μL anti-H3K9ac (Upstate Biotechnology, NY, USA), 5 μL anti-H3K4me2 (Upstate), 5 μL anti-H4ac (Upstate), 5 μL anti-H3K27me3 (Upstate), 5 μL anti-H4K20me3 (Upstate), 2.5 μL of a rabbit anti-mouse-IgG antiserum (negative-control) (Dako Inc, CA, USA) and 4 μL of anti-H3 (Abcam, MA, USA). After purification, DNA was resuspended in 80 μL TE solution. Quantification of precipitated DNA was performed using real-time qPCR (quantitative PRC) amplification. Histone's modification levels were normalized against total H3 detected and the ratio of modified-H3 to H3 was denoted as relative enrichment. ChIP experiments were performed twice.
Heterokaryons were generated by fusing either mouse ES, TS or XEN cells and human B-lymphocytes using 50% polyethylene glycol, pH 7.4 (PEG 1500, Roche, Hertfordshire, UK). Equal numbers of stem cells and B-lymphocytes were mixed, washed twice in phosphate buffered saline at 37°C and 1 mL of PEG at 37°C was added to the pellet of cells over 60 s followed by an incubation at 37°C for 90 s. Cell mixtures were washed with 10 mL of DMEM at 37°C added over 3 min. After centrifugation the pellet was allowed to swell in complete medium for 3 min before resuspension. In order to eliminate non-fused hB cells Ouabain (10-5 M) was added to the medium. Proliferating stem cells were eliminated by the addition of 10-5 M Ara-C 6 h after fusion and then removed after 12 h. Fused cells were cultured under conditions promoting the maintenance of undifferentiated mouse stem cells.
fetal calf serum
human B cells
inner cell mass
phosphate buffered saline
polymerase chain reaction
polycomb repressor complex 2
We thank Austin Smith, Neil Brockdorff and Janet Rossant for ES, TS and XEN cell lines, respectively. We are also grateful to Tatyana Nesterova for help and advice with TS cell culture and Eric O'Connor and Eugene Ng for FACS sorting. This work was supported by the Medical Research Council, UK. JS was supported by a doctoral grant from Fundação para a Ciência e a Tecnologia, Portugal.
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