ARTICLES Polycomb Cbx family members mediate the balance between haematopoietic stem cell self-renewal and differentiation Karin Klauke1, Vi²nja Radulovi¢1, Mathilde Broekhuis1, Ellen Weersing1, Erik Zwart1, Sandra Olthof1, Martha Ritsema1, Sophia Bruggeman1, Xudong Wu2, Kristian Helin2, Leonid Bystrykh1 and Gerald de Haan1,3 The balance between self-renewal and differentiation of adult stem cells is essential for tissue homeostasis. Here we show that in the haematopoietic system this process is governed by polycomb chromobox (Cbx) proteins. Cbx7 is specifically expressed in haematopoietic stem cells (HSCs), and its overexpression enhances self-renewal and induces leukaemia. This effect is dependent on integration into polycomb repressive complex-1 (PRC1) and requires H3K27me3 binding. In contrast, overexpression of Cbx2, Cbx4 or Cbx8 results in differentiation and exhaustion of HSCs. ChIP-sequencing analysis shows that Cbx7 and Cbx8 share most of their targets; we identified approximately 200 differential targets. Whereas genes targeted by Cbx8 are highly expressed in HSCs and become repressed in progenitors, Cbx7 targets show the opposite expression pattern. Thus, Cbx7 preserves HSC self-renewal by repressing progenitor-specific genes. Taken together, the presence of distinct Cbx proteins confers target selectivity to PRC1 and provides a molecular balance between self-renewal and differentiation of HSCs. Mature blood cells have a limited lifespan and are continuously unclear. Yet, expression patterns of PcG family members vary between replenished by the activity of HSCs, which have the potential to undergo distinct tissues and between various cell types within the same tissue4–7. both self-renewal and differentiation. Identifying the molecular basis For example, in the haematopoietic system, Bmi1 is specifically of this process will be of great value for a fundamental understanding expressed in immature haematopoietic cells, whereas the expression of normal and malignant haematopoiesis and holds promise for stem of the Bmi1 paralogue Mel18 is increased during differentiation6. This cell-based therapies in the future. argues for the existence of cell-type- and differentiation-stage-specific Proteins that maintain or modify the transcriptional profile of PRC1 and PRC2 subcomplexes with unique molecular functions8–11. stem cells by organizing chromatin structure can be considered as Analysis of the composition of the PRC1 complex showed that it key epigenetic candidates. Polycomb group (PcG) proteins were first contains a single representative of the Cbx family; either Cbx2, Cbx4, discovered as important developmental regulators in Drosophila1. Cbx7 or Cbx8 (refs 12,13). The amino-terminal chromodomain of According to the classical PcG model, they assemble into at least Cbx family members can bind H3K9me3 and H3K27me3 (refs 14,15), two complexes, PRC1 and PRC2, which collaborate to repress gene albeit with unequal affinities16,17. Therefore, the Cbx proteins are key transcription by catalysing histone modifications2,3. PRC2 is comprised components for targeting PRC1 to specific genomic loci to regulate of three core components (Ezh, Suz12 and Eed), of which the gene expression. Whereas several PcG proteins, such as Ezh2 and Bmi1, SET-domain containing Ezh protein contributes to gene repression by have shown to be important for HSC self-renewal6,18–22, no data are tri-methylating histone H3 on Lys 9 and 27 (H3K9me3, H3K27me3). available for how different Cbx subunits contribute to HSC regulation. PRC1 contains Pcgf (for example Bmi1 and Mel18), Phc, Ring1 and By overexpressing individual Cbx family members in HSCs, we Cbx, and catalyses mono-ubiquitylation of H2AK119. have interrogated the biological function of different Cbx-containing Whereas in Drosophila each polycomb component is encoded by PRC1 complexes. We show that the decision of HSCs to self-renew a single gene, the number of genes encoding for PcG proteins has or differentiate critically depends on the molecular composition increased in mammals. However, the relevance of this diversification is of the PRC1 complex. 1European Institute for the Biology of Ageing (ERIBA), Section Ageing Biology and Stem Cells, University Medical Centre Groningen, University of Groningen, Groningen 9700 AD, The Netherlands. 2Biotech Research and Innovation Centre (BRIC), Centre for Epigenetics and DanStem, University of Copenhagen, Copenhagen 2200, Denmark. 