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 complexes (PRCs) formed by polycomb group proteins play essential roles in development by mediating chromatin modification1,2,3,4,5. The polycomb repressive complex 2 (PRC2 complex) catalyzes histone H3 lysine 27 tri-methylation (H3K27me3) through its core components EZH1, EZH2, EED and SUZ126,7,8,9,10. In contrast, PRC1 contains RING1A and RING1B, E3 ubiquitin ligases that mono-ubiquitinylate histone H2A at lysine 119 (H2AK119ub1)11, 12. PRC1 and PRC2 coordinately mediate transcriptional repression through H3K27me3 modification. PRC2 is recruited to specific genomic locations and catalyzes deposition of H3K27me3, which in turn recruits PRC1, thus resulting in generation of H2AK119ub113,14,15. Whole-genome studies have revealed that PRC2 and its mark H3K27me3 occupy critical developmental genes in both human and mouse embryonic stem cells (ESCs)2, 3. Paradoxically, most genes occupied by H3K27me3 are also modified by H3 lysine 4 tri-methylation (H3K4me3)16,17,18, thus marking these loci with bivalent modifications to keep lineage genes in a poised state capable of responding rapidly to differentiation cues. Furthermore, these bivalent modifications are rapidly resolved during lineage specification to ensure the proper expression of lineage-specific genes19,20,21.
Loss-of-function studies on individual components of PRC2 have been performed and have been reported in Drosophila and mice10, 22,23,24,25,26. Deletion of three core PRC2 components (Eed, Suz12 and Ezh2) in mice results in severe defect in gastrulation23, 25, 27, 28, presumably because of the mis-expression of lineage-specific genes24, 25, 29, 30. Interestingly, deletion of PRC2 in mouse ESCs exhibits quite different phenotypes. For example, mouse ESCs (mESCs) with Suz12, Eed or Ezh2 deletion appear to be normal with little effect on self-renewal and morphology6, 7, 31,32,33. Transcriptionally, only a small subset of PRC2 target genes are affected in those mESCs. However, EZH2 −/− human ESCs (hESCs) have severe defects in self-renewal and differentiation34. Therefore, although PRC1 and PRC2 have been proposed to play critical roles in embryonic development, their exact roles in different pluripotent states (naive vs. primed) and in the differentiation toward three germ layers remain unresolved. In this report, we generated a panel of hESC lines with deletion of EZH1/EZH2, EED and SUZ12 and found that these cells underwent spontaneous differentiation to the meso-endoderm germ layers at the expense of the neural ectoderm. Furthermore, we found that PRC2 is required for maintaining pluripotency in only the primed state but not in the naive state.
PRC2 is required for pluripotency in hESCs
To gain insights into the role of PRC2 in cell fate decisions, we generated EED-, SUZ12-, EZH1- and EZH2 -knockout hESC lines by using CRISPR/Cas9 genome editing combined with homologous recombination (Fig. 1a)35,36,37. To ensure that the function of each PRC2 component was completely eliminated, we designed a targeting strategy to ablate the entire functional domain critical for each factor (Fig. 1a, b)7, 31, 38. We also included multiple hESC lines in our analysis to rule out the possible variations between cell lines (Fig. 1b). gRNA/Cas9 and the designed targeting vector were electroporated into H1 or H9 hESCs, and the positive clones were subsequently selected by neomycin or puromycin under defined culture conditions (Fig. 1b). Individual surviving colonies after drug selection were then isolated, expanded and analyzed by genomic PCR using different primer combinations shown in Fig. 1a. Using CRISPR/Cas9 combined with homologous recombination, we obtained a significantly high efficiency of homozygous gene targeting, varying from 10–40% across genes or cell lines (Fig. 1b). Thus, we successfully obtained multiple hESC clones with homozygous deletions of each individual core component of PRC2 (Fig. 1c, d, Supplementary Fig. 1a–e). To prevent potential batch variation between different hESC clones, we used at least two clones with homozygous targeting for each gene for further characterization.
We first observed that hESCs with deletion of different PRC2 components exhibited distinct morphology (Fig. 1c, d, Supplementary Fig. 1c). Whereas EZH1 −/− H1 or EZH2 −/− H1 hESCs remained undifferentiated and morphologically indistinguishable from wild-type (WT) H1 cells (Fig. 1c), EED −/− or SUZ12 −/− hESCs (H1 or H9 cells) exhibited a gradual and spontaneous differentiation phenotype (Fig. 1c, Supplementary Fig. 1c). Because EZH1 and EZH2 are functionally redundant7, 39, we performed an additional round of gene targeting in EZH1 −/− H1 or EZH2 −/− H1 cells by removing the antibiotic cassette removed by using Cre-LoxP (see Methods) to generate hESCs with double deletion of EZH1 and EZH2 (EZH1 −/−/EZH2 −/− H1). Regardless of whether the double knockout hESCs were originally generated from EZH1 −/− H1 (hereafter denoted EZH1 −/−/ EZH2 −/− H1) or from EZH2 −/− H1 (denoted EZH2 −/−/EZH1 −/− H1) cells, they exhibited gradual differentiation, as observed in EED −/− H1 or SUZ12 −/− H1 cells (Fig. 1c, Supplementary Fig. 1a, b, g–i). Together, these results suggested that PRC2 is essential for maintaining an undifferentiated state in hESCs.
