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EMBRYONIC STEM CELLS

Polycomb Repressive Complex 2 Is Dispensable for Maintenance of Embryonic Stem Cell Pluripotency

Department of Genetics and Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Key Words. Embryonic stem cell • Pluripotent • Epigenetics • Gene expression • Embryo

Correspondence: Correspondence: Terry Magnuson, Ph.D., Department of Genetics and Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA. Telephone: 919-843-6475; Fax: 919-843-4682; e-mail: trm4{at}med.unc.edu

Received on February 18, 2008; accepted for publication on March 19, 2008.

First published online in STEM CELLS EXPRESS  April 10, 2008.

	ABSTRACT

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Polycomb repressive complex 2 (PRC2) methylates histone H3 tails at lysine 27 and is essential for embryonic development. The three core components of PRC2, Eed, Ezh2, and Suz12, are also highly expressed in embryonic stem (ES) cells, where they are postulated to repress developmental regulators and thereby prevent differentiation to maintain the pluripotent state. We performed gene expression and chimera analyses on low- and high-passage Eed^null ES cells to determine whether PRC2 is required for the maintenance of pluripotency. We report here that although developmental regulators are overexpressed in Eed^null ES cells, both low- and high-passage cells are functionally pluripotent. We hypothesize that they are pluripotent because they maintain expression of critical pluripotency factors. Given that EED is required for stability of EZH2, the catalytic subunit of the complex, these data suggest that PRC2 is not necessary for the maintenance of the pluripotent state in ES cells. We propose a positive-only model of embryonic stem cell maintenance, where positive regulation of pluripotency factors is sufficient to mediate stem cell pluripotency.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
One of the earliest and most dynamic mechanisms of epigenetic gene regulation is covalent modification of histone tails by polycomb repressive complex 2 (PRC2). PRC2 is composed of three core components, EED, EZH2, and SUZ12. EZH2 is a histone methyltransferase and the catalytic subunit of the PRC2 complex. Although the functions of EED and SUZ12 remain unknown, all three core components are minimally required for robust PRC2 activity [1]. EED, however, is required for the stability of the EZH2 and SUZ12 proteins and global H3K27 methylation [2].

PRC2 methylates histone H3 at lysine 27 (H3K27). Trimethylated H3K27 (H3K27me3) recruits polycomb repressive complex 1 (PRC1) [3], which in turn mediates chromatin condensation [4] and may even recruit DNA methyltransferases [5] to specific genes during development. This process leads to transcriptional silencing and inheritance of the silenced state to daughter cells [6, 7]. PRC2, through this heritable mechanism of epigenetic gene repression, functions in the maintenance of cellular identity in hematopoietic stem cells [8, 9], differentiating trophoblast cells [10], embryonic mesoderm [11], and cancer stem cells [12, 13]. Reports also implicate PRC2 in the maintenance of the pluripotent state in embryonic stem cells [14–17].

PRC2 binds to and represses the transcription of many developmental regulators that are markers of differentiated cell lineages in both mouse and human embryonic stem (ES) cells [15, 16, 18]. In addition, H3K27me3 colocalizes with histone H3 lysine 4 trimethylation (H3K4me3), a chromatin modification associated with active genes, in bivalent domains at several promoters that mark genes that are silenced but poised for activation [15, 19, 20]. Remarkably, approximately 50% of bivalent domains coincide with binding sites for OCT4, NANOG, or SOX2, transcription factors required for maintaining pluripotency in ES cells. OCT4 itself was recently shown to bind to and activate the EED promoter in mouse ES cells [17]. These data point to an attractive hypothesis where ES cell identity is maintained by a careful balance between PRC2-mediated silencing and gene expression mediated by the transcription factors OCT4, NANOG, and SOX2.

We wanted to determine whether PRC2 was required to maintain ES cell identity and the pluripotent state by comparing gene expression and functional measures of pluripotency in low- and high-passage Eed^null ES cells. We report here that although developmental regulators are overexpressed in Eed^null ES cells, both low- and high-passage cells are functionally pluripotent. We hypothesize that they are pluripotent because they maintain expression of critical pluripotency factors and do not respond to differentiation signals. These data suggest that PRC2, and perhaps epigenetic silencing, is not necessary for maintaining the pluripotent state in embryonic stem cells. Rather, PRC2 may be important for transitions in cell fate (differentiation) and maintenance of multipotency in later progenitor cells. We propose a positive-only model of embryonic stem cell maintenance, where positive regulation of pluripotency factors is sufficient to mediate stem cell pluripotency.

