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. Author manuscript; available in PMC: 2015 Jan 2.
Published in final edited form as: Cell Stem Cell. 2013 Nov 14;14(1):68–80. doi: 10.1016/j.stem.2013.10.001

Polycomb repressive complex 2 regulates hematopoietic stem cell maintenance and differentiation in a developmental stage-specific manner

Huafeng Xie 1, Jian Xu 1, Jessie H Hsu 1, Minh Nguyen 1, Yuko Fujiwara 1,2, Cong Peng 1, Stuart H Orkin 1,2,*
PMCID: PMC3947409  NIHMSID: NIHMS536972  PMID: 24239285

Summary

Recent studies point to a pivotal role of polycomb repressive complex 2 (PRC2) in stem cell function and cancer. Loss of function approaches targeting individual PRC2 subunits have however generated findings that are difficult to reconcile. Here, we prevent assembly of both Ezh1- and Ezh2-containing PRC2 complexes by conditional deletion of Eed, a core subunit, and assess hematopoiesis. We find that deletion of Eed exhausts adult bone marrow HSCs, although fetal liver HSCs are produced in normal numbers. Eed null neonatal HSCs express HSC signature genes, but are defective in maintenance and differentiation. Comparative gene expression profiling revealed that neonatal and adult HSCs lacking Eed upregulated gene sets of conflicting pathways. Deletion of Cdkn2a, a PRC2 target gene, in Eed null mice enhances HSPC survival but fails to restore HSC functions. Taken together, our findings define developmental stage-specific requirements for canonical PRC2 complexes in normal HSC function.

Introduction

Gene expression and cell fate are controlled by transcription factors acting in concert with epigenetic regulators. Perturbation of epigenetic pathways is associated with altered stem cell function and oncogenesis (Margueron and Reinberg, 2011; Sauvageau and Sauvageau, 2010). Polycomb group (PcG) proteins represent a major class of epigenetic regulators in diverse developmental contexts and participate in transcriptional repression. Polycomb repressive complex 2 (PRC2) consists of 3 core subunits Eed (embryonic ectoderm development), Suz12 (suppressor of zeste 12 homolog) and Ezh2 (enhancer of zeste homolog 2), which possesses histone methyltransferase activity (Kuzmichev et al., 2002; Margueron and Reinberg, 2011). PRC2 is required for proper differentiation of embryonic stem (ES) cells and somatic stem cells, such as skin stem cells (Ezhkova et al., 2011; Ezhkova et al., 2009; Shen et al., 2008). The existence of Ezh1, a homolog of Ezh2, confounds simple interpretation of Ezh2 knockout experiments as Ezh1 associates with Eed and Suz12 to form an alternative PRC2 complex that partially compensates for the loss of Ezh2 in some cell contexts (Ezhkova et al., 2011; Margueron et al., 2008; Shen et al., 2008). Ezh2 expression generally correlates with cell proliferation, whereas Ezh1 expression is higher in non-proliferative adult tissues (Margueron et al., 2008). Although PRC2 was originally identified as a dedicated suppressor associated with the presence of the H3K27me3 mark at target genes, increasing evidence suggests that individual components may occupy active genes, a setting in which histone methyltransferase activity may or may not be required (Lee et al., 2011; Mousavi et al., 2012; Xu et al., 2012).

Differentiation and self-renewal of HSCs are tightly regulated to generate sufficient mature cells to replace aged cells and maintain the stem cell pool. In mouse and human, fetal liver (FL) and bone marrow (BM) are two major sites of HSC residence (Orkin and Zon, 2008). FL and neonatal BM HSCs actively self-renew to expand the HSC pool in the growing animal (Bowie et al., 2006; Harrison et al., 1997). In contrast, adult BM HSCs are mostly quiescent under steady-state conditions (Bowie et al., 2006; Cheng et al., 2000; Essers et al., 2009).

The requirements for PRC2 in hematopoiesis are inadequately defined and remain controversial. ENU-induced mutations in mice have suggested that Eed and Suz12 restrict proliferation of HSCs. Paradoxically, overexpression of Ezh2 enhances HSC self-renewal (Herrera-Merchan et al., 2012; Kamminga et al., 2006). Similar approaches have suggested that Polycomb Repressive Complex-1 (PRC1 (Bmi-1 PRC1)) is antagonistic to PRC2 in HSC proliferation (Majewski et al., 2010). The roles of PRC2 in hematopoiesis are of particular interest given the recent discoveries of loss-of-function and altered-specificity EZH2 mutants, as well as mutations in EED, SUZ12 and JARID2, in various hematopoietic malignancies (Lohr et al., 2012; McCabe et al., 2012; Morin et al., 2010; Nikoloski et al., 2010; Ntziachristos et al., 2012; Puda et al., 2012; Simon et al., 2012; Ueda et al., 2012; Zhang et al., 2012). Over-expression of PRC2 subunits, particularly EZH2, has also been linked to various cancers (Simon and Lange, 2008; Varambally et al., 2002).

Although several mouse models have been generated and utilized to study the role of PRC2 in hematopoiesis, its role remains elusive due to issues such as redundancy of Ezh homologs and difficulties in distinguishing effects of canonical versus noncanonical PRC2 functions, which are mediated by Ezh1/2 or Suz12 independent of H3K27me3 (Lee et al., 2011; Mousavi et al., 2012; Su et al., 2005; Xu et al., 2012). Initially, we observed that loss of Ezh2 alone results in limited effects on the maintenance of LT-HSCs, possibly due to in vivo compensation by Ezh1. To address the role of canonical PRC2 in hematopoiesis, while leaving its possible non-canonical functions minimally affected, we generated a conditional knockout allele of Eed in the mouse. This strategy circumvents the need to infer possible redundancy of Ezh1 in the setting of Ezh2 loss. Using this approach, we have defined complex and essential roles of canonical PRC2 functions in HSCs.

Results

Ezh2 is dispensable for maintenance of LT-HSCs

We first explored the requirement of Ezh2 for maintenance of both FL and BM LT-HSCs by utilizing Ezh2Fl/FlVavCre mice (referred to as Ezh2KO). VavCre is specifically expressed in hematopoietic cells, including HSCs (Stadtfeld and Graf, 2005). Ezh2KO embryos appeared morphologically normal and there were no significant differences in the frequencies and total numbers of FL LT-HSCs (Lin−CD48−CD150+Mac1+Sca-1+ cells) between Ezh2Fl/Fl and Ezh2KO embryos at embryonic day 14.5 (E14.5) and E17.5 (Figure 1A–C). Similarly, the frequencies and total numbers of BM LT-HSCs (Lin− Sca1+Kit+CD48−CD150+) were comparable between Ezh2Fl/Fl and Ezh2KO animals at 9-day and 15-day old, respectively (Figure 1D–F). Moreover, inducible deletion of Ezh2 by Mx1Cre (Kuhn et al., 1995) in adult HSCs did not impair HSC functions in a transplantation setting (data not shown). Taken together, these data indicate that Ezh2 is dispensable for the maintenance of both FL and BM LT-HSCs.

Figure 1. Loss of Ezh2 does not significantly affect the frequencies and numbers of LT-HSCs in FL and neonatal BM.

Figure 1

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(A) Sample plots of FACS analysis for FL LT-HSCs.

(B–C) Comparison of frequencies and total cell numbers of LT-HSCs from E14.5 (B) and E17.5 (C) FLs between Ezh2Fl/Fl (Ezh2Fl/FlVavWt) and Ezh2Δ/Δ (Ezh2KO) embryos. Data are shown as mean ± SD.

(D) Sample plots of FACS analysis for BM LT-HSCs.

(E–F) Comparison of BM LT-HSC frequencies and total cell numbers for 9-day-old (E) and 15-day-old (F) Ezh2Fl/Fl and Ezh2KO neonates. Data are shown as mean ± SD. (G–I) qRT-PCR analysis of Ezh1, Ezh2 and Eed mRNAs in LT-HSCs from E14.5 FLs and from different age mouse BM. After normalized with expression value of GAPDH, expression values of indicated genes in samples presented in each plot are compared to the smallest number within the group, which is set to 1. Data are shown as mean ± SD.

