PRC2 specifies ectoderm lineages and maintains pluripotency in primed but not naïve ESCs

Abstract

Polycomb repressive complex 2 and the epigenetic mark that it deposits, H3K27me3, are evolutionarily conserved and play critical roles in development and cancer. However, their roles in cell fate decisions in early embryonic development remain poorly understood. Here we report that knockout of polycomb repressive complex 2 genes in human embryonic stem cells causes pluripotency loss and spontaneous differentiation toward a meso-endoderm fate, owing to de-repression of BMP signalling. Moreover, human embryonic stem cells with deletion of EZH1 or EZH2 fail to differentiate into ectoderm lineages. We further show that polycomb repressive complex 2-deficient mouse embryonic stem cells also release Bmp4 but retain their pluripotency. However, when converted into a primed state, they undergo spontaneous differentiation similar to that of hESCs. In contrast, polycomb repressive complex 2 is dispensable for pluripotency when human embryonic stem cells are converted into the naive state. Our studies reveal both lineage- and pluripotent state-specific roles of polycomb repressive complex 2 in cell fate decisions.Polycomb repressive complex 2 (PRC2) plays an essential role in development by modifying chromatin but what this means at a cellular level is unclear. Here, the authors show that ablation of PRC2 genes in human embryonic stem cells and in mice results in changes in pluripotency and the primed state of cells.

Fig. 1

Deletion of polycomb repressive complex 2 in human embryonic stem cells. a Overview of the gene targeting strategy. gRNA was designed and validated for each polycomb repressive complex 2 (PRC2) component gene showing in box. To delete the critical domain for each factor, a homologous targeting vector containing puromycin or neomycin resistant cassette was constructed according to each gene. gRNA/Cas9 together with targeting vector were electroplated into H1 or H9 human embryonic stem cells (hESCs) and selected by the corresponding drug in defined condition. Positive clones were then isolated and expanded for further characterizations. b Targeting efficiencies of each gene. The functional domain that was deleted in each factor was shown. For SUZ12 and EED, gene targeting was performed in both H1 and H9 hESCs. c Morphology of H1 hESCs with targeted deletion of each gene. Scale bar, 200 μm. d qRT-PCR analysis on the expression level of each indicated gene in gene targeted hESCs. Wild-type H1 hESCs serve as control. Significance level was determined using unpaired two-tailed Student’s t tests. **, P < 0.01. The data represent mean ± SD from three biological repeats. e Total level of the indicated histone modification in gene targeted cells. The total histone modification level was analyzed by western-blot using the specific antibody on the whole-cell lysates from each indicated cell line. f qRT-PCR analysis on the expression level of the pluripotent genes, OCT4, SOX2, NANOG in gene targeted hESCs. Wild-type H1 hESCs serve as control. Significance level was determined using unpaired two-tailed Student’s t tests. **, P < 0.01. The data represent mean ± SD from three biological repeats. See also Supplementary Fig. 1

Fig. 2

PRC2^−/− hESCs exhibit spontaneous differentiation to meso-endoderm fate. a Morphology and alkaline phosphatase (ALP) activity staining on each indicated hESCs. Scale bar, 200 μm. b qRT-PCR analysis on the selected lineage genes in the indicated cell lines. Negative control: H1, positive control: H1 cells-derived embryonic bodies (H1-EB day 12). Significance level was determined using unpaired two-tailed Student’s t tests. **, P < 0.01. The data represent mean ± SD from three independent repeats. c Spearman rank correlation analysis on the whole-genome transcriptome of indicated cell lines. d Heatmap on the selected pluripotent and linage marker genes in the indicated hESCs. We set the expression level of genes in H1 hESCs as 1 and calculated the fold change (log2) of individual gene in none of core component of PRC2 in H1 hESCs, respectively. e Immunostaining on the pluripotency and lineage markers, OCT4 (pluripotency), CALPONIN (mesoderm), SOX17 (endoderm) in the indicated cell lines. Scale bar, 100 μm. See also Supplementary Fig. 2