3Correspondence should be addressed to G.d.H. (e-mail: [email protected]) Received 24 April 2012; accepted 1 February 2013; published online 17 March 2013; DOI: 10.1038/ncb2701 NATURE CELL BIOLOGY VOLUME 15 j NUMBER 4 j APRIL 2013 353 © 2013 Macmillan Publishers Limited. All rights reserved. ARTICLES RESULTS B-cell reconstitution (Supplementary Fig. S4a), suggesting that Cbx2 Distinct expression of Cbx family members during has a role in lymphopoiesis, but not myelopoiesis. However, HSPCs haematopoietic differentiation that overexpress Cbx2, Cbx4 or Cbx8 all fail to contribute to long-term First, we assessed the expression of Cbx family members in purified haematopoietic reconstitution (Fig. 2a), suggesting a rapid exhaustion haematopoietic subpopulations. Cbx7 is highly expressed in long-term of progenitors and HSCs. In contrast, cells that overexpress Cbx7 HSCs (LT-HSCs), and its transcript levels gradually decreases on lineage have a prominent competitive advantage over non-transduced cells, as commitment (Fig. 1a,b). However, Cbx7 is also highly expressed revealed by high chimaerism levels in peripheral blood (Fig. 2a). in differentiated lymphoid lineages (Fig. 1a and Supplementary Out of 22 mice, 20 reconstituted with Cbx7-overexpressing HSPCs Fig. S2). The Cbx8 transcript is equally expressed in various developed lethal leukaemias with variable latencies (Fig. 2b–d). Twelve primitive haematopoietic cells. However, at the protein level, Cbx8 mice were diagnosed with T-cell leukaemia. These mice showed levels increase on lineage commitment, indicating a possible role increased white blood cell (WBC) counts (Supplementary Fig. S4d for post-transcriptional regulation10,23. Cbx4 is abundant in most and Table S1), enlarged lymph nodes and splenomegaly (Fig. 2c and haematopoietic cell populations whereas Cbx2 is rather marginally but Supplementary Fig. S4d). Thymus and liver were variably affected C C ubiquitously expressed (Fig. 1a,b and Supplementary Fig. S2). Notably, (Supplementary Table S1). Cells were CD3e (Fig. 2d), CD4 and C of all Cbx family members, the Cbx7 transcript is most abundantly CD127 (Supplementary Fig. S4b and Table S1). Interestingly, Cbx7 expressed in LT-HSCs. These data suggest that the PRC1 complex in protein level was found to be elevated in human lymphoid tumours24. HSCs preferentially contains Cbx7. Previously, overexpression of Cbx7 in collaboration with c-Myc, in the lymphoid compartment has been shown to cause T-cell lymphomas24. Cbx family members affect in vitro self-renewal potential Here, we neither observed upregulation of c-Myc nor of other major As Cbx family members compete for integration into PRC1 (refs 12,13), oncogenes involved in lymphomagenesis (Supplementary Fig. S5). overexpression of individual Cbx family members can change the com- The second type of leukaemia developed with a short latency (∼4 position of PRC1 complexes in favour of inclusion of the overexpressed weeks for 3 out of 4 mice) and was classified as an immature leukaemia. Cbx protein. We overexpressed individual Cbx family members in Malignant cells did not express any of the lineage markers used for post-5FU-treated bone marrow (hereafter referred to as hematopoietic analysis (Fig. 2d and Supplementary Fig. S4c and Table S1). Peripheral stem and progenitors cells (HSPCs)). In cytokine-driven cultures, Cbx7 WBC counts were increased (Supplementary Fig. S4d and Table S1) and overexpression results in a strong proliferative advantage (Fig. 1c) and cells showed a blast-like morphology (Supplementary Fig. S4d). Finally, substantially increases the fraction of cells in the S–G2/M phase, without 2 out of 22 mice developed erythroid leukaemias. Spleens were mildly affecting apoptosis (Supplementary Fig. S3a,b). In contrast, Cbx4 or enlarged and one mouse showed haemorrhagic lesions in lymph nodes Cbx8 overexpression leads to decreased proliferation (Fig. 1c and (Fig. 2c). The numbers of peripheral WBCs and erythrocytes were Supplementary Fig. S3a,b), whereas Cbx2 overexpression does not lead decreased (Supplementary Fig. S4d and Table S1), whereas the number to any detectable changes in proliferation (Fig. 1c). In liquid cultures, of reticulocytes was increased (Supplementary Fig. S4d). In addition, Cbx7-overexpressing HSPCs retain an immature blast-like morphology bone marrow and spleen samples revealed numerous erythroid (Supplementary Fig. S3c), whereas most Cbx8 cells differentiate into precursors at variable stages of maturation (Supplementary Fig. S4d). myeloid cells within 10 days (Supplementary Fig. S3c). Malignant GFPC cells expressed Ter119 (Fig. 2d and Supplementary To measure self-renewal, cells were plated in methylcellulose-based Table S1). For two mice it was not possible to determine the leukemia colony assays, followed by whole-dish or single-colony replating. Over- subtype (mice number 2 and 20 in Supplementary Table S1). These expression of Cbx7 substantially increases the colony-forming ability. results show that Cbx7 has a strong oncogenic potential in vivo, whereas This effect is even more pronounced after