As expected, H3K27me3 modification on chromatin was completely abolished in EED −/− H1, SUZ12 −/− H1, and EZH2 −/−/EZH1 −/− H1 cells (Fig. 1e). In contrast, EZH1 −/− H1 or EZH2 −/− H1 cells that maintained an undifferentiated state (Fig. 1c) retained their H3K27me3 modifications, albeit at decreased levels compared with those in WT H1 cells (Fig. 1e). Known pluripotency marker genes such as OCT4, SOX2 and NANOG were inactivated in EED −/− H1, SUZ12 −/− H1 and EZH2 −/−/EZH1 −/− H1 cells (Fig. 1f, Supplementary Fig. 1f, j). These results demonstrated that PRC2-mediated H3K27me3 modification is required for maintaining hESCs in a pluripotent state.
PRC2−/− hESCs differentiated to default meso-endoderm fate
During gene targeting, hESCs with homozygous deletion of EED or SUZ12 or double deletion of both EZH2 and EZH1 were isolated and further cultured under defined conditions suitable for hPSCs. However, these cells subsequently underwent spontaneous differentiation, as indicated by the loss of typical hESC morphology and alkaline phosphatase (ALP) activity (Fig. 2a, Supplementary Fig. 2a). After examining the markers for the three germ layers using qRT-PCR, we found that these cell lines consistently expressed high levels of meso-endoderm genes but not neural ectoderm genes (Fig. 2b, Supplementary Fig. 2b). As controls, H1 cell-derived embryonic bodies (EBs) expressed genes corresponding to all selected lineages from the three germ layers (Fig. 2b). To further confirm the lineage fate of these differentiated cells, we performed whole-genome transcriptome analysis on EED −/− H1, SUZ12 −/− H1 or EZH2 −/−/EZH1 −/− H1 hESCs as well as EZH1 −/− H1 and EZH2 −/− H1 hESCs. Spearman’s rank correlation analysis on the global transcriptome clearly showed a more closely related differentiation phenotype among EED −/− H1, SUZ12 −/− H1 and EZH2 −/−/EZH1 −/− H1 hESCs, as compared with that of the WT H1 cells (Fig. 2c, Supplementary Fig. 2c). Moreover, EZH1 −/− H1 or EZH2 −/− H1 cells were similar to the undifferentiated H1 hESCs (Fig. 2c, Supplementary Fig. 2c). Furthermore, EED −/− H1, SUZ12 −/− H1 or EZH2 −/−/EZH1 −/− H1 hESCs did not express the selected marker genes indicative of pluripotency or neural ectoderm lineage (Fig. 2d)40, 41. These data suggested that disruption of PRC2 in hESCs leads to a default differentiation toward the meso-endoderm fate. Indeed, two well-known early mesoderm markers, (CALPONIN) and endoderm (SOX17), were detected at high levels by immunostaining in EED −/− H1, SUZ12 −/− H1 or EZH2 −/−/EZH1 −/− H1 hESCs but not in WT, EZH1 −/− or EZH2 −/− H1 hESCs (Fig. 2e, Supplementary Fig. 2d–f). Together, these data demonstrated that PRC2 regulates the loss of pluripotency by suppressing the meso-endoderm differentiation program.
EZH1 and EZH2 specify early neural ectoderm fate
hESCs with single deletion of EZH1 or EZH2 stayed in an undifferentiated state but had decreased levels of H3K27me3 modifications (Fig. 1c, e). Therefore, EZH1 −/− or EZH2 −/− H1 hESCs provide a good model to examine the role of PRC2 components in later lineage specifications. We first showed that EZH1 −/− or EZH2 −/− H1 hESCs cultured under defined conditions that support hPSCs retain typical pluripotency characteristics, as demonstrated by ALP staining, expression of well-known hESC markers, typical cell-cycle distribution for hESCs and low expression levels of differentiation markers (Fig. 3a–d, Supplementary Fig. 3a). To examine the differentiation potential of these cells, we injected EZH1 −/− or EZH2 −/− H1 hESCs into immuno-deficient mice and monitored teratoma formation. We observed normal teratoma formation for EZH1 −/− or EZH2 −/− H1 hESCs in all 3 injected mice. However, on the basis of H&E staining of the teratoma sections, we did not observe any ectoderm tissue from teratomas formed from EZH1 −/− or EZH2 −/− H1 cells (Fig. 3e, Supplementary Fig. 3b). In contrast, typical mesoderm and endoderm tissues were readily detected in EZH1 −/− or EZH2 −/− H1 teratomas and were similar to those in WT H1 hESC teratomas (Fig. 3e, Supplementary Fig. 3b). These data indicated that hESCs lacking EZH1 or EZH2 completely fail to specify neural ectoderm lineages in vivo. We then performed directed in vitro differentiation of EZH1 −/− or EZH2 −/− H1 cells into specific lineages representing the three embryonic germ layers. For neural ectoderm, we induced neural differentiation of hESCs through a well-established and efficient protocol based on dual-SMAD inhibition40. Through dual SMAD inhibition, WT H1 hESCs were efficiently converted into neural progenitor cells (NPCs) expressing known NPC markers and forming typical neural spheres in suspension (Fig. 3f). In contrast, EZH1 −/− or EZH2 −/− H1 hESCs differentiated into only epithelial-like cells without expressing any neural lineage markers and forming neural spheres (Fig. 3f). For mesoderm, we induced differentiation of blood cells by using a published protocol with stromal cell-free condition42. Similar percentages of CD34+ hematopoietic progenitor cells were obtained from the differentiation of both WT and EZH1 −/− or EZH2 −/− H1 hESCs through cytokine treatment (Fig. 3g)42. In addition, the known early and late mesoderm or blood lineage marker genes were also successfully induced in EZH1 −/− or EZH2 −/− H1 hESCs (Fig. 3g). Then, we demonstrated very similar endoderm differentiation between WT and EZH1 −/− or EZH2 −/− H1 hESCs, on the basis of the expression of SOX17 and several other endoderm markers (Fig. 3h), by using a previously published protocol43, 44. Together, our data demonstrate that EZH1 and EZH2 are required to specify the neural ectoderm lineage in hESCs but is dispensable for mesoderm or endoderm lineage.
