Ezh1 Is Required for Hematopoietic Stem Cell Maintenance and Prevents Senescence-Like Cell Cycle Arrest

Cell Stem Cell Article Ezh1 Is Required for Hematopoietic Stem Cell Maintenance and Prevents Senescence-like Cell Cycle Arrest Isabel Hidalgo,1,6 Antonio Herrera-Merchan,1,6 Jose Manuel Ligos,2 Laura Carramolino,3 Javier Nun˜ ez,4 Fernando Martinez,4 Orlando Dominguez,5 Miguel Torres,3 and Susana Gonzalez1,* 1Stem Cell Aging Group 2Cellomics Unit 3Genetic Control of Organ Development and Regeneration 4Bioinformatics Unit Centro Nacional de Investigaciones Cardiovasculares (CNIC), E-28029 Madrid, Spain 5Genomics Unit, Centro Nacional de Investigaciones Oncologicas (CNIO), E-28029 Madrid, Spain 6These authors contributed equally to this work *Correspondence: sgonzalez@cnic.es http://dx.doi.org/10.1016/j.stem.2012.08.001 SUMMARY known about how epigenetic mechanisms dictate the activities of Polycomb factors to ensure blood homeostasis. Polycomb group (PcG) proteins are key epigenetic PcG proteins assemble in multimeric complexes and induce regulators of hematopietic stem cell (HSC) fate. The transcriptional repression of target genes through chromatin PcG members Ezh2 and Ezh1 are important determi- changes such as methylation of lysine 27 on histone 3 (H3K27). nants of embryonic stem cell identity, and the tran- Biochemical analysis revealed that PcG proteins assemble in at script levels of these histone methyltransferases least two complexes, Polycomb repressive complexes 1 and 2 are inversely correlated during development. How- (PRC1 and PRC2). Studies of mouse mutants showed that these factors are essential for normal development, with deletion of ever, the role of Ezh1 in somatic stem cells is largely Polycomb genes resulting in prenatal or early postnatal death, unknown. Here we show that Ezh1 maintains repopu- depending on inactivated gene and how the inactivation is lating HSCs in a slow-cycling, undifferentiated state, achieved (Sauvageau and Sauvageau, 2010). The PRC2 complex protecting them from senescence. Ezh1 ablation contains one of two homologous enhancer of zeste (Ez) pro- induces significant loss of adult HSCs, with concom- teins (Ezh1 or Ezh2), which contain near identical catalytic SET itant impairment of their self-renewal capacity due domains (Laible et al., 1997). Ezh1 and Ezh2 are chromatin-modi- to a potent senescence response. Epigenomic and fying histone lysine methyltransferases (Margueron and Rein- gene expression changes induced by Ezh1 deletion berg, 2011). Most research on H3K27 has focused on the in senesced HSCs demonstrated that Ezh1-medi- trimethylated form (H3K27me3), while the monomethylated and ated PRC2 activity catalyzes monomethylation and dimethylated forms (H3K27me1 and H3K27me2, respectively) dimethylation of H3K27. Deletion of Cdkn2a on the remain poorly characterized (Margueron and Reinberg, 2011). Although Ezh2 knockout in embryonic stem cells (ESCs) reduces Ezh1 null background rescued HSC proliferation H3K27 dimethylation and trimethylation, H3K27 methylation at and survival. Our results suggest that Ezh1 is an PcG target promoters is not abolished. This has been attributed important histone methyltransferase for HSC mainte- to the activity of Ezh1, which forms an alternative PRC2 com- nance. plex (Shen et al., 2008) and has distinct chromatin-binding prop- erties (Margueron et al., 2008). The importance of the H3K27me3 mark has been characterized through conditional targeting of INTRODUCTION Ezh2 in somatic stem cells in the brain, pancreas, epidermis, and hematopoietic system (Chen et al., 2009; Ezhkova et al., Epigenetic regulation of chromatin structure is thought to con- 2009; Mochizuki-Kashio et al., 2011; Pereira et al., 2010; Su trol cell memory, and Polycomb group (PcG) proteins are impor- et al., 2003). Double knockout of Ezh1 and Ezh2 in mouse skin tant components of diverse epigenetic phenomena, including abolishes the H3K27me3 modification and severely compro- genomic imprinting, cell fate determination, stem-cell plasticity mises formation and maintenance of hair follicles (Ezhkova and renewal, and cancer development (Goldberg et al., 2007). et al., 2011). However, the role of PRC2-Ezh1 in the maintenance PcG-mediated epigenetic alteration of hematopoietic stem cells of HSCs is unknown. (HSCs) is considered a driving force behind many age-related Cell senescence is a permanent cell-cycle arrest that is re- HSC changes and is often dysregulated in human malignancies fractory to growth factors and other proliferation signals. Un- (Sauvageau and Sauvageau, 2010). Protection of the transcrip- like apoptotic cells, which rapidly disintegrate, senescent cells tional ‘‘stemness’’ network is thus essential for the maintenance remain viable in long-term culture and are found with increasing of a healthy HSC compartment throughout life. However, little is frequency in aged tissues and at sites of age-related pathology, Cell Stem Cell 11, 649–662, November 2, 2012 ª2012 Elsevier Inc. 649 Cell Stem Cell HSC Senescence Protection by Ezh1 A 4 young HSCs B C Ezh1 wt locus aged HSCs 14 15 16 17 18 3 19 20 21 Ezh1 Additional Additional 2 HindIII ScaI