Eed is dispensable for the development of FL HSCs

Since Ezh1 and Ezh2 partially overlap in their functions in ES and skin stem cells (Ezhkova et al., 2011; Ezhkova et al., 2009; Shen et al., 2008), we reasoned that Ezh1 might compensate for Ezh2 loss in HSCs. To test this, we first determined the expression of Ezh1, Ezh2 and Eed in LT-HSCs from E14.5 FL and BM at different ages. Ezh2 mRNA was 1.5-fold lower in neonatal BM compared to FL LT-HSCs, and was further decreased in the adult (Figure 1G). In contrast, Ezh1 mRNA was upregulated 5.4-fold from fetal to neonatal stage, and progressively increased with age (Figure 1H). Eed mRNA was modestly upregulated in LT-HSCs from the fetal to neonatal stage (1.8-fold), but remained largely constant thereafter (Figure 1I). These results suggest that the abundance of the PRC2 complex may be lower in FL HSCs than in postnatal BM HSCs, and that the ratio of Ezh1- versus Ezh2-PRC2 is higher in postnatal BM HSCs than in FL HSCs.

To assess directly the role of PRC2 during hematopoiesis, we generated Eed conditional knockout mice (Figure 2A–C). To validate the consequences of Eed loss, which is predicted to prevent the assembly of both Ezh1- and Ezh2-PRC2, we examined the levels of histone methylation in Eed−/− ES cells. In contrast to Ezh2−/− ES cells, which retained substantial H3K27me1, Eed−/− ES cells lacked both H3K27me1 and H3K27me3 (Figure 2D). Deletion of Eed did not alter the methylation levels at other H3 lysine residues (Figure 2D and data not shown). These results demonstrate that Eed is an essential core subunit of PRC2 and loss of Eed ablates canonical PRC2 function.

Figure 2. Eed deletion causes pancytopenia in neonates without affecting FL LT-HSCs.

Figure 2

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(A) Schematic diagram of the Eed locus, the targeting vector and the knockout allele. Light blue boxes depict exons of Eed; heavier lines represent introns of Eed gene and other genomic sequences.

(B) Southern blot validation of gene targeting.

(C) PCR validation of neomycin cassette excision.

(D) Western blot validation of a complete loss of H3K27 methylation in Eed−/− ES cells, compared to EedFl/Fl and Ezh2−/− ES cells.

(E) Comparison of frequencies (left) and total numbers per FL (right) of LT-HSCs between EedFl/FL (EedFl/FlVavWt) and EedΔ/Δ (EedKO) E17.5 FLs. Data are shown as mean ± SD.

(F) Representative photos of bones and spleens of 9-day-old WT and EedKO mice.

(G) Total white and red blood cell counts of 9-day-old WT (n=4) and EedKO (n=5) mice. Data are shown as mean ± SD.

See also Figure S1.

To study the role of Eed in HSC maintenance and function, we generated EedFl/FlVavCre (referred to as EedKO) animals. EedKO embryos developed normally (Figure S1A). No significant differences in FL LT-HSC frequencies (0.012±0.001% vs 0.009±0.002%, p=0.12) or cell numbers (7664±1259 HSCs/FL vs 6323±1640 HSCs/FL, p=0.28) were observed between E17.5 EedKO and control embryos, respectively (Figure 2E). Of note, EedKO embryos had a 3-fold reduction in B-lineage cells, suggesting a requirement of Eed for normal B-lineage cell development (Figure S1B).

Eed mutant neonates develop pancytopenia

EedKO pups were born at a normal Mendelian ratio and were indistinguishable from WT littermates at birth (data not shown). However, during the immediate postnatal period, mutants became pale and runted, and eventually succumbed to bacterial infection within 5–12 days after birth (Figure 6A and data not shown). Notably, bones from EedKO neonates were pale and hypocellular, and spleens were also pale and underdeveloped (Figure 2F and 3C). Complete blood counts revealed profound leukopenia and anemia in EedKO neonates (Figure 2G). Few myeloid or B-lineage cells were present in the EedKO BM, spleen or blood (Figure S1C and data not shown). These findings demonstrate that Eed is essential for normal hematopoiesis in postnatal animals.

Figure 6. Deletion of Cdkn2a partially rescues Eed loss in hematopoiesis.

Figure 6

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(A) Representative photo of 8-day-old WT, EedKO and DKO pups.

(B) WBC and RBC counts of 9-day-old control and mutant pups. Data are shown as mean ± SD.

(C–D) Deletion of Cdkn2a partially restores LSK and MyePro populations (C) as well as more primitive progenitors (D) in DKO. Data are shown as mean ± SD.

(E–F) Deletion of Cdkn2a enhances HSPCs survival. Different HSPC populations are stained with Annexin V and DAPI (E) and the percentages of early apoptotic cells (Annexin V+DAPI−) are shown in graph (F). Data are shown as mean ± SD.

See also Figure S6 and S7.

Figure 3. EedKO BM HSCs are defective in differentiation.

Figure 3

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(A–B) Sample FACS plots (A) showing 9-day-old WT (n=5), Ezh2KO (n=5) and EedKO (n=4) BM LSK and myeloid progenitor cells (MyePro, Lin−Sca-1−Kit+). The percentage of each population is plotted in graph (B). Data are presented as mean ± SD.

(C) Comparison of LT-HSC frequencies (as percentage of total BM cells, left), total live BM cells per mouse (middle) and total BM LT-HSCs per mouse (right) between WT, Ezh2KO and EedKO mice. Data are shown as mean ± SD.

(D–E) Sample FACS plots comparing BM LT-HSCs, ST-HSCs and restricted lineage progenitors (RLP) of 9-day-old WT, Ezh2KO, and EedKO BM (D) and the percentages are shown in the graph (E). Data are shown as mean ± SD.

(F–H) Graphs comparing Ezh1, Ezh2 and Eed mRNA levels in 9-day-old WT BM LT-, ST-HSCs, RLPs and MyePro cells. After normalized with expression value of GAPDH, expression values of indicated genes in samples presented in each plot are compared to the smallest number within the group, which is set to 1. Data are shown as mean ± SD.

See also Figure S2.

EedKO HSCs do not express c-Kit

The development of pancytopenia in EedKO neonates suggested defects in HSCs. We examined EedKO hematopoietic stem/progenitor cells (HSPCs) by flow cytometry. Strikingly, EedKO BM lacked c-Kit expressing cells within the lineage marker negative cell population (Lin−). Thus, LSK (Lin−Sca-1+Kit+) cells as well as the myeloid progenitor population (Lin−Sca-1−Kit+) were nearly absent in EedKO BM (Figure 3A–B). However, we observed a Lin−Sca-1+Kit− cell population, of which 33.1% expressed the LT-HSC characteristic marker profile, CD48−CD150+ (Figure S2C). In WT BM Lin−Sca-1+Kit− cell population was negligible and none of this subset was CD48−CD150+ (Figure S2B). These results suggest an alteration of the LT-HSC surface phenotype in EedKO BM. Specifically, EedKO BM Lin−Sca-1+c-Kit−CD48−CD150+ cells might be authentic LT-HSCs that lack c-Kit expression. In support of this, all Lin−Sca-1+CD48−CD150+ cells were c-Kit+ in WT neonatal BM (Figure S2A). More importantly, EedKO BM Lin−Sca-1+CD48− CD150+ cells expressed LT-HSC signature genes (Ivanova et al., 2002) at levels comparable to WT CD48−CD150+ LSK cells, as assessed by gene set enrichment analysis (GSEA) (Figure S2D). Furthermore, hierarchical clustering analysis of microarray expression data clustered EedKO BM Lin−Sca-1+CD48−CD150+ cells together with WT LT-HSCs, forming a distinct group separated from WT ST-HSCs (Figure S2E). Together, these data strongly support that Lin−Sca-1+Kit−CD48−CD150+ cells in EedKO BM correspond to LT-HSCs in WT BM.