PRC2 deletion preferentially induces BMP signalling in hESCs
Because the disruption of PRC2 in hESCs resulted in spontaneous differentiation, we then sought to investigate the underlying molecular events that drive differentiation. Because hESCs with PRC2 disruption (EED −/− H1, SUZ12 −/− H1 or EZH2 −/−/EZH1 −/− H1 hESCs) undergo differentiation and cannot be maintained in vitro, we designed an inducible system to rescue the self-renewal capacity of hESCs with PRC2 disruption (Fig. 4a, b). We first introduced an inducible system to over-express (OE) EED in hESCs and subsequently performed gene targeting to knockout the endogenous EED in these cells (Fig. 4a, b, Supplementary Fig. 4a) (see “Methods” section)45. This hESC line is referred to as H1-EED −/−/EED-OE. The EED level and PRC2 are maintained by DOX treatment during regular cell passaging (Fig. 4d). In the presence of DOX, H1-EED −/−/EED-OE maintained an undifferentiated state and could be normally passaged under defined conditions that support hPSCs (Fig. 4c). However, they underwent gradual and spontaneous differentiation after withdrawal of DOX, despite being kept in hESC medium containing high concentrations of FGF2 to support self-renewal46, 47. After DOX withdrawal, H1-EED −/−/EED-OE began to morphologically differentiate at day 16 and became fully differentiated at day 20 (Fig. 4c). Moreover, the pluripotency marker genes such as OCT4, SOX2 and NANOG were fully repressed at later stages of DOX withdrawal (Fig. 4e). In agreement with the data shown in Fig. 2, the meso-endoderm genes but not neural ectoderm genes were activated at later stages of DOX withdrawal (Fig. 4f). We then performed whole-genome transcriptome analysis on H1-EED −/−/EED-OE at different time points after DOX withdrawal. Spearman’s rank correlation analysis on the whole transcriptome clearly showed a gradual differentiation of H1-EED −/−/EED-OE after DOX withdrawal (Fig. 4g). Again, on the basis of the transcriptome data, meso-endoderm genes, but not ectoderm genes, were gradually activated during the time course of DOX withdrawal (Fig. 4h). The differentiation of H1-EED −/−/EED-OE into the meso-endoderm fate was further confirmed by immunostaining for marker genes specific for the three embryonic germ layers (Supplementary Fig. 4b). In searching for pathways that were responsible for the differentiation caused by PRC2 disruption, we identified that TGF-β/BMP signalling factors were clearly up-regulated at very early stages of DOX withdrawal (Fig. 4i, Supplementary Fig. 4c–f). BMPs and related factors such as BMP2, BMP4, BMP7, GDF6 and ID2 began to increase early at day 8 even when no obvious differentiation had been detected on the basis of morphology and the transcriptome data (Fig. 4c, g). Together, these data demonstrated that disruption of PRC2 in hESCs preferentially induces BMP signaling at early stage.
Inhibition of BMP signalling rescues PRC2 deficiency in hESCs
We then sought to examine whether the early induction of BMP signalling after PRC2 disruption might be the major reason for the differentiation of hESCs (Fig. 5a). To test this hypothesis, in addition to H1-EED −/−/EED-OE, we also prepared additional inducible systems to rescue the functional loss of other PRC2 components, for example, EZH1 or EZH2. Similarly, we first introduced the inducible system to over-express EZH1 or EZH2 in EZH2 −/− or EZH1 −/− H1 hESCs and then deleted endogenous EZH1 or EZH2 in the same cell lines (Fig. 5a, Supplementary Fig. 4a). These cells, denoted H1-EZH2 −/−/EZH1 −/−/EZH2-OE or H1-EZH2 −/−/EZH1 −/−/EZH1-OE, maintained a normal undifferentiated state in the presence of DOX (Fig. 5b, Supplementary Fig. 5a–d). As expected, after withdrawal of DOX, H1-EZH2 −/−/EZH1 −/−/EZH2-OE, H1-EZH2 −/−/EZH1 −/−/EZH1-OE and H1-EED −/−/EED-OE all displayed complete differentiation at 20 or more days of culture (Fig. 5b). However, the differentiation was blocked by treatment with dorsomorphin (DM), a BMP inhibitor (Fig. 5b)48. As a control, another compound, SB431542, that specifically inhibits TGF-β/Nodal signalling had no effect on the differentiation process (Fig. 5b)40. Moreover, other reported BMP inhibitors such as DMH149 and LDN19318949, 50 also completely blocked differentiation after DOX withdrawal in H1-EZH2 −/−/EZH1 −/−/EZH2-OE (Supplementary Fig. 6d). Further, using qRT-PCR, immunostaining and ALP staining (Fig. 5c, d, and Supplementary Fig. 6a–f), we confirmed that whereas OCT4 was down-regulated and the meso-endoderm genes SOX17 and CALPONIN were upregulated after DOX withdrawal, BMP inhibitor treatment reversed these phenotypes in all three examined hESC lines (Fig. 5c, d). These data together demonstrate that PRC2 regulates the loss of pluripotency by suppressing BMP signalling.