ScaI * HindIII * Ezh2 1 14 15 16 17 18 Neo 19 20 21 Relative Ezh1 levels mRNA WB Suz12 0 Ezh1 targeting vector 2 Eed 1 β-actin 0 13 kDa Relative Ezh2 levels mRNA wt HSCs (LSK) Lin- LSK LT-HSC MP CLP 350 pb 8.5 kDa 300 pb 7.6 kDa D HSCs E 5.7 kDa + - (LSKCD150 CD48 ) Ezh1 Ezh2 WB H3K27me3 H3K27me2 H3K27me1 H3 Ct (Ezh1-KO vs. Control) ΔΔ HSCs - INK4a INK4b (LSKCD150+CD48-) p15 Ezh2 Ezh1 p16 ARF F H&E Ki67 Ezh1 Bone marrow Ezh1-KO Control G Control Ezh1 MP LSK MP LSK MPP 46.7 % MPP 32.2 % 9.2 % CLP 3.7 % LT LT ST ST 38.8 % 43.1 % 23.4 % 26.2 % cKit cKit CD150 CD150 CD135 CD135 Sca1 CD34 Sca1 CD34 GMP CD48 CD48 CLP GMP 15.3 % 18.8% CLP MEP 32.9 % MEP 23.6 % CMP CMP 41.6 % 47.3 % CD16/32 68.4% CD16/32 63.7 % CD34 CD127 CD34 CD127 20 H Control I 15 Control Ezh1-KO Ezh1-KO cells) 10 6 (x10 5 Total in BM Total Total in BM cells Total 0 LSK LT ST MPP CD150+ CLP CMP MEP GMP CD48- Figure 1. Characterization of Primitive Hematopoietic Populations in Ezh1-KO Mice (A) Expression of total Ezh2 and Ezh1 mRNA was measured by qRT-PCR in sorted bone marrow (BM) subpopulations from young (8-week-old) and aged (50-week-old) WT mice: lineage negative (LinÀ); LinÀ, Sca1+, c-Kit+ (LSK); long-term repopulating HSCs (LSKCD34lowFlk-2low; LT-HSCs), myeloid progenitors (LinÀKit+Sca1À; MPs), and common lymphoid progenitors (LinÀKitlowSca1lowCD127+; CLPs). Results are standardized to b-actin and are expressed as the fold change of the indicated subpopulations from aged mice compared with the same gated subpopulations from young mice (means ± SD; n = 12, *p < 0.05). 650 Cell Stem Cell 11, 649–662, November 2, 2012 ª2012 Elsevier Inc. Cell Stem Cell HSC Senescence Protection by Ezh1 including preneoplastic lesions. Cells senesce in response to the regulation of histone H3K27 methylation. Our results indicate many potentially oncogenic stressors, including severe DNA that Ezh1 promotes a slow cycling state in primitive HSCs and damage, dysfunctional telomeres, chromatin alterations, oxida- prevents differentiation and senescence of adult HSCs. tive stress, and strong mitogenic signals such as those delivered by some oncogenes (Collado et al., 2007). Induction of the RESULTS senescence program normally requires activation of tumor sup- pressor pathways, including the p19ARF (p14ARF in humans), p53, Expression of the Histone Methyltransferase Ezh1 and RB-p16INK4a signaling cascades, and the senescence pro- Increases during HSC Aging gram is now regarded as a potent cell-autonomous barrier to Since expression of several PcG genes involved in chromatin tumorigenesis (Prieur and Peeper, 2008). Accordingly, loss of organization becomes dysregulated during the aging of HSCs the senescence response increases the incidence of cancer in (Chambers et al., 2007b), we hypothesized that histone methyl- humans and mice (Nardella et al., 2011). Moreover, cell se- transferase activity might be coordinately distributed between nescence also entails increased secretion of proinflammatory Ezh1 and Ezh2 in young and old HSC populations. Ezh1 and proteins, inducing the senescence-associated (SA) secretory Ezh2 mRNAs were detected in primitive long-term HSCs (LT- phenotype (SASP), and might contribute significantly to chronic HSCs) isolated from the bone marrow (BM) of young (8-week- inflammation (Freund et al., 2010). old) wild-type C57BL/6 mice; but while levels of Ezh1 mRNA Epigenetic mechanisms play an important role in senescence. were 3-fold higher in LT-HSCs isolated from BM of 50-week- In senescing human fibroblasts, heterochromatic regions con- old mice, Ezh2 transcript levels did not change with age (p < dense to form senescence-associated heterochromatic foci 0.05; Figure 1A). A similar divergence was seen with protein that limit the DNA damage response (Di Micco et al., 2011; Narita expression (Figure 1B), as noted in other studies on human et al., 2003). Expression of Jhdm1b, a demethylase specific for and mouse HSCs (Mochizuki-Kashio et al., 2011; Wagner the H3K36me2 modification, causes cell immortalization or et al., 2009) and mouse kidney (Margueron et al., 2008). Protein leukemic transformation depending on its demethylase activity levels of the other PRC2 components, Suz12 and Eed, did not on p15INK4b, and its knockdown results in cell senescence (He change with age (Figure 1B). Given this pattern of Ezh1 expres- et al., 2008). In human and mouse fibroblasts, the Cdkn2a region sion in repopulating HSCs, we hypothesized that Ezh1 might act (encoding p16INK4a and ARF) is repressed by PcG-mediated primarily as a homeostatic regulator of HSCs. H3K27me3, and the repressive mark is lost during oncogene- To test this, we generated a conditional knockout Ezh1 allele in induced senescence, resulting in Cdkn2a expression (Bracken which exons encoding the SET catalytic domain were flanked et al., 2007). with loxP sequences (ckoEzh1fl/fl)(Figure 1C). To induce spe- The incomplete loss of H3K27me3 in Ezh2À/À ESCs, the rela- cific Ezh1 deletion in the hematopoietic system, we crossed tively mild phenotypes of tissues lacking Ezh2, and the inverse ckoEzh1fl/fl mice with vavCre transgenic mice.

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