EedKO neonatal BM LT-HSCs are defective in differentiation

The frequency of EedKO neonatal BM LT-HSCs (as percentage of total BM cells) was significantly increased relative to WT (0.715±0.311% vs 0.015±0.003%, p<0.01) (Figure 3C). However, after adjustment for the markedly reduced number of total viable cells, no significant difference in cell number of BM LT-HSCs was observed (11371.4±5462 (EedKO) versus 5841.7±206.0 (control), p=0.39, Figure 3C). Thus, a block in differentiation and/or defects in cell proliferation/cell death of EedKO HSPCs may account for the marked reduction in mature lineage cells.

To explore these possibilities, we examined the frequencies of LT-HSCs, ST-HSCs and restricted lineage progenitor cells (RLPs) (Kiel et al., 2005), which consisted of 7.74±0.64%, 8.33±0.78% and 46.04±3.67% of WT LSK cells, respectively (Figure 3D–E). Percentages of LT-, ST-HSCs and RLPs in EedKO BM (percent of Lin-Sca-1+ cells) were 4.9-fold, 1.8-fold and 0.08-fold of their WT correspondences, respectively (Figure 3D–E). The ratio of LT-HSC vs ST-HSC was markedly increased from 0.93:1 in WT to 2.46:1 in EedKO BM, and the RLP population was nearly absent. Taken together, these data indicate that loss of Eed in HSPCs perturbs the normal differentiation of LT-HSCs to ST-HSCs and, more dramatically, the differentiation of ST-HSCs into restricted lineage progenitor cells. Of note, the expression levels of Ezh2, Ezh1 and Eed mRNAs were the highest in ST-HSCs relative to LT-HSCs, RLPs and myeloid progenitors (Figure 3F–H), suggesting a higher activity and dependency of PRC2 in ST-HSCs than in other HSPCs.

Eed mutant BM cells are out-competed by WT cells in transplantation

The early lethality of EedKO mice precludes direct assessment for the requirements of Eed in adult HSCs. Therefore, we crossed EedFl/Fl mice with an M×1Cre strain and the Rosa26R–EYFP reporter mice (Ye et al., 2003). The resulting mutant (EedFl/FlM×1CreRosa26R–EYFPFl/Wt) or control (EedFl/WtM×1CreRosa26R–EYFPFl/Wt) mice were used to conduct competitive BM transplantation assays (Figure 4A). Following polyI:polyC (pIpC) administration, the percentage of EYFP+ donor cells in mice receiving control cells increased over time (Figure 4B). In contrast, the percentage of EYFP+ Eed mutant cells increased transiently after pIpC administration, and progressively decreased thereafter to a baseline of 20% (Figure 4B). The residual EYFP+ mutant cells retained incompletely excised Eed allele, in contrast to a complete excision of floxed allele in the control EYFP+ cells (Figure S3B). Starting at a ratio of 2:1 (CD45.2 donors vs CD45.1 competitors), it increased to 3.8:1 in 5 months in the control cell recipients. In contrast, this ratio was markedly reduced to 0.3:1 in the mutant cell recipients (Figure 4C), indicating that Eed mutant BM cells were out-competed by WT cells in the recipients.

Figure 4. Loss of Eed in adult HSCs leads to their exhaustion.

Figure 4

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(A) Scheme of competitive BM transplantation for experiments conducted for (B) and (C).

(B) Changes in percentage of EYFP positive cells (of CD45.2+ donor cells) before and at different time points after administration of pIpC.

(C) Graph represents ratios of donor versus competitor derived cells (CD45.2 vs CD45.1) in the blood at different time points after pIpC administration. Data are shown as mean ± SD.

(D–E) Graph (D) showing the relative ratios of CD45.1 vs CD45.2 BM cells, LSK cells and LT-HSCs at different time points after pIpC administration. Relative ratio is defined as the CD45.1 vs CD45.2 ratio of BM cells, LSK cells or LT-HSCs at certain time point post pIpC administration relative to the CD45.1 vs CD45.2 ratio of blood cells of the corresponding mouse before pIpC administration. Grey bars represent EedWt/WtM×1CreRosa26R–EYFPFl/Wt cell (WT) recipients and black bars represent EedFl/FlM×1CreRosa26R–EYFPFl/Wt cell (EedKO) recipients. Data are shown as mean ± SD. Representative FACS plots distinguishing donor (CD45.1) versus competitor (CD45.2) derived BM LT-HSCs are shown in (E).

See also Figure S3.

Deletion of Eed leads to loss of adult HSCs

To assess directly whether Eed is essential for adult HSC maintenance, we compared relative ratios between Eed mutant and WT HSCs (CD45.1) over time in a competitive repopulation assay. Relative ratio was defined as the ratio of CD45.1 donor versus CD45.2 competitor cells (BM, LSK cells or LT-HSCs) of a recipient mouse measured after pIpC administration relative to the ratio of CD45.1 donor versus CD45.2 competitor blood cells of the same mouse determined before pIpC injection. The relative ratio of mutant LSK cells and LT-HSCs was reduced by 35.2% and 38.2%, respectively, one week after pIpC administration. These values for WT cells were 0.92±0.06 and 0.92±0.16, respectively. At 4 weeks post pIpC injection, the relative ratio of Eed mutant LT-HSCs fell to 0.07±0.02 and donor derived LT-HSCs were nearly absent (Figure 4D). In contrast, the relative ratio for control LSK cells and LT-HSCs remained constant. These data demonstrate that Eed is required for maintenance of adult BM HSCs.

Loss of Eed enhances expression of proliferation and differentiation genes in LT-HSCs

To investigate the molecular basis underlying PRC2-regulated HSC differentiation and maintenance, we compared gene expression profiles of EedKO versus control LT-HSCs. Consistent with a role for PRC2 in transcriptional repression, Eed loss in HSCs was associated largely with gene derepression in both neonatal and adult BM HSCs. Eed loss led to up- and down-regulation of 339 and 108 genes, respectively, in EedKO neonatal LT-HSCs relative to controls at a 3-fold cutoff. At a 10-fold cutoff, 53 and 2 genes were up- or down-regulated, respectively (Figure 5B).

Figure 5. Loss of Eed in LT-HSCs derepresses PRC2 target genes.

Figure 5

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(A–B) Heatmap of the top up- or down-regulated genes (A) and overall gene expression changes upon Eed loss (B) in EedKO BM LT-HSCs compared to controls.

(C) Folds change of mRNA levels (mutant relative to WT) of indicated genes after loss of Ezh2 or Eed in neonatal BM LT-HSCs. Data are shown as mean ± SD.

(D) Similar to (C) but comparing the expression of indicated genes after Eed loss in adult LT-HSCs. Data are shown as mean ± SD.

(E) Percentages of apoptotic BM LT-, ST-HSCs and RLPs in neonatal WT, Ezh2KO or EedKO BM. Data are shown as mean ± SD.

(F–G) ChIP-qPCR analysis of H3K27me3 (F) and H3K9me3 (G) at the proximal promoter regions of indicated genes in FACS-sorted WT and Eed mutant LSK cells from adult BMs. Data are shown as mean ± SD.

See also Figure S4 and S5.

By integrated pathway analysis (IPA), we observed that the differentially expressed genes (2-fold cutoff) are highly associated with hematopoietic development and function, cellular growth and proliferation, cell death and survival (Figure S4E). GSEA analysis comparing neonatal EedKO and control BM LT-HSCs revealed enrichment of genes involved in negative regulation of cell cycle progression (Figure S4C). The established PRC2 target gene Ckdn2a (Jacobs et al., 1999), along with other cell cycle inhibitor genes, Cdkn1a and Cdkn2b, were among the most highly up-regulated genes in EedKO HSCs (Figure 5A, C–D and S5D). Inhibitors of HSPC differentiation, Id2 and Sox7 (Costa et al., 2012; Gandillet et al., 2009; Ji et al., 2008; Li et al., 2010), were also among the top up-regulated genes upon Eed loss (Figure 5A and C–D). Additionally, HoxC4, a homeobox gene that promotes self-renewal of HSCs (Auvray et al., 2012; Daga et al., 2000), was also highly upregulated in EedKO HSCs (Figure 5A and C–D). Thus, loss of Eed in HSCs leads to enhanced expression of a set of genes involved in counteracting pathways, such as cell cycle inhibitors and proliferation enhancers, as well as genes that function to block HSC differentiation. Importantly, loss of Eed enhanced expression of these genes in both neonatal and adult LT-HSCs (Figure 5C and D).