PRC2 is required for pluripotency in primed not naive state
The apparent discrepancy between our results and those reported in mESCs may reflect a differential requirement of PRC2 in naive and primed states51,52,53,54,55. To test this possibility, we generated PRC2 component gene knockout cell lines in conical OG2 mESCs with GFP controlled by the Oct4 promoter (Oct4: GFP)56. Two critical PRC2 genes, Suz12 and Eed, were selected for gene targeting in mESCs (Fig. 6a, Supplementary Fig. 7a–e). As expected, and in agreement with previous reports, mESCs deficient in Suz12 (mESCs-Suz12 −/−) or Eed (mESCs-Eed −/−) exhibited a normal phenotype and expressed Oct4 when they were maintained under typical conditions for mESC growth (see Methods) (Fig. 6a, Supplementary Fig. 7d) despite the upregulation of Bmp4 (Fig. 6c). However, when converted into the primed state through a well-established protocol52, mESCs-Suz12 −/− and mESCs-Eed −/− but not WT mESCs displayed a gradual and spontaneous differentiation, as indicated by morphology changes and a loss of Oct4: GFP (Fig. 6a, b). The differentiation phenotype was further confirmed by the downregulation of pluripotency marker genes and upregulation of differentiation genes and TGF-β/BMPs signalling factors in mESCs-Suz12 −/− and mESCs-Eed −/− (Fig. 6c, Supplementary Fig. 7f). These results suggested that PRC2 is dispensable for maintaining pluripotency in naive but is required for primed mouse ESCs.
Additionally, hESCs are regarded as being in a primed state that can be converted to a naive state by over-expressing NANOG and KLF2 or switching to a medium containing specific growth factors and small molecules57, 58. To test whether PRC2 functions similarly in naive and primed states in human cells, we first knocked down EZH1 by shRNA in H1-EZH2 −/− hESCs, thus resulting in an expected differentiation when the cells were in a primed state (Fig. 6d, e, Supplementary Fig. 6g, h). However, when the cells were converted to a naive state by over-expression of NANOG/KLF2, H1-EZH2 −/− hESCs became resistant to differentiation after EZH1 knockdown (Fig. 6d, e, Supplementary Fig. 6g, h)57. Furthermore, pluripotency marker genes such as OCT4, SOX2, NANOG were well maintained in the converted naive state of H1-EZH2 −/− hESCs, whereas the same genes were significantly downregulated in H1-EZH2 −/− hESCs after EHZ1 knockdown in an “unconverted” primed state (Fig. 6e, Supplementary Fig. 6h). These data suggested that in human pluripotent stem cells, PRC2 is also required for pluripotency in the primed state and not the naive state. To further confirm whether this phenomenon is a general mechanism of PRC2 function in naive pluripotency, we also examined other reported protocols to obtain hESCs in a naive state58. As reported58, we converted WT H1 cells, H1-EED −/−/EED-OE or H1-SUZ12 −/−/SUZ12-OE cells into naive state by switching them to medium containing hLIF/ACTIVIN A plus 5 small molecules (5i/L/A), as demonstrated by the up-regulation of marker genes of naive pluripotency (Fig. 6f, Supplementary Fig. 6i). In agreement with the data above, after DOX withdrawal, H1-EED −/−/EED-OE or H1-SUZ12 −/−/SUZ12-OE maintained in a primed state underwent differentiation (Fig. 6f, Supplementary Fig. 6i). In contrast, H1-EED −/−/EED-OE or H1-SUZ12 −/−/SUZ12-OE in naive state maintained their undifferentiated phenotype, as indicated by morphology and marker gene expression (Fig. 6f, Supplementary Fig. 6i). Together, our data demonstrate that PRC2 is required for pluripotency in primed but not naive ESCs.
PRC complexes are essential regulators of cell lineage decisions during development in different species, such as Drosophila and mice10, 24,25,26, 59. At the molecular level, PRCs repress gene expression by mediating histone modifications such as H3K27me3 deposition and through other epigenetic mechanisms11, 14, 33. Mutations in each individual core component such as Eed, Suz12 and Ezh2 in mice result in early lethality due to gastrulation defects23, 25, 27. However, the molecular mechanisms underlying the gastrulation defects caused by PRC2 deficiency have not been fully elucidated. In this report, we showed that PRC2-deficient hESCs undergo spontaneous differentiation toward the meso-endoderm germ layers without neural ectoderm, thus suggesting that PRC2 is required for the specification of the ectoderm fate at early stages of differentiation. Furthermore, our data demonstrated that PRC2 is required for maintaining pluripotency in a primed state but is dispensable in the naive state. Therefore, our analyses provide more insights into the understanding of cell fate decisions mediated by PRC complexes during lineage specification at very early stages of development (Fig. 6g).
PRC2 is part of a large network of chromatin regulators that cooperatively govern the genomic architecture. In this work, we sought to begin to understand their exact roles in the dynamic regulation of cell fate decisions. For example, the PRC2-driven H3K27me3 landscape together with H3K4me3 is considered to be critical in repressing lineage genes in a “poised” state that can respond quickly to the differentiation stimuli for lineage commitment16, 17, 60, 61. However, our data revealed a specific role of PRC2 in lineage specification as well as their differential requirement in maintaining naive or primed state of pluripotency. Thus, it is not clear how the epigenetic state such as the “poised state” plays a critical role during cell fate decisions into specific lineages. To answer that question, a similar approach as ours could be used to investigate the mediators of H3K4me3 deposition. Given the preliminary data on the role of PRC2 in lineage and cell fate determination, we were particularly surprised by the degree of its specificity toward the ectoderm lineage through the suppression of the meso-endoderm. This phenotype indicates the possibility that signalling pathways and developmental factors for the meso-endoderm lineage are preferentially de-repressed after PRC2 disruption. Indeed, we showed that BMP signalling plays a critical role in this process. Mechanistically, more work is needed to further delineate PRC2 and BMP pathways.