	MATERIALS AND METHODS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
ES Cells and Culture
Eed^null ES cell lines and their wild-type sibling ES lines were derived from the Eed^17Rn53354SB strain of mice carrying the ROSA26 β-geo transgene [21]. These ES cells carry a homozygous point mutation in the Eed gene that results in a functionally null allele [22], as well as a constitutively expressed β-geo gene that serves as a reporter and a selectable marker. Images and a detailed description of mutant ES cell morphology can be found in supplemental online Figure 1.

Eed^null ES cells were maintained on irradiated murine embryonic fibroblasts (MEFs) using standard ES culture conditions. Specifically, cells were grown in ES medium, consisting of minimal essential medium- (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) with 15% fetal calf serum (Invitrogen) supplemented with nonessential amino acids, glutamate, sodium pyruvate, β-mercaptoethanol, penicillin-streptomycin, and leukemia inhibitory factor (LIF). MEF-conditioned medium was also produced by growing irradiated MEFs in ES medium for 48 hours and collecting the medium. To generate RNA, ES cells were passaged onto a gelatinized plate and cultured with 50% MEF-conditioned medium/50% ES medium.

To generate high passage (pass) ES cells, both Eed^null and wild-type ES cells were cultured for 25 additional passages. Low-pass refers to ES cells at pass 7 (p7), whereas high-pass refers to ES cells at pass 32 (p32) or higher. Eed^null ES cells can be maintained with good morphology (supplemental online Fig. 1). All ES lines used in this study were feeder- and LIF-dependent. For microarray analysis, p32 cells were used and for chimera analysis, and p35 cells were used for high-pass cultures.

Immunocytochemistry
ES cells were cultured on gelatin-coated coverslips with feeders as described above. Coverslips were treated with CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, and 10 mM 1,4-piperazinediethanesulfonic acid [PIPES] [pH 6.8]) containing 0.5% Triton X-100, fixed in 4% paraformaldehyde/1 x phosphate-buffered saline (PBS), and stored in 70% ethanol. Coverslips were washed in 1x PBS and incubated in a humid chamber with blocking buffer (1x PBS, 5% goat serum, 0.2% Tween-20, and 0.2% fish skin gelatin). Blocked samples were incubated with primary antibodies (anti-1mH3K27 [Upstate, Charlottesville, VA, http://www.upstate.com], anti-2mH3K27 [Upstate], anti-3mH3K27 [Upstate], anti-OCT4 [Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com], and anti-NANOG [Santa Cruz Biotechnology]) diluted 1:200 in blocking buffer. The coverslips were then washed in 1x PBS/0.2% Tween-20, blocked again in blocking buffer, and incubated with the appropriate secondary antibody (goat anti-rabbit Alexa 594, goat anti-rabbit Alexa 488, goat anti-mouse Alexa 594, or goat anti-mouse Alexa 488 [Molecular Probes, Eugene, OR, http://probes.invitrogen.com]). Coverslips were washed in 1x PBS/0.2% Tween-20 and mounted with Vectashield (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Stained slides were visualized by fluorescence microscopy.

Microarray Analysis
Eed^null and wild-type ES cells were cultured in triplicate for microarray analysis. Samples were harvested and RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA was further purified using RNeasy columns (Qiagen, Hilden, Germany, http://www1.qiagen.com). The quality of the RNA was confirmed prior to labeling using the Agilent Nano RNA Lab-on-a-Chip and the 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, http://www.agilent.com).

RNAs were combined with RNA spike-in control RNAs from the RNA Spike-In kit (two-color; Agilent) and labeled using the RNA Low-Input Linear Amp Kit PLUS (two-color; Agilent) with cyanine 3-cytosine triphosphate (CTP) and cyanine 5-CTP (Perkin Elmer, Waltham, MA, http://www.perkinelmer.com). The labeled RNAs were again purified using RNeasy columns (Qiagen). Quality and labeling fidelity of the labeled RNAs was assessed using the Nano RNA Lab-on-a-Chip and the 2100 Bioanalyzer (Agilent).

