Polycomb repressive complex 2 is dispensable for maintenance of embryonic stem cell pluripotency

Abstract

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.

Figure 1. H3K27 methylation in low pass Eed^null ES cells

(A.)Wild-type, low pass Eed^null, and high pass Eed^null ES cells were stained with an antibody against H3K27me1. Monomethylation is robust in wild-type ES cells and detectable in low pass Eed^null ES cells, but absent in high pass mutant ES cells. (B.) Low pass Eed^null ES cells were stained with antibodies against H3K27me1, me2, and me3. Low pass mutant ES cells stain positively for H3K27me1, but not for H3K27me2 or me3. Positively staining feeder cells serve as internal controls. Corresponding gray-scale images of DAPI stains are shown to the right.

Figure 2. Developmental regulators are aberrantly expressed in Eed^null 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 standard error calculated from 3 technical replicates. (A.) Microarray data reveals that Polycomb-bound Gata genes (Gata3, Gata4, and Gata6) show increased expression in Eed^null ES cells. Gata1 and Hprt, which are not Polycomb bound are shown as controls. Gray bars represent low pass and black bars represent high pass Eed^null ES cells. (B.) Microarray data from low pass Eed^null ES cells show an increase in expression compared to wild-type ES cells for many Polycomb bound developmental regulators. These genes were also surveyed by Boyer et al. (C.) Microarray data from high pass Eed^null ES cells shows a further increase in expression levels for the same genes shown in B.

Figure 3. Expression of pluripotency factors in Eed^null 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 as wild-type counterparts. (A.) The relative expression levels of several pluripotency markers in low (gray bars) and high (black bars) pass Eed^null ES cells was determined by microarray analysis. While most factors have lower expression levels than wild-type ES cells, gene expression is maintained. With the exception of Klf4, low and high pass mutant cells have similar expression levels. Error bars indicate standard error calculated from 3 technical replicates. (B.) Real-time RT-PCR verification of selected genes represented in panel A shows that expression levels are reproducible. Real-time RT-PCR was carried out in duplicate on RNA generated from pooled cell samples (3 different samples per pool). Error bars represent standard error from 2 replicate pools. (C.) Wild-type and high pass Eed^null ES cells were stained with antibodies against OCT4 and NANOG. Robust staining for is observed for both factors. Corresponding DAPI images are found in Figure S2.

Figure 4. Expression of downstream mediators of pluripotency

(A.) Relative expression levels for transcripts identified by Palmqvist et al. as being closely correlated with functional pluripotency in low (yellow) and high (red) pass Eed^null ES cells. Most genes are expressed at wild-type levels or higher in mutant ES cells. (B.) Real-time RT-PCR verification of selected genes represented in panel A. Real-time RT-PCR was carried out in duplicate on RNA generated from pooled cell samples (3 different samples per pool). Error bars for panel D represent standard error from 2 replicate pools.

Figure 5. Chimeric contribution of Eed^null ES cells

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

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. PRC2, and perhaps other repressive factors are dispensable for the maintenance of pluripotency in mouse ES cells.