H3K27me3 modifications were completely abolished after deletion of PRC2 components. These data indicated that the roles of PRC2 and H3K27me3 in regulating lineage specification is specific and not as broad as previously thought23, 31, 34, 62. Interestingly, hESCs with single deletion of EZH1 or EZH2 maintained a certain level of H3K27me3 and were in a typical undifferentiated state, in agreement with functional redundancy between EZH1 and EZH2 in catalyzing histone modifications7, 39. However, these cells were completely defective in the generation of neural ectoderm lineages, but the specifications of other lineages remained unaffected (Fig. 3). The detailed molecular mechanisms underlying neural ectoderm defect in H1-EZH1 −/− or H1-EZH2 −/− cells remain unknown. One possibility is that the decreased level of H3K27me3 in H1-EZH1 −/− or H1-EZH2 −/− failed to fully repress BMPs that might dominantly switch the cell fate to meso-endoderm lineages during differentiation. The detailed molecular mechanisms underlying neural ectoderm defect in H1-EZH1 −/− or H1-EZH2 −/− cells require further investigation.
We initially encountered an intriguing dichotomy for PRC2 disruption in hESCs and mESCs. We observed a clear differentiation phenotype in hESCs with deletion of each core component of PRC2, but mESCs-Suz12 −/− or mESCs-Eed −/− maintained a relatively normal undifferentiated phenotype. hESCs and mESCs have been considered to represent different states of pluripotency, primed vs. naive53,54,55. Interestingly, after conversion into a primed state, mESCs-Suz12 −/− or mESCs-Eed −/− clearly exhibited spontaneous differentiation, as observed in hESCs (Fig. 6)52. Moreover, disruption of PRC2 in “naive” hESCs that were generated in vitro resulted in substantially less impairment of pluripotency (Fig. 6)57, 58. Therefore, PRC2 is differentially required for maintaining pluripotency for cells in different states, i.e., it indispensable for the primed state but unnecessary for the naive state. The molecular mechanisms of how PRC complexes maintain the cellular identity of naive and primed PSCs remain to be fully elucidated. Nonetheless, the differential requirement of PRC2 may serve as a molecular signature for distinguishing naive vs. primed states in hESCs.
Human ESC lines H1 (Wi Cell), H9 (Wi Cell) and knockout cell lines were maintained in mTeSR1 (STEMCELL Technologies) on matrigel (Corning)-coated plates. Mouse ESC cell line OG2 with GFP controlled by Oct4 promoter, was kindly provided by Dr. Jiekai Chen. OG2 mESCs and knockout mESCs based on OG2 mESCs were maintained on feeder layers in mESC + 2iL medium (DMEM/ high glucose (Hyclone), 15% FBS (Gibco), NEAA (Gibco, 100×), GlutaMAX (Gibco, 100×), Sodium Pyruvate (Gibco, 100×), 1 μM PD0325901 (Selleck), 3 μM CHIR99021 (Selleck), 1000 units/mL mLIF). OG2 mESCs and knockout mESCs based on OG2 mESCs were maintained on gelatin (Millipore)-coated plate in mouse N2B27 + 2iL medium (50% DMEM/High glucose (Hyclone), 50% Knockout DMEM (Gibco), N2 (Gibco, 200×) + B27 (Gibco, 100×), NEAA (Gibco, 100×), GlutaMAX (Gibco, 100×), Sodium Pyruvate (Gibco, 100×), 1 μM PD0325901 (Selleck), 3 μM CHIR99021 (Selleck), 100 μM β-mercaptoethanol (Gibco), 1000 units mL−1 mLIF). All cell types were maintained at 5% CO2.
Gene knockout in human and mouse ESCs
pX330 (Addgene) can express Cas9 protein and guide RNA. Guide RNAs (gRNAs) for EZH1, EZH2, EED, SUZ12, mouse Suz12 and Eed were designed on the website (crispr.mit.edu)37. Donor DNAs of these genes containing left and right homology arms, a LoxP-flanked PGK-puromycin cassette or a LoxP-flanked PGK-neomycin cassette, and they were used for targeting. For targeting, 1 × 106 hESCs were electroporated with 2 μg of donor DNA and 4 μg of pX330 plasmid containing gRNA for each gene respectively. Then the electroporated hES cells were plated onto matrigel-coated six-well plates with Y-27632 (10 μM, Sigma) for 1 day. Positive clones were selected by puromycin (1 μg mL−1, Gibco) or G418 (100 μg mL−1, Sigma) in mTeSR1. To get double deletion of EZH1 and EZH2, the antibiotic cassettes in H1-EZH1 −/− or H1-EZH2 −/− were deleted using Cre-LoxP system. 1 × 106 hESCs (H1-EZH1 −/− or H1-EZH2 −/−) were electroporated with 400 ng of Cre mRNA, respectively. Then 500 electroporated hES cells were plated onto matrigel-coated six-well plates with Y-27632 (10 μM, Sigma) for 2 days. After deletion of antibiotic cassette in H1-EZH1 −/− or H1-EZH2 −/−, the protocol of knockout EZH2 based on H1-EZH1 −/− or knockout EZH1 based on H1-EZH2 −/− followed previous procedure. For deletion of mouse Suz12 or Eed, 1 × 106 mESCs were electroporated with 2 μg of donor DNA and 4 μg of pX330 plasmid containning gRNA for Suz12 or Eed. Then the electroporated mES cells were plated onto feeder in mESC + 2iL medium. Positive clones were selected by puromycin (1 μg mL−1, Gibco) or G418 (100 μg mL−1, Sigma) in mESC + 2iL medium. All guide RNA sequences and primer sequences are listed in Supplementary Table 1.