The following head-to-head experiments were performed using both dye directions (Cy3 vs. Cy5, and then swapped): low-pass mutant versus low-pass wild-type and high-pass mutant versus high-pass wild-type for each of the three replicates. In total, 12 microarrays were completed. Labeled RNAs were hybridized to the 4X44K mouse whole-genome oligo microarray for at least 17 hours in Hi-RPM hybridization buffer (Agilent), according to the manufacturer's protocols. Microarray slides were washed according to manufacturer's instructions and scanned on an Agilent microarray scanner. The microarray images were interpreted using Feature Extraction 9.5 software (Agilent) and further normalized using GeneSpring GX software (Agilent). Default normalizations were performed that included Lowess normalization and dye swap transformation on appropriate arrays. Data were averaged only for the three replicates for any one dye direction. The dye swaps verified that the data did not suffer from dye bias.

Interpretation of Microarray Data
To identify genes overexpressed in Eed^null ES cells, GeneSpring GX was used to sort genes in which the expression level of mutant relative to wild-type ES cells was greater than 2.0 in at least 6 of 12 instances. This would take into account both low- and high-pass comparisons but ensure that the results were technically repeatable. Correlation of H3K27me3-bound genes and overexpressed genes was determined by merging the list of overexpressed genes with the chromatin structure status of the promoters included in the supplementary data of Mikkelsen et al. [20].

Graphs comparing expression levels for developmental regulators and pluripotency genes were performed from the average of three technical replicates. All unique transcripts representing individual genes were included in the analysis. The data were plotted using the graph function of Excel (Microsoft, Redmond, WA, http://www.microsoft.com). Error bars shown represent the SE from the three technical replicates.

Real-Time Reverse Transcription-Polymerase Chain Reaction
Samples were prepared for real-time reverse transcription (RT)-polymerase chain reaction (PCR) by pooling three wells each of low-pass wild-type, high-pass wild-type, low-pass Eed^null, and high-pass Eed^null ES cells and harvesting RNA using Trizol reagent (Invitrogen). RNAs were purified using RNeasy columns (Qiagen). Two samples from each condition were provided for RT-PCR analysis. Real time RT-PCR was carried out on the RNA samples by Dr. Hyung-suk Kim in the Animal Clinical Chemistry and Gene Expression Facility at the University of North Carolina at Chapel Hill using TaqMan technology (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com).

Generation and Analysis of Chimeras
Chimeric embryos were generated by the Animal Models Core at the University of North Carolina at Chapel Hill. We provided the core facility with either low-pass or high-pass ES cells that were grown on an MEF feeder layer for 48 hours and harvested by trypsinization. The Animal Models Core performed blastocyst injections using standard procedures. Pregnant females were dissected at 9.5, 10.5, or 12.5 days post coitum (dpc), where the date of blastocyst injection was considered 3.5 dpc. Embryos were dissected in 1x PBS and fixed with 0.2% glutaraldehyde. Embryos were processed for 5-bromo-4-chloro-3-indolyl-β-D-galactoside staining with ferric salts. Stained embryos were rinsed three times in 1x PBS, postfixed in 4% paraformaldehyde, and cleared using a glycerol gradient. Following photography, embryos were embedded in paraffin or Tissue-Tek OCT Compound (Sakura Finetek, Torrance, CA, http://www.sakura.com) for sectioning. Embryos prepared for paraffin sections were dehydrated through an ethanol gradient and incubated with xylenes and permeating paraffin prior to embedding in paraffin. Paraffin embedded embryos were sectioned at 8 µM, deparaffinized, and counterstained with nuclear fast red before dehydration and mounting. Embryos prepared for frozen sections were cryoprotected using a sucrose gradient and OCT Compound prior to embedding in OCT Compound and freezing. Cryosections were 10 µM and were not counterstained prior to dehydration and mounting.

	RESULTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Eed^Null ES Cells Retain H3K27 Monomethylation
Our previous observation that Eed^null ES cells lacked monomethylated H3K27 (H3K27me1), dimethylated H3K27 (H3K27me2), and H3K27me3 pertained to high-passage Eed^null ES cells [2]. We repeated the immunocytochemistry using antibodies specific for each of the three forms of H3K27 methylation on low- and high-passage Eed^null ES cells to confirm this observation. Surprisingly, we found that H3K27me1 was readily detectable in low-passage Eed^null ES cells but not in high-passage mutant ES cell lines (Fig. 1A, 1B). Figure 1A shows wild-type along with low- and high-pass Eed mutant ES cells stained with an antibody against H3K27me1. The loss of H3K27me1 was observed in two independent high-passage Eed^null ES cell lines (data not shown), as well as cells newly passaged for these experiments, indicating that loss of H3K27me1 was not an artifact of clonal variation. Figure 1B shows Eed^null ES cells at a low passage number, stained with polyclonal antibodies against H3K27me1, H3K272me, and H3K273me. 4,6-Diamidino-2-phenylindole stains in the right panel of Figure 1B identify the ES cell colony. Both low- and high-passage Eed^null ES cells lacked H3K27me2 and H3K27me3, consistent with previous reports.

View larger version (26K): [in this window] [in a new window]   Figure 1. H3K27 methylation in low-pass Eednull ES cells. (A): Wild-type, low-pass Eednull, and high-pass Eednull ES cells were stained with an antibody against H3K27me1. Monomethylation is robust in wild-type ES cells and detectable in low-pass Eednull ES cells, but absent in high-pass mutant ES cells. (B): Low-pass Eednull ES cells were stained with antibodies against H3K27me1, H3K27me2, and H3K27me3. Low-pass mutant ES cells stained positively for H3K27me1 but not for H3K27me2 or H3K27me3. Positively staining feeder cells served as internal controls. Corresponding gray-scale images of 4,6-diamidino-2-phenylindole stains are shown to the right. Abbreviations: H3K27me1, monomethylated H3K27; H3K27me2, dimethylated H3K27; H3K27me3, trimethylated H3K27.

PRC2 is purported to maintain pluripotency through repression of developmental regulators [16, 18, 23, 24]. We wondered whether we could detect previously observed changes in gene expression between mutant and wild-type Eed^null ES cells. We used microarray expression analysis to directly compare passage-matched Eed^null and wild-type ES cells. We compared low-passage mutant and wild-type ES cells, as well as high-passage mutant and wild-type ES cells. In all, 2,037 transcripts, or 4.9% of the transcripts represented on the microarray, were determined to be upregulated by twofold or more in low and/or high-pass Eed^null ES cells. Only 106 of the 2,037 genes were overexpressed in high-passage versus low-passage Eed^null ES cells, reinforcing the lack of large-scale differences between low- and high-pass mutant ES cells.

Data using single molecule-based sequencing technology for profiling histone modifications identified promoters containing H3K27me3, either alone or in a bivalent domain with the H3K4me3 modification in mouse ES, neural precursor (NPC), or MEF cells [20]. We correlated our expression data with these histone modification profiles and found that 27% of the promoters modified with H3K27me3 or the bivalent marks in ES cells were upregulated by twofold or more in Eed^null ES cells. Transcription factors required for the expression of some H3K27me3-bound genes may not be present in ES cells, which may explain why all PRC2-bound promoters are not activated in Eed mutant ES cells. Only 6% of promoters containing H3K27me3 alone are upregulated in Eed^null ES cells, suggesting that an activating chromatin modification is largely required for expression of PRC2 silenced genes. These data are summarized in supplemental online Table 1.

Of the 2,037 transcripts overexpressed in Eed^null ES cells, 747 transcripts, representing almost 37% of all overexpressed transcripts, harbor bivalent promoters in ES cells. When we considered the histone modification profiles of the upregulated genes in lineage-committed cells, we found that 3% and 16% of upregulated genes were marked by bivalent promoters in NPCs and MEFs, respectively. These data are summarized in supplemental online Table 2. All overexpressed transcripts are not marked with H3K27me3, suggesting that many transcripts overexpressed in Eed^null ES cells are not directly regulated by PRC2 and may be secondary effects.