Fig. 3

EZH1 and EZH2 specify early neural ectoderm fate. a Morphology and alkaline phosphatase (ALP) activity staining on each indicated hESCs. Scale bar, 200 μm. b FACS analysis on the expression of indicated pluripotent markers in the indicated hESCs. c Cell cycle of the indicated hES cell lines. The data represent mean ± SD from three biological repeats. d qRT-PCR analysis on lineage genes in the indicated hESCs. Wild-type H1 hESCs serve as control. The data represent mean ± SD from three biological repeats. e H&E staining on sections of teratomas formed by the indicated hESC cell lines. Scale bar, 200 μm. Significance level was determined using unpaired two-tailed Student’s t tests. ***, P < 0.001. The data represent mean ± SD from three biological repeats. f Neural differentiation of the indicated hESC cell lines. hESCs were treated by SB431542/Dorsomorphin (DM) (5/5 μM) in N2B27 medium in monolayer condition and analysed by qRT-PCR on lineage markers and neural sphere formation. Scale bar, 100 μm. Significance level was determined using unpaired two-tailed Student’s t tests. *, P < 0.05. ***, P < 0.001. The data represent mean ± SD from three independent repeats. g Blood differentiation. The indicated hESCs were treated in the cocktail of indicated cytokines in monolayer condition and analyzed by qRT-PCR on indicated blood lineage markers or FACS analysis on CD34⁺ cells. The data represent mean ± SD from three independent repeats. h Endoderm differentiation. The indicated hESCs were treated by activin A (100 ng mL⁻¹) in defined condition. The endoderm marker, SOX17 was analyzed by immunostaining and other endoderm lineage markers were analyzed by qRT-PCR. Scale bar, 20 μm. The data represent mean ± SD from three independent repeats. DE, definitive endoderm cell; ES embryonic stem cell; HPC, hematopoietic progenitor cell; NPC, neural progenitor cell. See also Supplementary Fig. 3

Fig. 4

PRC2 deletion preferentially induces BMP signalling in hESCs. a Diagram of lentiviral-based inducible system for EED expression. EED expression was controlled by DOX treatment. The morphology of H1 hESCs with EED over-expression is shown. Scale bar, 200 μm. b Strategy of EED knockout in H1 hESCs with EED over-expression. CRISPR/Cas9 and targeting vector were transfected into H1 hESCs with DOX inducible EED overexpression (H1-EED-OE). The positive cell clones were then selected by puromycin in the absence of DOX for the first 10 days and expanded in the presence of DOX). The positive clones were then isolated and maintained in defined medium with DOX. The correctly targeted cells, referred as H1-EED ^−/−/EED-OE were confirmed by genomic PCR and qRT-PCR on endogenous EED. c Gradual differentiation of H1-EED ^−/−/EED-OE upon withdrawal of DOX, indicated by morphology and ALP staining. Scale bar, 200 μm. d qRT-PCR examination on the expressions of EED in H1-EED ^−/−/EED-OE at 28 days after DOX withdrawal. Significance level was determined using unpaired two-tailed Student’s t tests. **, P < 0.01. The data represent mean ± SD from three independent repeats. e, f qRT-PCR examination on the expressions of indicated pluripotent and lineage genes in H1-EED ^−/−/EED-OE at different time points after DOX withdrawal. Significance level was determined using unpaired two-tailed Student’s t tests. **, P < 0.01. The data represent mean ± SD from three independent repeats. g Spearman’s rank correlation analysis on the whole-genome transcriptome of H1-EED ^−/−/EED-OE at different time points after DOX withdrawal. h Heatmap on the selected pluripotent and lineage marker genes in H1-EED ^−/−/EED-OE at different time points after DOX withdrawal. We set the expression level of genes in H1 hESCs as 1 and calculated the fold change (log2) of individual gene in H1-EED ^−/−/EED-OE hESCs, respectively. i Heatmap on TGF-β/BMPs signaling genes in H1-EED ^−/−/EED-OE at different time points after DOX withdrawal. We set the expression level of genes in H1 hESCs as 1 and calculated the fold change (log2) of individual gene in H1-EED ^−/−/EED-OE hESCs, respectively. See also Supplementary Fig. 4