Inducible system for gene knockout in hESCs
In human ES cell line (H1), we firstly induced an inducible over-expression (OE) system45 for over-expressing EED or SUZ12 respectively, hereafter referred as H1-EED-OE, H1-SUZ12-OE. In human knockout cell line H1-EZH2 −/− or H1-EZH1 −/−, we induced an inducible over-expression (OE) system for over-expressing EZH2 or EZH1 respectively, hereafter referred as H1-EZH2 −/−/EZH2-OE or H1-EZH1 −/−/EZH1-OE. These cells were selected with 2 μg mL−1 doxycycline (DOX) and puromycin. And then endogenous core components of PRC2 were deleted in these inducible OE cell lines respectively, For targeting, 1 × 106 hESCs were electroporated with 2 μg of donor DNA and 4 μg of pX330 plasmid containing gRNA for each gene respectively. Then the electroporated hES cells were plated onto matrigel-coated 6-well plates with Y-27632 (10 μM, Sigma) for 1 day. Positive clones were selected by puromycin (1 μg mL−1, Gibco) in mTeSR1. And we selected positive clones using puromycin for 3 days, and then these clones were picked and cultured in mTeSR1 plus 2 μg mL−1 doxycycline (DOX) on matrigel-coated plates. All primer sequences are listed in Supplementary Table 2.
PCR detection of knockout cell clones
Genomic DNA of knockout cell clones was extracted with TIANamp Genomic DNA Kit (Tiangen) for PCR analysis. 30–50 ng of genomic DNA templates and KOD PLUS (Toyobo) were used in all PCR reactions. Primer set of each gene including F1 and R1 was used to amplify a 2.5–3.2 Kb product of the targeted integration. Primer set of each gene including F2 and R2 was used to amplify a 2.1–2.8 Kb product product to identify whether random integration occurred. All primers sequences are listed in Supplementary Table 1.
Western blot analysis
To detect knockout efficiency of these genes and the methylation of H3K27 and H3K4, cells were lysed on ice in RIPA buffer (Beyotime). Whole-cell extracts were resolved by 10% SDS-PAGE to get knockout efficiency of these genes and by 15% SDS-PAGE to get the methylation of H3K27 and H3K4, and then transferred to PVDF membranes (Millipore), and incubated with primary antibodies over-night at 4 °C. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies. After the membranes were washed in TBST, HRP was detected by ECL (Beyotime) and visualized by SmartChemi Image Analysis System (Sagecreation). The antibodies were used according to the manufacturer’s recommendations. All uncropped western blots can be found in Supplementary Figs. 8 and 9. The information for antibodies used is listed in Supplementary Table 3.
Flow cytometry analysis
The cells were trypsined for single cells with 0.25% trypsin-EDTA (Gibco) and fixed with 1% paraformaldehyde for about 20 min at room temperature, washed twice with 2% fetal bovine serum (FBS, Natocor) in PBS. And then cells were permeabilizated with 90% methanol for 30 min at 4 °C. After washed, cells were incubated with primary antibodies and isotype control antibodies for 30 min at 37 °C. After washed, cells were incubated with secondary antibodies for 30 min at 37 °C. The cells were washed twice and resuspended in 200 μL PBS, and then analyzed with Accuri C6 (BD Biosciences). The information for antibodies is listed in Supplementary Table 3.
Quantitative real-time PCR
Total RNA was extracted with Trizol (Invitrogen), and reverse transcribed with oligo dT (Takara) and RT ACE (Toyobo), and then Quantitative real-time PCR qPCR was performed with CFX96 machine (BIO-RAD) and SsoAdvanced SYBR Green Supermix (BIO-RAD) following the manufacturer’s recommendations. GAPDH was used for quantitative real-time PCR (qRT-PCR) normalization of human sample, and Gapdh was used for qRT-PCR normalization of mouse sample. All the data were measured in three repeats. All primer sequences are listed in Supplementary Table 2.
Alkaline phosphatase staining
The cells were plated on matirgel-coated 6-well plates for alkaline phosphatase (ALP) assay. The cells were fixed with 4% paraformaldehyde for 20 min at room temperature. After washed thrice with 1 × TBST, the cells were processed with ALP buffer (Beyotime) for 5 min. And then, the sample were added with BCIP (Beyotime) and NBT (Beyotime) for 15 min. The usage of regents was followed with the manufacturer’s recommendations (Beyotime).
Undifferentiated and differentiated ESCs were seeded onto matrigel-coated 24-well plates. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, washed thrice with PBS for 5 min each time. Cells were permeabilized with 0.3% triton X-100 (sigma) and 10% goat serum in PBS, meanwhile cells were incubated with corresponding primary antibodies overnight at 4 °C. The cells were washed thrice with PBS for 5 min each time. And then, cells were incubated with secondary antibodies for 1 h at room temperature. The cells were washed thrice with PBS for 5 min each time. The cells were stained with DAPI (Sigma) for 5 min at room temperature. Images were captured with Leica DMI6000B microscope (Leica Microsystems, GmbH). The information for antibodies is listed in Supplementary Table 3.