We then identified developmental regulators that were previously shown to be marked by H3K27me3 and upregulated by real-time PCR [18] in our microarray data. Consistent with previous results, Gata genes that are Polycomb-bound (Gata3, Gata4, and Gata6) were upregulated in mutant ES cells, but genes not bound by Polycomb (Gata1 and Hprt) had expression levels similar to those of wild-type ES cells (Fig. 2A). Three unique Gata6 transcripts were represented on the microarray. Interestingly, one of these is not upregulated in mutant ES cells, suggesting that the particular splice form may be regulated by tissue- or developmental stage-specific splicing that does not occur in ES cells. Many other Polycomb-bound developmental regulators were also found to be upregulated in Eed^null ES cells (Fig. 2B, 2C). In addition, we found that expression levels of these genes were lower in low-pass ES cells (Fig. 2B) and higher in high-pass ES cells (Fig. 2C). These data support the previous finding that PRC2 is required for the repression of important developmental regulators in ES cells.

View larger version (26K): [in this window] [in a new window]   Figure 2. Developmental regulators are aberrantly expressed in Eednull ES cells. Multiple listings for the same gene indicate independent transcripts represented on the microarray and may represent alternate splice forms. A horizontal black line on each graph indicates a value of 1, indicating that mutant and wild-type ES cells have the same expression levels. Error bars indicate SE calculated from three technical replicates. (A): Microarray data reveal that Polycomb-bound Gata genes (Gata3, Gata4, and Gata6) show increased expression in Eednull ES cells. Gata1 and Hprt, which are not Polycomb-bound, are shown as controls. Gray bars represent low-pass Eednull ES cells, and black bars represent high-pass Eednull ES cells. (B): Microarray data from low-pass Eednull ES cells show an increase in expression compared with wild-type ES cells for many Polycomb-bound developmental regulators. These genes were also surveyed by Boyer et al. [18]. (C): Microarray data from high-pass Eednull ES cells show a further increase in expression levels for the same genes shown in (B). Abbreviation: ES, embryonic stem.

We assessed expression levels of previously known pluripotency factors in low- and high-passage Eed^null ES cells to determine whether they are expressed in mutant ES cells. Figure 3A shows the microarray expression levels of known pluripotency factors in low- and high-pass Eed^null ES cells relative to wild-type, passage-matched counterparts. Real-time PCR was used to verify the expression levels of some of these genes (Fig. 3B). We also analyzed protein expression using immunocytochemistry for OCT4 and NANOG (Fig. 3C). Most of the known pluripotency factors show reduced expression in mutant ES cells compared with wild-type ES cells (Fig. 3A, 3B); however, only Sox2, Zfp42, and Oct4 in high-passage cells are expressed at levels less than 50% of wild-type levels, denoted by the dotted line. ES cells were cultured for 48 hours off of feeder cells prior to collection of RNA. Since Eed^null ES cells are feeder-dependent, reduced levels of pluripotency factors may reflect early differentiation resulting from feeder-free culture conditions. Supporting this notion, immunocytochemistry for OCT4 on cells maintained on feeders showed intense staining in mutant ES cells, similar to their wild-type counterparts.

View larger version (26K): [in this window] [in a new window]   Figure 3. Expression of pluripotency factors in Eednull ES cells. Multiple listings for the same gene indicate independent transcripts represented on the microarray and may represent alternate splice forms. Horizontal black lines indicate a value of 1, where mutant and wild-type ES cells have the same expression levels. Dashed black lines represent a value of 0.5, where mutant cells have half of the expression level of wild-type counterparts. (A): The relative expression levels of several pluripotency markers in low-pass (gray bars) and high-pass (black bars) Eednull ES cells were determined by microarray analysis. Although most factors have lower expression levels than wild-type ES cells, gene expression was maintained. With the exception of Klf4, low- and high-pass mutant cells had similar expression levels. Error bars indicate SE calculated from three technical replicates. (B): Real-time reverse transcription (RT)-polymerase chain reaction (PCR) verification of selected genes represented in (A) showed that expression levels are reproducible. Real-time RT-PCR was carried out in duplicate on RNA generated from pooled cell samples (three different samples per pool). Error bars represent SE from two replicate pools. (C): Wild-type and high-pass Eednull ES cells were stained with antibodies against OCT4 and NANOG. Robust staining was observed for both factors. Corresponding 4,6-diamidino-2-phenylindole images are found in supplemental online Figure 2. Abbreviation: ES, embryonic stem.