Deletion of Eed in BM LT-HSCs activates expression of pro-apoptotic genes and induces cell death

By GSEA analysis we also observed enrichment of hypoxia response genes, most are targets of Hif-1, in EedKO LT-HSCs relative to controls (Figure S4A–B and data not shown). Noxa, a direct target of Hif-1 and a BH3 domain only pro-apoptotic gene (Kim et al., 2004), was the most highly up-regulated gene in EedKO LT-HSCs relative to control LT-HSCs (Figure 5A, C–D). In addition, p21 and Wig1, targets of Arf/P53 that have been suggested to induce cell death in Bmi-1 null HSCs (Park et al., 2003), were also markedly up-regulated in EedKO neonatal and adult BM HSCs (Figure S5A–B). GSEA analysis comparing EedKO and WT BM LT-HSCs confirmed the enrichment of pro-apoptotic genes in EedKO HSCs (Figure S5C). Together, these data suggest that EedKO HSCs are predisposed to apoptosis. To test this, we performed Annexin V and DAPI staining on HSPCs and observed that 12.9±1.9% of EedKO BM LT-HSCs were apoptotic (Annexin V+DAPI−), a percentage that was 5.9-fold greater than in WT LT-HSCs and 3.7-fold that of Ezh2−/− LT-HSCs, respectively (Figure 5E). More profound differences in the rate of cell death were observed in restricted lineage progenitors, 31.6±10.8% in EedKO BM vs 2.4±0.9% in WT BM, a striking 13.2-fold increase in EedKO cells relative to WT cells (Figure 5E). Overall, these results demonstrate that loss of Eed activates expression of pro-apoptotic genes and induces cell death in the HSPC compartments, which may also partly account for the decreased mature hematopoietic cells in EedKO mice.

Derepressed genes in EedKO HSCs are enriched for H3K27me3 targets

PRC2-mediated gene repression is heralded by the presence of the H3K27me3 mark on, or surrounding, target genes. Since expression of Cdkn1a, Cdkn2a/b, Ho×C4, Id2, Sox7 and Noxa was markedly elevated in Eed mutant HSCs, we asked whether these genes are direct targets of PRC2 and marked by H3K27me3. By chromatin immunoprecipitation (ChIP) analysis, we observed that in WT LSK cells the proximal promoter regions of these genes were significantly enriched for H3K27me3, which was largely depleted upon Eed loss (Figure 5F). These observations are consistent with the increased mRNA expression of these genes in EedKO HSPC cells (Figure 5A and C–D). These data strongly suggest that these genes are directly targeted by PRC2 and marked by H3K27me3 in wild type HSPCs. Importantly, loss of Eed and H3K27me3 mark had minimal influence on the levels of other histone marks, such as H3K9me3, at the promoter regions of these genes (Figure 5G).

Compound deletion of Cdkn2a partially rescues Eed mutant HSPCs

Deletion of Eed activated transcription of both Ink4a and Arf from the Cdkn2a locus (Figure 5SD), an established Polycomb target (Jacobs et al., 1999; Kotake et al., 2007). Deletion of Cdkn2a has been reported to rescue the defects of some Polycomb gene knockouts, such as Bmi1 and Ezh1 (Hidalgo et al., 2012; Molofsky et al., 2005; Oguro et al., 2006). Therefore, we examined whether Cdkn2a deletion also rescues the hematopoietic defects upon Eed loss. Compound deletion of Cdkn2a and Eed (DKO) failed to rescue the neonatal lethality. Similar to the EedKO neonates, DKO pups became pale within a couple of days after birth (Figure 6A). White and red blood cell counts in DKO pups were only 12.3±8.2% and 14.2±4.0% of WT littermates, and were not significantly different from EedKO neonates (p values=0.44 and 0.75 for WBC and RBC, respectively) (Figure 6B). The number of total BM cells in 7–9 days old pups was 4.5-fold lower in EedKO than in WT animals, and was not restored upon deletion of Cdkn2a (Figure 3C and data not shown). However, Cdkn2a deletion restored c-Kit expression in HSPCs (Figure S6). Thus, LSK cells, which were nearly absent in EedKO BM, consisted of 11.4±3.4% of Lin− cells in DKO BM (Figure 3A and 6C), a frequency that was 2-fold greater than that of WT LSK cells (5.6±1.7%). Cdkn2a deletion also partially restored the myeloid progenitor population to 13.2±3.2% of Lin− cells in DKO (Figure 6D), which was still substantially lower than that of WT myeloid progenitors (36.4±4.5%). Therefore, the ratio of LSK versus myeloid progenitor cells in the DKO BM was 5.5-fold higher than that in the WT BM, suggesting an accumulation of HSPCs in more immature stages and likely caused by a block in cell differentiation.

Deletion of Cdkn2a in EedKO mice also enhanced HSPC survival. Apoptotic LT-and ST-HSCs were reduced 35.8% and 33.9% in DKO BM relative to EedKO BM, respectively. More strikingly, apoptotic restricted lineage progenitor cells decreased by 67.3% (Figure 6E–F). As a result, the percentage of restricted lineage progenitor cells increased 4.5 fold in DKO BM relative to EedKO BM, though it remained 28.2% lower than in WT BM (Figure 6D).

The above results are in stark contrast to previous findings that Cdkn2a deficiency rescued hematopoietic defects in Bmi1 and Ezh1 KO mice, enabling double KO bone marrow cells to efficiently reconstitute recipients the hematopoietic system in a competitive transplantation setting (Hidalgo et al., 2012; Oguro et al., 2006). Deletion of Cdkn2a was unable to rescue defects of EedKO BM in reconstituting recipient hematopoiesis. EedKO BM cells were unable to form CFU-S or contribute to hematopoiesis in recipients (Figure S7). Thus, deletion of Cdkn2a only partially enhanced the survival of EedKO HSPCs without restoring HSC functions.

Discussion

Here we have investigated the requirement of PRC2 in hematopoiesis through conditional inactivation of its essential core subunit, Eed. By this approach we circumvented confounding issues related to possible redundancy between Ezh1 and Ezh2, as well as noncanonical functions of some PRC2 components. We found that loss of Eed leads to multiple and profound defects in hematopoiesis. Eed mutant HSCs fail to differentiate into mature blood cells and are prone to cell death. PRC2 suppresses the expression of numerous genes in HSCs, including differentiation inhibitors, cell cycle inhibitors, the self-renewal enhancer and the pro-apoptotic gene. Therefore, we infer that PRC2 coordinates diverse pathways to ensure proper self-renewal and differentiation of HSCs. Deletion of Cdkn2a enhances HSPC survival without relieving the differentiation blockage, further suggesting that PRC2 governs multiple pathways in controlling normal HSC functions. While loss of Eed leads to derepression of similar genes in both neonatal and adult BM LT-HSCs, different cellular phenotypes ensued. These contrasting outcomes are most likely due to intrinsic differences in HSC proliferation and differentiation at different stages (Figure 7). The enrichment of the H3K27me3 mark at genes upregulated in Eed mutant HSCs and depletion upon Eed loss demonstrate that they are direct targets of PRC2. Therefore, relief of PRC2-mediated transcriptional repression upon inactivation of Eed leads to the complex and developmental-stage specific hematopoietic defects.

Figure 7.

Figure 7

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PRC2 regulates HSC differentiation and survival.