Teratoma formation and analysis
The experiments involving animal research for teratomas formation had been reviewed and approved by IACUC at GIBH (NO. 2010012). The size of tumour growth that was acceptable for ethical approval was about 2.5 × 2.5 cm. In our experiments, EZH1 −/−, EZH2 −/− and wild-type H1 hES cells that were cultured on matrigel-coated 6-well plates were digested by Accuatse (Sigma) for 8 min at 37 °C and resuspended in 30% matrigel (Corning) in DMEM/F12 (Hyclone), and then injected subcutaneously into immuno-deficient mice. We used that the age of mice were about 4 weeks, and the sex of mice were both male and female, and strain of mice were NOD-SCID mice. Teratomas were detected after 8 weeks and fixed in 4% paraformaldehyde, and then stained with hematoxylin/eosin (H&E). The size of tumour growth was about 2 × 2 cm.
Neural progenitor cells differentiation
To initiate neural differentiation, hESC were plated onto matrigel-coated 12-well plates with 95–100% of cell confluence, and then these cells were cultured in N2B27 medium plus SB431542 and Dorsomorphin (50% DMEM/F12 (Hyclone), 50% Neurobasal (Gibco), N2 (Gibco, 200×), B27 (Gibco, 100×), Glutamax (Gibco, 200×), NEAA (Gibco, 200×), 5 μg mL−1 insulin(Gibco, 200×), 1 μg mL−1 heparin (Sigma), 5 μM SB431542 (Selleck), 5 μM Dorsomorphin(DM, Selleck))40. Every 2 days changing fresh culture medium. After 8 days, the cells were passaged on matrigel-coated 6-well plates in N2B27 medium. After 16 days, canonical neural rossettes of wild-type hESCs appeared and these cells were suspended for neural sphere formation.
Hematopoietic progenitor cells differentiation
To initiate hematopoietic differentiation42, the hESCs were passaged with dispase (2 mg mL−1) onto matrigel-coated 12-well plates. These cells were cultured in E6 medium46 plus ACTIVIN A and BMP4 (DMEM/F12 (Hyclone), 64 mg L−1 Lascorbic acid (Sigma), NaCl (Sigma, adjusting the osmolarity to 340 mOsm), ITS –G (Gibco, 100×) and 50 ng mL−1 ACTIVIN A (Sino biological, 10429-HNAH-50), 50 ng mL−1 BMP4 (Peprotech, 120-05ET)) for 2 days. And then cells were cultured in E6 medium plus 40 ng mL−1 VEGF (Sino biological, 10008-HNAB-50) and 50 ng mL−1 bFGF (Sino biological, 10014-HNAE-50) for next two days. For next 3 days, these cells were cultured in E6 medium plus 40 ng mL−1 VEGF, 50 ng mL−1 bFGF, 10 μM SB431542 (Selleck). After 7 days, these cells were collected for extracting total RNA and FACS analysis for CD34 expression. CD34 antibody conjugated with PerCP-Cy5.5 were used according to the manufacturer’s recommendations.
Definitive endoderm cells differentiation
To initiate definitive endoderm cells (DE cells) differentiation43, 44, hESCs were cultured for 3 days in RPMI1640 (Gibco) /B27 medium (Insulin minus, Gibco) and 100 ng mL−1 Activin A (Peprotech) on matrigel-coated 24-well plate. After 3 days, these cells were collected for extracting total RNA and immunostaining for SOX17 expression.
Conversion of primed mouse ESCs
To induce naive mouse ESCs into primed mouse ESCs (mEpiSCs), conversion of WT, Suz12 −/−, Eed −/−mESCs into the primed state according to Qilong Ying’s paper52. 3 × 105 mESCs were seeded on gelatin-coated 6-well plate and cultured in mouse N2B27 + 2iL medium for day. And then these cells were cultured in switched medium (50% DMEM/F12 (Hyclone), 50% Neurobasal (Gibco), N2 (Gibco, 200×), B27 (Gibco, 100×), Glutamax (Gibco, 100×), NEAA (Gibco, 100×), 100 μM β-mercaptoethanol (gibco), 3 μM CHIR99021 (Selleck), 2 μM XAV939 (Sigma), 10 ng mL−1 Activin A (Peprotech), 10 ng mL−1 FGF2 (R&D systems)) for 5 days. And then WT mESCs maintained typical primed state.
Conversion of naive hESCs
To induce primed hESCs into naive hESCs, we used methods that have been reported by Austin Smith and Rudolf Jaenisch57, 58. At first, conversion of WT or H1-EZH2 −/− hESCs into the naive state according to Austin Smith’s paper. NANOG and KLF2 were overexpressed in hESCs though lentiviral-based inducible vectors. hESCs with NANOG/KLF2 expression were further cultured in switched medium (human N2B27 + 2iL medium: 50% DMEM/F12 (Hyclone), 50% Neurobasal (Gibco), N2 (Gibco, 200×), B27 (Gibco, 100×), Glutamax (Gibco, 200×), NEAA (Gibco, 200×), Sodium Pyruvate (Gibco, 100×), 1 μM PD0325901 (Selleck), 3 μM CHIR99021 (Selleck), 100 μM β-mercaptoethanol (gibco), 1000 units mL−1 mLIF) in the presence of DOX induced transgene expression. These cells were passaged for every week, and cultured on feeder in N2B27 + 2iL medium plus DOX. At about 3–4 passages, these cells maintained typical naive state and expressed the naive marker TFCP2L1. And then these naive cells were maintained on feeder in N2B27 + 2iL medium plus 2 μM + Gö6983.