View larger version (30K): [in this window] [in a new window]   Figure 4. Expression of downstream mediators of pluripotency. (A): Relative expression levels for transcripts identified by Palmqvist et al. [28] as being closely correlated with functional pluripotency in low-pass (yellow) and high-pass (red) Eednull ES cells. Most genes are expressed at wild-type levels or higher in mutant ES cells. (B): Real-time reverse transcription (RT)-polymerase chain reaction (PCR) verification of selected genes represented in (A). Real-time RT-PCR was carried out in duplicate on RNA generated from pooled cell samples (three different samples per pool). Error bars in (D) represent SE from two replicate pools. Abbreviation: ES, embryonic stem.

View larger version (78K): [in this window] [in a new window]   Figure 5. Chimeric contribution of Eednull embryonic stem (ES) cells. Eednull cell contribution in chimeric embryos recovered at 9.5 days post coitum (dpc) was visualized by β-galactosidase staining. Whole-mount embryos from chimeric embryos made with low-pass Eednull ES cells (A–C) showed fairly normal development of even moderately high-contribution chimeras (A, B). Eednull cells could occasionally be seen in the embryonic forebrain and heart field (C), although these areas are reported to be relatively devoid of mutant cells in chimeric embryos [21]. Paraffin sections ([D], with further magnification in [E, F]) revealed that low-pass Eednull ES cells contribute to tissues derived from all three embryonic germ layers, including neurepithelium ([D, E], arrows), mesenchyme ([E, F], asterisks), and gut endoderm ([F], dashed arrow). Results were similar to those previously reported [21]. (G–L): High-pass Eednull cell contribution in chimeric embryos recovered at 9.5 dpc was visualized by β-galactosidase staining. Whole-mount images representing high-contribution (G) and moderate-contribution (H) chimeric embryos are shown. High-contribution chimeras were almost exclusively Eednull ES cell-derived, as indicated by blue staining in the whole-mount embryo (G) and cryosections (J). As reported previously [21], high-contribution chimeras suffer defects also seen in homozygous Eednull embryos, including an overgrown allantois ([J], boxed area), poorly developed neurepithelium ([J], arrow), and severe sparsity of embryonic mesoderm ([J], asterisk). Moderate range chimeras showed relatively normal development (G, I, L), with Eednull cells contributing seamlessly to neurepithelium ([I, L], solid arrows), surface epithelium ([L], open arrow), mesenchyme ([I, L], asterisk), and endoderm ([L], dashed arrow). Even relatively well-differentiated cell types, such as embryonic blood (K), can be populated by Eednull cells.

To determine whether terminally differentiated Eed^null cells were capable of survival, we isolated MEFs from 10.5-dpc chimeric embryos. We chose this stage because it preceded the wave of lethality of high- to moderate-contribution chimeras but produced larger embryos with more cells. Whole 10.5-dpc embryos were dissociated by passage through a needle and plated. Eed^null cells can be selected for using G418 because the mutant ES cells contain a β-geo cassette at the Rosa26 locus, which confers resistance to G418. Twenty-four hours after plating, G418 was added to some of the MEFs in culture. Although wild-type MEFs could be recovered from embryos without selection, Eed^null MEFs were not recovered from dissociated embryos plated with or without G418. In some cases, poor cellular growth occurred from plated embryos, even in the absence of G418. In these cases, few cells attached to the tissue culture dish, and they failed to divide further. One similar MEF outgrowth was stained for β-galactosidase activity, and positive staining confirmed that the cells were Eed^null. A few (total of four) LacZ-positive cells were also seen in a robustly growing MEF culture without G418 selection, 4 days after initial plating. However, upon splitting this culture, Eed^null (LacZ-positive) cells could not be found. These data suggest that Eed^null MEFs cannot be maintained in culture.

	DISCUSSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Previous studies demonstrated a role for PRC2 and H3K27me3 in the repression of developmental regulators [15, 16, 18, 24]. Lee et al. [16] and Boyer et al. [18] extended this observation to suggest that PRC2 was required for maintenance of the pluripotent state because Eed^null ES cells aberrantly express lineage-specific markers and spontaneously differentiate in culture. However, the same Eed^null ES cells were previously shown to contribute to all tissue lineages in embryoid bodies and chimeric embryos and are therefore functionally pluripotent [21]. These disparate results were obtained with high- and low-passage Eed^null ES cells, respectively. We hypothesized that high-passage Eed^null ES cells are not pluripotent and that differences between low- and high-passage ES cells would reveal the factors responsible for the loss of pluripotency.