Fig. 5

Inhibition of BMP signalling rescues PRC2 deficiency in hESCs. a Strategy of PRC2 disruption rescue experiments. H1-EZH2 ^−/−/EZH1 ^−/−/EZH2-OE or H1-EZH2 ^−/−/EZH1 ^−/−/EZH1-OE was prepared as described in Fig. 4a, b. H1-EED ^−/−/EED-OE, H1-EZH2 ^−/−/EZH1 ^−/−/EZH2-OE and H1-EZH2 ^−/−/EZH1 ^−/−/EZH1-OE were cultured in defined medium in the absence of DOX, but with adding with BMP or TGF-β inhibitors (1 μM DM or 5 μM SB431542) for 20 more days. b Morphology of the indicated hESCs cultured in defined medium with indicated condition. DM while not SB431542 treatment rescued morphological change triggered by DOX withdrawal. Scale bar, 200 μm. c Expression of pluripotent genes OCT4, SOX2 and NANOG in the indicated hESCs with different treatments. Significance level was determined using unpaired two-tailed Student’s t tests. **, P < 0.01. The data represent mean ± SD from three independent repeats. d Immunostaining on the pluripotency and lineage markers, OCT4 (pluripotency), CALPONIN (mesoderm), SOX17 (endoderm) in the indicated hESCs with different treatments. Scale bar, 100 μm. See also Supplementary Figs. 5 and 6

Fig. 6

PRC2 is required for maintaining pluripotency in primed not naive state. a Conversion of WT, Suz12 ^−/− , or Eed ^−/− naive mESCs into the primed state. WT mESCs is OG2 mESCs with GFP expression controlled by Oct4 promoter (Oct4: GFP). For conversion into the primed state, WT, Suz12 ^−/− , or Eed ^−/− naive mESCs were treated with indicated conditions in the absence of feeder cells. Scale bar, 100 μm. b FACS analysis on Oct4: GFP in WT, Suz12 ^−/− , or Eed ^−/− mESCs at different state. c qRT-PCR analysis on Oct4, Sox2, Tfcp2l1, and Bmp4 in WT, Suz12 ^−/− , or Eed ^−/− mESCs at different state. Significance level was determined using unpaired two-tailed Student’s t tests. **, P < 0.01. The data represent mean ± SD from three independent repeats. d Conversion of WT or H1-EZH2 ^−/− hESCs into the naive state. hESCs with NANOG/KLF2 expression were further cultured in switched medium with indicated condition. TFCP2L1, a marker gene of naive pluripotency, was detected by qRT-PCR. Significance level was determined using unpaired two-tailed Student’s t tests. **, P < 0.01. The data represent mean ± SD from three independent repeats. Scale bar, 100 μm. e Knockdown of EHZ1 in primed or naive state WT or H1-EZH2 ^−/− hESCs. OCT4, TFCP2L1, and EZH1 were examined by qRT-PCR in the indicated hESCs with EZH1 knockdown. Significance level was determined using unpaired two-tailed Student’s t tests. **, P < 0.01. *, P < 0.05. The data represent mean ± SD from three independent repeats. Scale bar, 100 μm. f Conversion of WT or H1-EED ^−/−/EED-OE hESCs into the naive state. hESCs were further cultured in switched medium (5i/L/A) with indicated condition in the presence or absence of DOX. Left: Morphology of H1, H1-EED ^−/−/EED-OE with DOX or without DOX in primed or naive state. Right: OCT4, SOX2, NANOG, and TFCP2L1 were examined by qRT-PCR in the indicated hESCs. Significance level was determined using unpaired two-tailed Student’s t tests. **, P < 0.01. The data represent mean ± SD from three independent repeats. Scale bar, 100 μm. g The model for the requirement of PRC2 in primed and naive ESCs. See also Supplementary Fig. 7