Secondly, conversion of WT or H1-EED −/−/EED-OE or H1-SUZ12 −/−/SUZ12-OE hESCs into the naive state according to Rudolf Jaenisch’s paper. 2 × 105 WT hESCs were seeded on feeder and cultured in KSR medium (80% DMEM/F12 (Hyclone), 15% FBS (Hyclone), 5% KSR (Gibco), Glutamax (Gibco, 200×), NEAA (Gibco, 200×), 100 μM β-mercaptoethanol (gibco), and 4 ng mL−1 FGF2 (R&D systems), 10 μM Y-27632 (Sigma)) for 4 days. 2 × 105 H1-EED −/−/EED-OE or H1-SUZ12 −/−/SUZ12-OE were seeded on feeder and cultured in KSR medium plus DOX for 4 days. Then these cells were trypsinized for single-cell dissociation and cultured on feeder in 5i/L/A medium (50% DMEM/F12 (Hyclone), 50% Neurobasal (Gibco), N2 (Gibco, 200×), B27 (Gibco, 100×), Glutamax (Gibco, 200×), NEAA (Gibco, 200×), 50 μg mL−1 BSA (Sigma), 100 μM β-mercaptoethanol (gibco), 1 μM PD0325901 (Selleck), 1 μM IM-12 (Enzo), 0.5 μM SB590885 (R&D systems), 1 μM WH-4-023 (A Chemtek), 10 μM Y-27632 (Sigma), 20 ng mL−1 hLIF, 20 ng mL−1 Activin A (Peprotech)) for 10 days. We converted H1-EED −−/EED-OE or H1-SUZ12 −/−/SUZ12-OE hESCs to naive state in the presence or absence of DOX. And then these cells maintained typical naive state. These cells were passaged for every week, and cultured on feeder in 5i/L/A medium.
RNA-seq and spearman’s rank correlation and heatmap analysis
After the digestion of cultured cells, WT hESCs and the target cells were collected and lysed with 400 μL Trizol (Invitrogen)45. Total RNA was extracted using a Directzol RNA MiniPrep kit (Zymo Research) and sequencing libraries was established using a TruSeq RNA Sample Preparation Kits v2 (48 samples) (Illumina) according to the manufacturer’s protocol. The samples were run on an NextSeq system with NextSeq 500 Mid Output Kit v2 (150 cycles).
The number of raw reads mapped to human mRNA reference sequence for GRCh38/hg38 using RSEM (rsem-1.2.4)63, Bowtie2 (v2.2.5), and normalized with EDASeq (v2.2.0)64. Gene expression is expressed as “normalized tag count.” Other downstream analyses were performed using glbase65. In brief, differential expression between differentiation state and the ESC control was analyzed using the edgeR package66, where raw read counts per gene were normalized using Trimmed Mean of M-values (TMM). Differentially expressed genes were extracted at the cutoff of false discovery rate (FDR) of <=1% and fold change of > =3. 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. These selected pluripotent genes and lineage genes were analyzed for heatmap, and expression level of genes in H1 hESCs were set as 1 and the fold change (log2) of individual gene were calculated in other cell lines, respectively. Spearman’s correlation coefficients were then computed with TPM values at log2 scale and all correlation coefficients among samples were represented as a heatmap in R.
In general, Results were presented as mean ± SD calculated using Microsoft Excel and GraphPad Prism at least three biological repeats. Significance level between samples was determined using unpaired two-tailed Student’s t tests. P value < 0.05 was considered statistically significant in the figures. No samples were excluded for any analysis.
The RNA-Seq data have been deposited in the Gene Expression Omnibus database under the accession code GSE92625. PCR, qRT-PCR and the RNA-seq data have also been deposited in figshare (http://dx.doi.org/10.6084/m9.figshare.5146789). The authors declare that all the data supporting the findings of this study are available within the article and its supplementary information files or from the corresponding upon reasonable request.
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We thank the lab members in GIBH for their kindly help. This work was supported by the National Key Research and Development Program of China, Stem Cell and Translational Research (2017YFA0102601); the Frontier and Key Technology Innovation Special Grant from the Department of Science and Technology of Guangdong Province (2014B020225006, 2014B020225002, 2014B050504008, 2015B020228003, 2016B030230002, 2016B030229008); the National Basic Research Program of China, 973 Program of China (2015CB964900, 2014CB965200); the National Natural Science Foundation of China (31371514, 31421004, 31500948); Cooperation Grant of Natural Science Foundation of Guangdong Province (2014A030313801, 2014A030312012, 2015A030310229); the Science and Information Technology of Guangzhou Key Project (201508020258, 201506010092, 2015A030310254); the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2015293); International Science & Technology Cooperation Program of China (2014DFA30180); the “Hundred Talents Program” of Chinese Academy of Science (to X.Z.); Science and Technology Planning Project of Guangdong Province, China (2014B030301058), the Guangdong Province Special Program for Elite Scientists in Science and Technology Innovation (to B.L., 2014TQ01R746; to G.P., 2015TX01R203); Natural Science Foundation of Guangdong Province, China (2016A030313167); Science and Technology Planning Project of Guangdong Province, China (2013B050800004).
The authors declare no competing financial interests.
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