Since previously published characterization of H3K27 methylation in Eed^null ES cells was carried out on high-passage cells, we first wanted to verify that low-passage mutant ES cells lacked global H3K27 methylation. Previous data suggested that H3K27me1 may be mediated by a complex other than the canonical PRC2 complex and that EED is also a member of the alternate complex [2, 29] (S.J.C. and T.M., unpublished data). To our surprise, low-passage Eed^null ES cells retained H3K27me1, whereas high-passage mutant cell lines lacked this modification. H3K27me2 and H3K27me3 are found at the promoters of genes silenced by PRC2, but the function of H3K27me1 is poorly understood. H3K27me1 is thought to be a dynamic modification that is widely distributed across euchromatin, except for the transcriptional start sites of active genes [30]. We speculate that EED likely mediates monomethylation since it is absent in high-passage mutant ES cells. However, the removal of H3K27me1 may occur passively, as opposed to H3K27me3, which can be actively removed by the histone demethylases UTX and JMJD3. Upon comparing cellular morphology and expression profiles of low- and high-passage Eed^null ES cells, we found few changes, suggesting that global expression changes do not result from the loss of H3K27 monomethylation.

The changes in expression between Eed^null ES cells and passage-matched wild-type ES cells that we observed agreed with previously published reports [15, 18, 23] that demonstrated increased expression levels of developmental regulators in Eed^null ES cells. Ectopic expression of lineage-specific genes was observed in both low- and high-passage Eed mutant ES cells. Expression levels of many genes became further increased in high-passage mutant ES cells. These data support a role for PRC2 in the repression of developmental regulators.

Ectopic expression of developmental regulators can drive in vitro differentiation of ES cells. For instance, forced expression of Gata4 and Gata6 directs ES cell differentiation to primitive endoderm [31], and overexpression of Cdx2 is sufficient to differentiate ES cells to trophectoderm [32, 33]. Differentiation induced by overexpression of Cdx2, Gata4, and Gata6 was reinforced by downregulation of Oct4 and Nanog. Furthermore, depletion of OCT4, NANOG, or SOX2 alone can result in the functional loss of pluripotency in embryos and ES cells [25–27, 34, 35]. Expression levels of Oct4, Nanog, and Sox2 were reduced in Eed^null ES cells; however, mutant ES cells retained expression of the pluripotency-associated transcription factors. Reduced expression levels of Oct4, Nanog, and Sox2 may reflect early differentiation due to the brief feeder-free culture condition. Robust staining for OCT4 and NANOG proteins in Eed^null ES cells maintained on feeders supports this possibility.

We also considered genes that were downregulated during the first 18 hours of differentiation by LIF removal [28]. The reduced expression of these genes most closely correlated with loss of functional pluripotency, suggesting that they were the downstream mediators of the pluripotent state. Many of these factors have promoters that are themselves bound by OCT4, NANOG, or SOX2 in mouse [35] or human ES cells [36] and thus are likely to represent part of the same transcriptional network. Eed^null ES cells retain expression of these pluripotency markers. In addition, low-passage Eed mutant ES cells had higher expression levels than wild-type ES cells. Although transcription factors known to mediate pluripotency are reduced in Eed^null ES cells, genes most closely correlated with functional pluripotency are not. This could occur because the level of transcription of pluripotency-associated transcription factors, although reduced, is sufficient to direct proper expression of downstream markers. Interestingly, recently reported induced pluripotent cell lines also show severely reduced levels of Oct4 and Sox2 but are functionally pluripotent [37].

Eed^null ES cells were previously shown to be pluripotent by their ability to form three germ layers and differentiate in vitro into various tissue types, including neurons, in embryoid bodies, and by incorporation into most tissues of chimeric embryos [21]. Furthermore, primordial germ cells are also specified in Eed^null embryos, suggesting that mutant cells can even contribute to the germline [11]. Although these experiments were performed with the earliest passages of Eed^null ES cells, we have demonstrated here that both low- and high-passage Eed^null ES cells are pluripotent by chimeric embryo analyses, the most stringent test of pluripotency for mouse ES cells.

