Epigenetic regulation of Nanog expression by Ezh2 in pluripotent stem cells

Abstract

Nanog levels in pluripotent stem cells are heterogeneous and this is thought to reflect two different and interchangeable cell states, respectively poised to self-renew (Nanog-high subpopulation) or to differentiate (Nanog-low subpopulation). However, little is known about the mechanisms responsible for this pattern of Nanog expression. Here, we have examined the impact of the histone methyltransferase Ezh2 on pluripotent stem cells and on Nanog expression. Interestingly, induced pluripotent stem (iPS) cells lacking Ezh2 presented higher levels of Nanog due to a relative expansion of the Nanog-high subpopulation, and this was associated to severe defects in differentiation. Moreover, we found that the Nanog promoter in embryonic stem (ES) cells and iPS cells coexists in two alternative univalent chromatin configurations, either H3K4me3 or H3K27me3, the latter being dependent on the presence of functional Ezh2. Finally, the levels of expression of Ezh2, as well as the amount of H3K27me3 present at the Nanog promoter, were higher in the Nanog-low subpopulation of ES/iPS cells. Together, these data indicate that Ezh2 directly regulates the epigenetic status of the Nanog promoter affecting the balance of Nanog expression in pluripotent stem cells and, therefore, the equilibrium between self-renewal and differentiation.

Figure 1

Generation and validation of Ezh2-null ipS. (A) Strategy to obtain Ezh2-null ipS. Ezh2-floxed MeFs were infected with the three reprogramming factors, oct4, Klf4 and Sox2, and two weeks later ipS^f/f colonies were pooled and replated in the absence or presence of 4OHT. Colonies of iPS^f/f (−4OHt) or iPS^Δ/Δ (+4OHt) cells were picked one week later. (B) Morphology of iPS^f/f and iPS^Δ/Δ colonies. Cells were cultured on feeder fibroblasts. Both genotypes presented a typical morphology and did not differentiate spontaneously. (C) qRT-pCR analysis of stemness markers. Relative endogenous expression of each gene was normalized to actin and error bars represent standard deviation. The figure shows the data for two independent clones. A total of six clones were analyzed per genotype yielding similar results. (D) Protein levels of Ezh2, H3K27me3 and Nanog in iPS^f/f and iPS^Δ/Δ cells. The figure shows the data for two independent iPS^Δ/Δ clones. Additional clones (5 clones of iPS^f/f and 8 clones of iPS^Δ/Δ) are shown in Supplemental Figure 1c. (E) Confocal immunofluorescence of H3K27me3. The figure is representative of a total analysis of six iPS^f/f clones and five iPS^Δ/Δ clones. Note that the positive H3K27me3 signal in the iPS^Δ/Δ panel corresponds to the feeder fibroblasts.

Figure 2

Impaired differentiation of Ezh2-null iPS cells. (A) Cultures of iPS cells were treated with retinoic acid (RA) in the absence of LIF for four days and differentiation was assessed by the loss of the stemness marker alkaline phosphatase detected by a histochemical reaction. The figure is representative of a total analysis of three clones of each genotype. (B) Expression by qRT-PCR of the indicated neural markers before (−RA) or after (+RA) RA-induced differentiation. Values correspond to the average and standard deviation of a single clone per genotype. The figure is representative of a total analysis of six clones of each genotype. (C) Levels of stemness proteins Nanog and Oct4 upon RA-induced differentiation. The figure is representative of a total of three clones of each genotype. (D) FACS analysis of Nanog four days after RA-induced differentiation. The figure is representative of a total of three clones of each genotype. Values correspond to the average and standard deviation of the percentage of Nanog-positive cells in a total of three clones per genotype. (E) Embryoid bodies formed by ipS^f/f and ipS^Δ/Δ cells. Top panels, low magnification images of three embryoid bodies of each genotype. Bottom panels, sections of embryoid bodies stained with hematoxylin and eosin at two different magnifications. Images are representative of a total of six clones of each genotype. (F) Teratomas formed by iPS^f/f and iPS^Δ/Δ cells. Sections of teratomas were stained for Nanog by immunohistochemistry and counterstained with hematoxylin. Insets show low magnification views of the entire teratomas. Images are representative of a total of two clones of each genotype, and each clone was used to generate six independent teratomas.

Figure 3

Ratio between Nanog-high and Nanog-low sub-populations in Ezh2-null iPS cells. (A) Expression by qRT-PCR of Nanog. Values are relative to iPS^f/f and correspond to the average and standard deviation. Student’s t-test was used to determine statistical significance. (B) Levels of Nanog protein by confocal immunofluorescence. Fluorescence was quantified per cell in arbitrary fluorescence units (AFUs) and colored according to the indicated ranges. The figure is representative of a total analysis of three iPS clones of each genotype. (C) FACS analysis of Nanog. The upper part shows the direct Nanog profile, and the lower part the deconvolution of the profile into two peaks. Deconvoluted peaks were quantified in six clones of each genotype and values correspond to the average and standard deviation. Student’s t-test indicated that the two Nanog peaks (low and high) in iPS^Δ/Δ cells were significantly different from the corresponding Nanog peaks in iPS^f/f cells (p < 0.001). (D) Expression by qRT-PCR of Nanog and Ezh2 in Nanog-low and Nanog-high subpopulations of wild-type ES cells sorted by cytometry using anti-Nanog staining. Two independent experiments were performed. Each qRT-PCR determination was done in triplicate. Student’s t-test was used to determine statistical significance. (E) Expression by qRT-PCR of Nanog, Ezh2 and Oct4 in Nanog-low and Nanog-high subpopulations of TNG-A ES cells sorted by cytometry according to the GFP fluorescence signal. Student’s t-test indicated that the levels of Ezh2 in GFP-cells were significantly different from the parental TNG-A cells and from GFP⁺ cells. (F) Proliferation of iPS in gelatin-coated plates. Values correspond to the average and standard deviation of a total of three clones per genotype. (G) Analysis of cell cycle phases measured by the incorporation of propidium iodide. Values correspond to the average and standard deviation of a total of three clones per genotype. (H) Quantification of DNA replication by incorporation of BrdU for 30 min. The figure is representative of a total of three clones per genotype and values correspond the average and standard deviation of the three clones.

Figure 4

Epigenetic marks at the Nanog promoter in Ezh2-null iPS cells. (A) Chromatin immunoprecipitation (ChIP) of Ezh2 and qPCR of the Nanog promoter in the indicated cells under standard culture conditions. Values correspond to the average and standard deviation of triplicate qPCR reactions for each immunoprecipitation. Student’s t-test was used to determine statistical significance. The data are representative of a total of two clones per genotype. (B) Sequential ChIP, first of H3K27me3 and then of H3K4me3, at the Nanog promoter in wild-type ES cells. Irx2 and Tcf4 promoters were used as positive and negative controls of bivalency, respectively. Values correspond to the average and standard deviation of triplicate qPCR reactions for each immunoprecipitation. (C) ChIP of H3K4me3 and H3K27me3 and q-PCR of the Nanog promoter in the indicated cells in standard growth conditions (−RA) or four days after differentiation (+RA). Values correspond to the average and standard deviation of triplicate q-PCR reactions of a single clone per genotype. Student’s t-test was used to determine statistical significance. The data are representative of a total of three clones per genotype. (D) ChIP of H3K27me3 and q-PCR of the Nanog promoter in TNG-A ES cells sorted by cytometry according to the GFP fluorescence signal. Values correspond to the average and standard deviation of triplicate q-PCR reactions. Student’s t-test was used to determine statistical significance. (E) Model of Nanog regulation by Ezh2 protein.