(A) PRC2 suppresses target genes in WT HSCs. Purple ovals represent methyl groups.

(B) Some PRC2 target genes are selectively derepressed in EedKO BM HSCs.

(C–D) WT (C) and EedKO (D) neonatal BM HSCs self-renew and differentiate in a BM niche. Eed loss leads to a differentiation block (panel D, red bars) and apoptosis (panel D, cells with black dots) of some HSCs without significantly affecting HSC number. (E–F) Similar to C–D but showing WT (E) and Eed mutant (F) adult BM HSCs. Apoptosis becomes the dominant effect of Eed loss in adults, resulting in HSC failure.

Overlapping function of Ezh1 and Ezh2 in HSCs

Ezh1 partially compensates for Ezh2 loss in embryonic and skin stem cell (Ezhkova et al., 2011; Ezhkova et al., 2009; Shen et al., 2008). A similar mechanism is likely also at play in HSCs. We note that loss of Ezh2 affects a smaller proportion of genes than the loss of Eed (Table S1 and Figure S4D). Of note, Ezh2 loss fails to elicit derepression of cell cycle and differentiation inhibitor genes, and thus preserves largely normal differentiation and functions of HSPCs. Moreover, expression of Noxa, Wig1 and Perp is derepressed to a much lesser extent in Ezh2KO LT-HSCs compared to EedKO LT-HSCs. Surprisingly, Ezh2 loss fails to derepress expression of Cdkn2a in LT-HSCs (Figure 5C), which is highly upregulated in Ezh1 mutant HSCs (Hidalgo et al., 2012). Consistent with these gene expression changes, apoptosis is also only slightly increased in Ezh2KO relative to WT LT-HSCs (Figure 5C and E). Such differences may account for a smaller reduction in the number of BM HSCs in Ezh2KO relative to EedKO (Figure 3C). Together, these results suggest that Ezh1- and Ezh2-containing PRC2 complexes have selective targets and that Ezh1 compensates for Ezh2 loss at some, but not all, PRC2 targets. In adult HSCs, Ezh2 loss exerts even fewer effects on HSCs, in consistent with elevated Ezh1 level and decreased Ezh2 expression at this stage (Figure 1G and H). Of note, Ezh1 mRNA does not increase upon the loss of Ezh2 in BM HSCs (data not shown).

Ezh2 was reported to be essential for FL but not adult BM HSCs on the basis of conditional deletion mediated by Tie2Cre (Mochizuki-Kashio et al., 2011). In our study, VavCre-mediated Ezh2 excision failed to reproduce a similar phenotype in FL HSCs, most likely due to the application of different Cre lines. While VavCre mediates gene deletion specifically in hematopoietic cells, Tie2Cre also induces excision in endothelial cells (Kisanuki et al., 2001). Therefore, the described FL HSC defects may not be due to a cell-autonomous effect.

Roles of canonical versus non-canonical PRC2 in hematopoiesis

Canonical PRC2 consists of 3 core subunits Eed, Suz12 and Ezh1/2 and methylates H3K27. H3K27me3 is considered to be a mark for repressed or poised genes, depending on the coexistence of activation marks (Bernstein et al., 2006). More recently, core subunits of PRC2 have been found to bind to regions where H3K27me3 is absent and associate with active genes (Lee et al., 2011; Mousavi et al., 2012; Xu et al., 2012), suggesting that they may have non-canonical functions that are independent of PRC2 histone substrate. Eed associates almost exclusively with methylated H3K27 (Xu and Orkin, unpublished data). Hence, Eed deletion likely abolishes all canonical PRC2 function without compromising non-canonical PRC2 activities. Close comparisons between Ezh1, Ezh2, Eed knockouts and Ezh1/Ezh2 double knockout mice will be very informative in distinguishing canonical versus non-canonical PRC2 functions and their roles in differentiation and development. For example, the ratio of up- versus downregulated genes, comparing Ezh1−/− to WT HSCs, is 1.56:1 (Hidalgo et al., 2012). This value is 3.13:1 in EedKO HSCs relative to WT HSCs (Figure 5B, Figure 3-fold cutoff), indicating that more genes are upregulated upon the loss of Eed than Ezh1. Importantly, deletion of Ezh1 resulted in downregulation of many genes crucial for HSC functions, such as Gata3, Runx1, Meis1, Pten, and Pbx1, which may in part be responsible for the HSC defects (Hidalgo et al., 2012). Strikingly, none of these genes is affected in EedKO HSCs. Therefore, Eed likely acts through the canonical PRC2 complex regulating genes to ensure normal HSC maintenance and differentiation. In contrast, Ezh1 may participate in control through both canonical and non-canonical PRC2 functions.

Complex consequences of Eed loss in HSCs

Gene expression is controlled by the availability of appropriate transcription factors plus the accessibility of their corresponding binding sites on the genome (Reik, 2007). In HSCs, Eed loss, enhances expression of genes of various pathways, including differentiation inhibitors, apoptotic genes, proliferation enhancers, and cell cycle inhibitors. These genes exert conflicting effects in cellular phenotypes (Figure 5A, C–D and S4E), and may account for the profound and complex consequences of Eed loss during hematopoiesis. In addition, intrinsic differences in neonatal and adult BM HSCs integrate with the effects of Eed loss, resulting in developmental-stage specific phenotypes in HSCs. Neonatal BM HSCs actively proliferate for 2–3 weeks after birth and become quiescent thereafter (Bowie et al., 2006). EedKO neonatal HSCs are likely impaired in differentiation, in part due to the derepression of differentiation inhibitors, thereby trapping them within the HSC pool. Meanwhile, elevated HoxC4 expression upon Eed loss may further enhance HSC self-renewal. On the other hand, Eed loss also promotes quiescence through derepression of CDK inhibitor expression. Moreover, upregulation of Noxa and other cell death genes favors apoptosis of HSCs. In essence, Eed mutant HSCs face conflicting signals to expand the HSC pool, block proliferation and trigger cell death. The net outcome of Eed inactivation may depend on the relative strength of these interacting conflicting signals. Furthermore, the deleterious effects of Eed loss for HSCs also depend on cellular context. Specifically, neonatal BM HSCs undergoes a high rate of self-renewal divisions to achieve a steady-state adult HSC pool. Concomitantly, more neonatal HSCs are called into differentiation than in the adults in order to supply the growing individual with sufficient intermediate progenitors. Hence, inhibition of HSC differentiation in EedKO neonates, together with potentially enhanced proliferation, contributes sufficient cells to the HSC pool to compensate for those lost by increased apoptosis and cell cycle inhibition (Figure 7C–D). In contrast, few adult LT-HSCs are in cell cycle or initiating differentiation under normal circumstances (Wilson et al., 2008). Therefore, in this setting, apoptosis becomes the dominant effect of Eed inactivation in adult LT-HSCs (Figure 7E–F).

Cdkn2a deletion only partially rescues defective EedKO HSPCs

Cdkn2a deletion partially rescued Eed-deficient HSPCs from cell death, most dramatically within the restricted lineage progenitor population. Reduced cell death was not as significant in Cdkn2a/Eed DKO LT-HSCs (Figure 6F). Derepression of Cdkn2a expression in HSPCs upon loss of Polycomb proteins has been suggested to induce cell death though downstream targets of p53, including P21 and Wig1 (Park et al., 2003). In EedKO BM, Cdkn2a and Wig1 were upregulated the highest in restricted lineage progenitor cells relative to LT- and ST-HSCs (Figure S5A). In contrast, Noxa was upregulated the highest in LT-HSCs (Figure 5SA). Noxa is a direct target of both PRC2 and Hif-1, the latter of which is expressed at its highest level in LT-HSCs among other HSPCs (Takubo et al., 2010). Therefore, inactivation of Cdkn2a is unable to curtail Noxa expression in DKO LT-HSCs, and consequently only slightly decreases apoptosis of DKO LT-HSCs compared to EedKO HSCs. Moreover, knockout of Cdkn2a fails to significantly downregulate inhibitors of HSPC differentiation (Figure S5B) and to rescue the differentiation block in DKO HSPCs. Together, our data indicate that PRC2 suppresses genes of diverse pathways ensuring normal HSC functions, and inactivation of a single target gene, such as Cdkn2a, is insufficient to rescue the defective EedKO HSCs.