Neurons can be generated from in vitro differentiation of Eed^null ES cells [21], but MEFs cannot. Consistent with this finding, high-contribution Eed^null chimeras have a paucity of mesoderm but form neuroectoderm. This suggests that although EED is dispensable for the maintenance of a pluripotent stem cell population, it is still required for differentiation and/or maintenance of multipotent progenitors. Interestingly, very few of the genes that are upregulated in Eed^null ES cells are marked with bivalent chromatin modifications in NPCs compared with MEFs [20]. The bivalent modifications mark promoters that are silenced but poised for expression in that cell type [19]. The overexpression of genes that are required to be repressed in MEFs may explain why we cannot derive that cell type from chimeric embryos. Conversely, few of the overexpressed genes are required to be silenced in NPCs, in agreement with the relatively normal development of neural precursor cells in embryoid bodies.

SUZ12-deficient ES cells have also been derived, and although it is not known whether they are pluripotent, they can be maintained in culture and stain positively for OCT4 and NANOG [38]. In contrast to EED-deficient ES cells, SUZ12-deficient ES cells cannot form neurons after in vitro differentiation. Although it is formally possible that a function for SUZ12 outside of the canonical PRC2 complex could explain the disparate results, we think that this is unlikely because SUZ12 protein is absent from EED-deficient ES cells [2]. One possible explanation is that the retinoic acid (RA)-induced differentiation scheme used by Pasini et al. [38] induced PRC2-regulated genes that otherwise remained repressed in the EED-deficient embryoid bodies that were not differentiated using RA. A recent report demonstrates that RA directly induces expression of the JMJD3 histone demethylase, suggesting that RA induces activation of PRC2-regulated genes via JMJD3 [39]. A better understanding of the interplay between PRC2 and its corresponding histone demethylases during transcriptional activation is needed to fully understand this dilemma. In any case, SUZ12-and EED-deficient ES cells each maintain expression of key pluripotency factors while simultaneously expressing known differentiation factors. This creates a situation where factors both positively and negatively influencing stem cell self-renewal coexist.

A minimal set of pluripotency factors may promote the necessary gene expression and cell proliferation that is required to support stem cell self-renewal. The forced coexpression of four transcription factors, Oct4, Sox2, Klf4, and Myc, can reprogram terminally differentiated fibroblasts into pluripotent ES-like cells that contribute to the germline [37, 40, 41]. OCT4, SOX2, and KLF4 function as transcription factors or coactivators [26, 42, 43], whereas MYC promotes the G1- to S-phase transition [44]. Although OCT4 may mediate gene repression by binding and activating the Eed promoter [17], we have demonstrated that gene repression mediated by EED is not necessary for the maintenance of pluripotent ES cells.

To our knowledge, no known epigenetic repressor is required for the maintenance of pluripotency. In fact, a histone lysine demethylase that reverses a repressive histone modification is essential for pluripotency [45]. We propose a positive-only model for the maintenance of pluripotency in ES cells (Fig. 6). Expression of pluripotency factors or their downstream targets may be sufficient to sustain self-renewal of pluripotent embryonic stem cells, even when lineage-specific factors are aberrantly expressed. The notion that epigenetic repression is dispensable for embryonic stem cell self-renewal is further supported by observations that ES cells maintain an open chromatin conformation [15, 46, 47] and transcribe a large number of genes [48–51]. These data support the hypothesis that pluripotency is the default state for genetic systems [45, 52].

View larger version (19K): [in this window] [in a new window]   Figure 6. Positive-only model for the maintenance of stem cell pluripotency. This cartoon demonstrates how pluripotency factors might regulate stem cell self-renewal in the absence of repressive factors. Polycomb repressive complex 2 and perhaps other repressive factors are dispensable for the maintenance of pluripotency in mouse ES cells. Abbreviation: ES, embryonic stem.

	CONCLUSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
We thank Dr. Hyung-Suk Kim for quantitative RT-PCR support, Jade Zhang and the Animal Models Core for accommodating the blastocyst injections, Jessica Nadler for gene expression microarray assistance, and Andy Fedoriw for critical reading of the manuscript. This work is supported by a National Research Service Award Fellowship (to S.J.C.) and a grant from the NIH (to T.M.).

	FOOTNOTES

 
Author contributions: S.J.C.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; D.Y.: collection and/or assembly of data, data analysis and interpretation; T.M.: conception and design, financial support, provision of study material, data analysis and interpretation, manuscript writing, final approval of manuscript.

	REFERENCES

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 

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