EedΔ/WT BM HSCs exhibit a growth advantage in competitive transplantation

Previous studies have shown that BM cells heterozygous for an ENU-induced, loss of function mutation of Suz12 exhibit enhanced competitive reconstitution capacity (Majewski et al., 2008), suggesting a suppressive role of PRC2 in HSC activity. Consistent with this finding, we observed that adult BM HSCs heterozygous for Eed (EedFl/WtM×1CreRosa26R–EYFPFl/Wt, referred to as EedΔ/WT HSCs) out-competed WT HSCs in a competitive transplantation setting (Figure 4C and S3A). However, a complete loss of Eed gene leads to HSC exhaustion in the adult BM (Figure 4 C–D). On the other hand, overexpression of Ezh2 has been reported to augment HSC proliferation and forestalls its exhaustion in serial transplantation assays (Herrera-Merchan et al., 2012; Kamminga et al., 2006). Taken together, these data suggest that proper HSC function necessitates fine-tuned control of PRC2.

Despite a selective growth advantage of EedΔ/WT HSCs, no leukemia was observed in the recipients in a one-year period after BM transplantation and pIpC administration (data not shown). Similarly, no leukemia was observed in mice with heterozygous loss of Ezh2 or Eed by VavCre for at least 18 months (data not shown), although inactivation of both alleles of Ezh2 by M×1Cre leads to T-ALL in mice (Simon et al., 2012). Thus, inactivation of a single copy of Eed enhances cell proliferation, but is insufficient to trigger leukemogenesis. Additional mutations are likely needed for generation of hematopoietic malignancies in individuals with heterozygous mutation of PRC2 (Makishima et al., 2010; Ntziachristos et al., 2012).

Conclusions

Loss of Eed in BM HSCs produces devastating and complex consequences by triggering counteracting and conflicting signals that control cell cycle inhibition, selfrenewal, differentiation and apoptosis. The effects of Eed loss in the hematopoietic system far exceed the loss of Ezh2 or Ezh1 alone. Current efforts of targeting PRC2 for therapy have centered primarily on inhibition of Ezh2, as its overexpression characterizes many cancers, both hematopoietic and non-hematopoietic in origin (Sauvageau and Sauvageau, 2010; Simon and Lange, 2008). In those circumstances, in which oncogenesis strictly depends on Ezh2, this strategy is predicted to be successful (Wilson et al., 2010). In other settings, such as in some leukemias, inactivation of all canonical PRC2 function may be required (Neff et al., 2012; Shi et al., 2012). Our study reveals the complex consequences of PRC2 loss for hematopoiesis and demonstrates its essential roles in HSC differentiation, self-renewal and survival. Further work is needed to elucidate regulation of Ezh1 and Ezh2 expression during development, as well as how Ezh1- and Ezh2-containing PRC2 complexes attain their target specificity.

Experimental Procedures

Generation of Eed conditional allele and mice

Genomic sequences spanning intron 1–2, intron 2–6, and intron 6–8 were generated by PCR and subcloned into the modified pFlexible vector at the sites of ClaI/AscI, Pac1 and NotI, respectively. Positive clones were validated by Southern Blot analysis. The neomycin cassette was removed by transient expression of Flp recombinase and validated by PCR. Two karyotypically normal ES cell clones were injected into blastocysts to generate chimeras.

Flow cytometry

Cells were first incubated with Fc-Block then with biotin-conjugated lineage marker antibodies (CD3e (145–2C11), CD4 (GK1.5), CD5 (53–7.3), CD8a (53–6.7), CD11b (M1/70), B220 (RA3–6B2), Gr-1 (RB6–8C5) and Ter119 (TER119)) and followed by Percp-Cy5.5 conjugated streptavidin, PE-Cy7 conjugated Sca-1 (D7), APC-Cy7 conjugated c-Kit (2B8), PE conjugated CD48 (HM48-1) and APC conjugated CD150 (TC15-12F.2) antibodies. After washing, labeled cells were run on LSR II/LSR Fortessa for analysis or on FACSAria II for cell sorting. FACS data were analyzed with Flowjo software (Tree Star).

Global gene expression profiling

Total RNA was isolated using the RNeasy Micro Plus Kit (Qiagen), reverse transcribed, amplified with the WT-Ovation Pico RNA Amplification System V2 (NuGen Technologies), labeled (NuGen; Encore Biotin Module) and hybridized to Affymetrix mouse 430A 2.0 arrays. Data were analyzed with GenePattern, GSEA and IPA.

ChIP-qPCR

ChIP was performed as described with modifications (Xu et al., 2010). Briefly, 2.5–5 × 105 of FACS-sorted LSK cells were crosslinked with 1% formaldehyde (Sigma) for 5 min at room temperature. Chromatin was sonicated in RIPA buffer with 0.3 M NaCl to ∼500 bp. Dynabeads Protein A (Invitrogen) was used for collection of chromatin. 1∼2 µg of antibodies per IP were incubated with beads for 3∼5 hours before incubating with sonicated chromatin for overnight. ChIP DNA was purified and quantified by real-time PCR using the iQ SYBR Green Supermix (Bio-Rad). Anti-H3K27me3 (07–449, Millipore) and anti-H3K9me3 (17–625, Millipore) antibodies were used for the analyses.

Supplementary Material

01
02

Highlights.

  1. Ezh2 is dispensable for HSC formation and maintenance.

  2. Loss of Eed leads to HSC exhaustion in adults.

  3. PRC2 suppresses diverse classes of genes with conflicting functions.

  4. Deletion of Cdkn2a partially rescues HSC defects of Eed loss.

Acknowledgments

We gratefully acknowledge Matthias Stadtfeld, Jennifer Trowbridge and Boris Wilson for critical review of the manuscript. This work was supported in part by NIH U01 grant CA105423 and the HSCI Blood Program. S.H.O. is an Investigator of the HHMI. H.X. was a Leukemia and Lymphoma Society fellow. J.X., an HHMI-Helen Hay Whitney Foundation fellow, is supported by an NIDDK Career Development Award K01DK093543.

Footnotes

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Accession Numbers

Gene expression data have been deposited in the Gene Expression Omnibus under accession number GSE51084

References

  1. Auvray C, Delahaye A, Pflumio F, Haddad R, Amsellem S, Miri-Nezhad A, Broix L, Yacia A, Bulle F, Fichelson S, et al. HOXC4 homeoprotein efficiently expands human hematopoietic stem cells and triggers similar molecular alterations as HOXB4. Haematologica. 2012;97:168–178. doi: 10.3324/haematol.2011.051235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, Fry B, Meissner A, Wernig M, Plath K, et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell. 2006;125:315–326. doi: 10.1016/j.cell.2006.02.041. [DOI] [PubMed] [Google Scholar]
  3. Bowie MB, McKnight KD, Kent DG, McCaffrey L, Hoodless PA, Eaves CJ. Hematopoietic stem cells proliferate until after birth and show a reversible phase-specific engraftment defect. J Clin Invest. 2006;116:2808–2816. doi: 10.1172/JCI28310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cheng T, Rodrigues N, Shen H, Yang Y, Dombkowski D, Sykes M, Scadden DT. Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science. 2000;287:1804–1808. doi: 10.1126/science.287.5459.1804. [DOI] [PubMed] [Google Scholar]
  5. Costa G, Mazan A, Gandillet A, Pearson S, Lacaud G, Kouskoff V. SOX7 regulates the expression of VE-cadherin in the haemogenic endothelium at the onset of haematopoietic development. Development. 2012;139:1587–1598. doi: 10.1242/dev.071282. [DOI] [PubMed] [Google Scholar]
  6. Daga A, Podesta M, Capra MC, Piaggio G, Frassoni F, Corte G. The retroviral transduction of HOXC4 into human CD34(+) cells induces an in vitro expansion of clonogenic and early progenitors. Exp Hematol. 2000;28:569–574. doi: 10.1016/s0301-472x(00)00135-1. [DOI] [PubMed] [Google Scholar]
  7. Essers MA, Offner S, Blanco-Bose WE, Waibler Z, Kalinke U, Duchosal MA, Trumpp A. IFNalpha activates dormant haematopoietic stem cells in vivo. Nature. 2009;458:904–908. doi: 10.1038/nature07815. [DOI] [PubMed] [Google Scholar]
  8. Ezhkova E, Lien WH, Stokes N, Pasolli HA, Silva JM, Fuchs E. EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair. Genes Dev. 2011;25:485–498. doi: 10.1101/gad.2019811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ezhkova E, Pasolli HA, Parker JS, Stokes N, Su IH, Hannon G, Tarakhovsky A, Fuchs E. Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells. Cell. 2009;136:1122–1135. doi: 10.1016/j.cell.2008.12.043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gandillet A, Serrano AG, Pearson S, Lie ALM, Lacaud G, Kouskoff V. Sox7-sustained expression alters the balance between proliferation and differentiation of hematopoietic progenitors at the onset of blood specification. Blood. 2009;114:4813–4822. doi: 10.1182/blood-2009-06-226290. [DOI] [PubMed] [Google Scholar]
  11. Harrison DE, Zhong RK, Jordan CT, Lemischka IR, Astle CM. Relative to adult marrow, fetal liver repopulates nearly five times more effectively long-term than short-term. Exp Hematol. 1997;25:293–297. [PubMed] [Google Scholar]
  12. Herrera-Merchan A, Arranz L, Ligos JM, de Molina A, Dominguez O, Gonzalez S. Ectopic expression of the histone methyltransferase Ezh2 in haematopoietic stem cells causes myeloproliferative disease. Nat Commun. 2012;3:623. doi: 10.1038/ncomms1623. [DOI] [PubMed] [Google Scholar]
  13. Hidalgo I, Herrera-Merchan A, Ligos JM, Carramolino L, Nunez J, Martinez F, Dominguez O, Torres M, Gonzalez S. Ezh1 Is Required for Hematopoietic Stem Cell Maintenance and Prevents Senescence-like Cell Cycle Arrest. Cell Stem Cell. 2012;11:649–662. doi: 10.1016/j.stem.2012.08.001. [DOI] [PubMed] [Google Scholar]
  14. Ivanova NB, Dimos JT, Schaniel C, Hackney JA, Moore KA, Lemischka IR. A stem cell molecular signature. Science. 2002;298:601–604. doi: 10.1126/science.1073823. [DOI] [PubMed] [Google Scholar]
  15. Jacobs JJ, Kieboom K, Marino S, DePinho RA, van Lohuizen M. The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature. 1999;397:164–168. doi: 10.1038/16476. [DOI] [PubMed] [Google Scholar]
  16. Ji M, Li H, Suh HC, Klarmann KD, Yokota Y, Keller JR. Id2 intrinsically regulates lymphoid and erythroid development via interaction with different target proteins. Blood. 2008;112:1068–1077. doi: 10.1182/blood-2008-01-133504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kamminga LM, Bystrykh LV, de Boer A, Houwer S, Douma J, Weersing E, Dontje B, de Haan G. The Polycomb group gene Ezh2 prevents hematopoietic stem cell exhaustion. Blood. 2006;107:2170–2179. doi: 10.1182/blood-2005-09-3585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kiel MJ, Yilmaz OH, Iwashita T, Terhorst C, Morrison SJ. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell. 2005;121:1109–1121. doi: 10.1016/j.cell.2005.05.026. [DOI] [PubMed] [Google Scholar]
  19. Kim JY, Ahn HJ, Ryu JH, Suk K, Park JH. BH3-only protein Noxa is a mediator of hypoxic cell death induced by hypoxia-inducible factor 1alpha. J Exp Med. 2004;199:113–124. doi: 10.1084/jem.20030613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, Yanagisawa M. Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev Biol. 2001;230:230–242. doi: 10.1006/dbio.2000.0106. [DOI] [PubMed] [Google Scholar]
  21. Kotake Y, Cao R, Viatour P, Sage J, Zhang Y, Xiong Y. pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16INK4alpha tumor suppressor gene. Genes Dev. 2007;21:49–54. doi: 10.1101/gad.1499407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kuhn R, Schwenk F, Aguet M, Rajewsky K. Inducible gene targeting in mice. Science. 1995;269:1427–1429. doi: 10.1126/science.7660125. [DOI] [PubMed] [Google Scholar]
  23. Kuzmichev A, Nishioka K, Erdjument-Bromage H, Tempst P, Reinberg D. Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein. Genes Dev. 2002;16:2893–2905. doi: 10.1101/gad.1035902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee ST, Li Z, Wu Z, Aau M, Guan P, Karuturi RK, Liou YC, Yu Q. Contextspecific regulation of NF-kappaB target gene expression by EZH2 in breast cancers. Mol Cell. 2011;43:798–810. doi: 10.1016/j.molcel.2011.08.011. [DOI] [PubMed] [Google Scholar]
  25. Li H, Ji M, Klarmann KD, Keller JR. Repression of Id2 expression by Gfi-1 is required for B-cell and myeloid development. Blood. 2010;116:1060–1069. doi: 10.1182/blood-2009-11-255075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lohr JG, Stojanov P, Lawrence MS, Auclair D, Chapuy B, Sougnez C, Cruz-Gordillo P, Knoechel B, Asmann YW, Slager SL, et al. Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing. Proc Natl Acad Sci U S A. 2012;109:3879–3884. doi: 10.1073/pnas.1121343109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Majewski IJ, Blewitt ME, de Graaf CA, McManus EJ, Bahlo M, Hilton AA, Hyland CD, Smyth GK, Corbin JE, Metcalf D, et al. Polycomb repressive complex 2 (PRC2) restricts hematopoietic stem cell activity. PLoS Biol. 2008;6:e93. doi: 10.1371/journal.pbio.0060093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Majewski IJ, Ritchie ME, Phipson B, Corbin J, Pakusch M, Ebert A, Busslinger M, Koseki H, Hu Y, Smyth GK, et al. Opposing roles of polycomb repressive complexes in hematopoietic stem and progenitor cells. Blood. 2010;116:731–739. doi: 10.1182/blood-2009-12-260760. [DOI] [PubMed] [Google Scholar]
  29. Makishima H, Jankowska AM, Tiu RV, Szpurka H, Sugimoto Y, Hu Z, Saunthararajah Y, Guinta K, Keddache MA, Putnam P, et al. Novel homo- and hemizygous mutations in EZH2 in myeloid malignancies. Leukemia. 2010;24:1799–1804. doi: 10.1038/leu.2010.167. [DOI] [PubMed] [Google Scholar]
  30. Margueron R, Li G, Sarma K, Blais A, Zavadil J, Woodcock CL, Dynlacht BD, Reinberg D. Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms. Mol Cell. 2008;32:503–518. doi: 10.1016/j.molcel.2008.11.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Margueron R, Reinberg D. The Polycomb complex PRC2 and its mark in life. Nature. 2011;469:343–349. doi: 10.1038/nature09784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McCabe MT, Graves AP, Ganji G, Diaz E, Halsey WS, Jiang Y, Smitheman KN, Ott HM, Pappalardi MB, Allen KE, et al. Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27) Proc Natl Acad Sci U S A. 2012;109:2989–2994. doi: 10.1073/pnas.1116418109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mochizuki-Kashio M, Mishima Y, Miyagi S, Negishi M, Saraya A, Konuma T, Shinga J, Koseki H, Iwama A. Dependency on the polycomb gene Ezh2 distinguishes fetal from adult hematopoietic stem cells. Blood. 2011;118:6553–6561. doi: 10.1182/blood-2011-03-340554. [DOI] [PubMed] [Google Scholar]
  34. Molofsky AV, He S, Bydon M, Morrison SJ, Pardal R. Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16Ink4a and p19Arf senescence pathways. Genes Dev. 2005;19:1432–1437. doi: 10.1101/gad.1299505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Morin RD, Johnson NA, Severson TM, Mungall AJ, An J, Goya R, Paul JE, Boyle M, Woolcock BW, Kuchenbauer F, et al. Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nat Genet. 2010;42:181–185. doi: 10.1038/ng.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mousavi K, Zare H, Wang AH, Sartorelli V. Polycomb protein Ezh1 promotes RNA polymerase II elongation. Mol Cell. 2012;45:255–262. doi: 10.1016/j.molcel.2011.11.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Neff T, Sinha AU, Kluk MJ, Zhu N, Khattab MH, Stein L, Xie H, Orkin SH, Armstrong SA. Polycomb repressive complex 2 is required for MLL-AF9 leukemia. Proc Natl Acad Sci U S A. 2012;109:5028–5033. doi: 10.1073/pnas.1202258109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nikoloski G, Langemeijer SM, Kuiper RP, Knops R, Massop M, Tonnissen ER, van der Heijden A, Scheele TN, Vandenberghe P, de Witte T, et al. Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nat Genet. 2010;42:665–667. doi: 10.1038/ng.620. [DOI] [PubMed] [Google Scholar]
  39. Ntziachristos P, Tsirigos A, Van Vlierberghe P, Nedjic J, Trimarchi T, Flaherty MS, Ferres-Marco D, da Ros V, Tang Z, Siegle J, et al. Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia. Nat Med. 2012;18:298–301. doi: 10.1038/nm.2651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Oguro H, Iwama A, Morita Y, Kamijo T, van Lohuizen M, Nakauchi H. Differential impact of Ink4a and Arf on hematopoietic stem cells and their bone marrow microenvironment in Bmi1-deficient mice. J Exp Med. 2006;203:2247–2253. doi: 10.1084/jem.20052477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Orkin SH, Zon LI. Hematopoiesis: an evolving paradigm for stem cell biology. Cell. 2008;132:631–644. doi: 10.1016/j.cell.2008.01.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Park IK, Qian D, Kiel M, Becker MW, Pihalja M, Weissman IL, Morrison SJ, Clarke MF. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature. 2003;423:302–305. doi: 10.1038/nature01587. [DOI] [PubMed] [Google Scholar]
  43. Puda A, Milosevic JD, Berg T, Klampfl T, Harutyunyan AS, Gisslinger B, Rumi E, Pietra D, Malcovati L, Elena C, et al. Frequent deletions of JARID2 in leukemic transformation of chronic myeloid malignancies. Am J Hematol. 2012;87:245–250. doi: 10.1002/ajh.22257. [DOI] [PubMed] [Google Scholar]
  44. Reik W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature. 2007;447:425–432. doi: 10.1038/nature05918. [DOI] [PubMed] [Google Scholar]
  45. Sauvageau M, Sauvageau G. Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer. Cell Stem Cell. 2010;7:299–313. doi: 10.1016/j.stem.2010.08.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Shen X, Liu Y, Hsu YJ, Fujiwara Y, Kim J, Mao X, Yuan GC, Orkin SH. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell. 2008;32:491–502. doi: 10.1016/j.molcel.2008.10.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shi J, Wang E, Zuber J, Rappaport A, Taylor M, Johns C, Lowe SW, Vakoc CR. The Polycomb complex PRC2 supports aberrant self-renewal in a mouse model of MLL-AF9;Nras(G12D) acute myeloid leukemia. Oncogene. 2012 doi: 10.1038/onc.2012.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Simon C, Chagraoui J, Krosl J, Gendron P, Wilhelm B, Lemieux S, Boucher G, Chagnon P, Drouin S, Lambert R, et al. A key role for EZH2 and associated genes in mouse and human adult T-cell acute leukemia. Genes Dev. 2012;26:651–656. doi: 10.1101/gad.186411.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Simon JA, Lange CA. Roles of the EZH2 histone methyltransferase in cancer epigenetics. Mutat Res. 2008;647:21–29. doi: 10.1016/j.mrfmmm.2008.07.010. [DOI] [PubMed] [Google Scholar]
  50. Stadtfeld M, Graf T. Assessing the role of hematopoietic plasticity for endothelial and hepatocyte development by non-invasive lineage tracing. Development. 2005;132:203–213. doi: 10.1242/dev.01558. [DOI] [PubMed] [Google Scholar]
  51. Su IH, Dobenecker MW, Dickinson E, Oser M, Basavaraj A, Marqueron R, Viale A, Reinberg D, Wulfing C, Tarakhovsky A. Polycomb group protein ezh2 controls actin polymerization and cell signaling. Cell. 2005;121:425–436. doi: 10.1016/j.cell.2005.02.029. [DOI] [PubMed] [Google Scholar]
  52. Takubo K, Goda N, Yamada W, Iriuchishima H, Ikeda E, Kubota Y, Shima H, Johnson RS, Hirao A, Suematsu M, et al. Regulation of the HIF-1alpha level is essential for hematopoietic stem cells. Cell Stem Cell. 2010;7:391–402. doi: 10.1016/j.stem.2010.06.020. [DOI] [PubMed] [Google Scholar]
  53. Ueda T, Sanada M, Matsui H, Yamasaki N, Honda ZI, Shih LY, Mori H, Inaba T, Ogawa S, Honda H. EED mutants impair polycomb repressive complex 2 in myelodysplastic syndrome and related neoplasms. Leukemia. 2012 doi: 10.1038/leu.2012.146. [DOI] [PubMed] [Google Scholar]
  54. Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, Sanda MG, Ghosh D, Pienta KJ, Sewalt RG, Otte AP, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature. 2002;419:624–629. doi: 10.1038/nature01075. [DOI] [PubMed] [Google Scholar]
  55. Wilson A, Laurenti E, Oser G, van der Wath RC, Blanco-Bose W, Jaworski M, Offner S, Dunant CF, Eshkind L, Bockamp E, et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell. 2008;135:1118–1129. doi: 10.1016/j.cell.2008.10.048. [DOI] [PubMed] [Google Scholar]
  56. Wilson BG, Wang X, Shen X, McKenna ES, Lemieux ME, Cho YJ, Koellhoffer EC, Pomeroy SL, Orkin SH, Roberts CW. Epigenetic antagonism between polycomb and SWI/SNF complexes during oncogenic transformation. Cancer Cell. 2010;18:316–328. doi: 10.1016/j.ccr.2010.09.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Xu J, Sankaran VG, Ni M, Menne TF, Puram RV, Kim W, Orkin SH. Transcriptional silencing of {gamma}-globin by BCL11A involves long-range interactions and cooperation with SOX6. Genes Dev. 2010;24:783–798. doi: 10.1101/gad.1897310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Xu K, Wu ZJ, Groner AC, He HH, Cai C, Lis RT, Wu X, Stack EC, Loda M, Liu T, et al. EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycomb-independent. Science. 2012;338:1465–1469. doi: 10.1126/science.1227604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Ye M, Iwasaki H, Laiosa CV, Stadtfeld M, Xie H, Heck S, Clausen B, Akashi K, Graf T. Hematopoietic stem cells expressing the myeloid lysozyme gene retain long-term, multilineage repopulation potential. Immunity. 2003;19:689–699. doi: 10.1016/s1074-7613(03)00299-1. [DOI] [PubMed] [Google Scholar]
  60. Zhang J, Ding L, Holmfeldt L, Wu G, Heatley SL, Payne-Turner D, Easton J, Chen X, Wang J, Rusch M, et al. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature. 2012;481:157–163. doi: 10.1038/nature10725. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

01
NIHMS536972-supplement-01.pdf (8.8MB, pdf)
02
NIHMS536972-supplement-02.xlsx (36.2KB, xlsx)

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