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Poised Lineage Specification in Multipotential Hematopoietic Stem

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Poised Lineage Specification in Multipotential Hematopoietic Stem Cell Stem Cell Short Article Poised Lineage Specification in Multipotential Hematopoietic Stem and Progenitor Cells by the Polycomb Protein Bmi1 Hideyuki Oguro,1,2 Jin Yuan,1,2 Hitoshi Ichikawa,4 Tomokatsu Ikawa,5 Satoshi Yamazaki,3,6 Hiroshi Kawamoto,5 Hiromitsu Nakauchi,3,6 and Atsushi Iwama1,2,* 1Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan 2JST, CREST 3JST, ERATO Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan 4Genetics Division, National Cancer Center Research Institute, Tokyo, 104-0045, Japan 5Laboratory for Lymphocyte Development, RIKEN Research Center for Allergy and Immunology, Yokohama 230-0045, Japan 6Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Sciences, University of Tokyo, Tokyo 108-8679, Japan *Correspondence: [email protected] DOI 10.1016/j.stem.2010.01.005 SUMMARY Pietersen and van Lohuizen, 2008). They reside in two main complexes, termed Polycomb repressive complex 1 and 2 Polycomb group (PcG) proteins are essential regula- (PRC1 and PRC2). PRC2 and trithorax group (trxG) proteins tors of stem cells. PcG and trithorax group proteins mark developmental regulator gene promoters with bivalent mark developmental regulator gene promoters with domains consisting of overlapping repressive and activating bivalent domains consisting of overlapping repres- histone modifications to keep developmental regulators sive and activating histone modifications to keep ‘‘poised’’ for activation in embryonic stem cells (ESCs) (Bernstein them poised for activation in embryonic stem cells. et al., 2006; Spivakov and Fisher 2007; Mendenhall and Bern- stein, 2008). Likewise, in adult stem cells, developmental regula- Bmi1, a component of PcG complexes, maintains tors that govern lineage specification are supposedly repressed the self-renewal capacity of adult stem cells, but its epigenetically to maintain their multipotency (Pietersen and van role in multipotency remains obscure. Here we Lohuizen, 2008). Among PcG proteins, Bmi1, a component of show that Bmi1 is critical for multipotency of hemato- PRC1, and its role in the inheritance of the stemness of adult poietic stem cells (HSCs) and multipotent progeni- stem cells have been well characterized (Molofsky et al., 2003; tors (MPPs). B cell lineage developmental regulator Park et al., 2003; Iwama et al., 2004). Bmi1 maintains the self- genes, Ebf1 and Pax5, appeared to be transcription- renewal capacity of adult stem cells, at least partially, by repres- ally repressed by bivalent domains before lineage sing the Ink4a/Arf locus, which encodes a cyclin-dependent Ink4a Arf commitment. Loss of Bmi1 resulted in a resolution kinase inhibitor, p16 , and a tumor suppressor, p19 of bivalent domains at the Ebf1 and Pax5 loci, leading (Jacobs et al., 1999; Oguro et al., 2006). Deletion of both Ink4a to their premature expression in HSC/MPPs accom- and Arf in Bmi1-deficient mice substantially restored the defec- tive self-renewal capacity of HSCs (Oguro et al., 2006) and neural panied by accelerated lymphoid specification and stem cells. On the other hand, comprehensive genome-wide a marked reduction in HSC/MPPs. Thus, Bmi1 is analyses demonstrated that PcG proteins directly repress a large required to reinforce bivalent domains at key devel- cohort of developmental regulators in ESCs and, by doing so, opmental regulator gene loci to maintain lineage maintain ESCs in an undifferentiated pluripotent state (Boyer specification poised for activation in adult stem cells. et al., 2006; Lee et al., 2006). In adult stem cells, however, target genes for Bmi1 involved in stem cell functions other than Ink4a/ Arf have not been well documented, and the contribution of Bmi1 INTRODUCTION to the multipotency and lineage specification of stem cells remains unclear. Here we show that Bmi1 plays a crucial role All hematopoietic cells are generated from hematopoietic stem in preventing premature lineage specification of HSCs and multi- cells (HSCs) via a hierarchy of progenitor populations with potent progenitors (MPPs) through reinforcement of bivalent restricted differentiation potential. Although many transcription domains at key developmental gene loci. factors that play key roles in the lineage commitment of HSCs have been identified, the precise regulation of their expression RESULTS in HSCs remains to be elucidated (Dias et al., 2008; Laslo et al., 2008; Orkin and Zon, 2008). Polycomb group (PcG) Premature Derepression of B Cell Lineage-Regulator proteins are involved in the maintenance of gene silencing Genes in Bmi1-Deficient Multipotent Progenitors through chromatin modifications and have been implicated in Target genes for Bmi1 involved in stem cell functions other than stem cell self-renewal (Sparmann and van Lohuizen, 2006; Ink4a/Arf have not been well documented. To address this issue, Cell Stem Cell 6, 279–286, March 5, 2010 ª2010 Elsevier Inc. 279 Cell Stem Cell Bmi1 Inhibits Premature Differentiation of HSCs -/- Figure 1. Premature Derepression of B -/- À/À AB Lineage Regulator Genes in Bmi1 Ink4a- -/- À/À -/- Arf HSCs/MPPs Ink4a-Arf -/- -/- Ink4a-Arf -/- -/- (A) Heat map of the expression of B lineage- O Wild-type Ink4a-Arf Bmi1 Bmi1 2 associated genes in a microarray analysis of H Wild-typeInk4a-ArfBmi1 Bmi1 À Pou2af1 (OBF1) IL-7Ra KSL HSCs/MPPs. Red, higher expression; Sfpi1 Igll1 (l5) green, lower expression than wild-type average. Vpreb1 Ebf1 Tcfe2a (B) Semiquantitative RT-PCR analysis of B lineage Rag1 St6gal1 (CD75) Il7r developmental genes, Bmi1, Ink4a, and Arf in wild- Ms4a1 (CD20) À/À À/À À/À Ebf1 type, Ink4a-Arf , Bmi1 , and Bmi1 Ink4a- Cd19 À/À À Vpreb3 Arf IL-7Ra KSL cells. Pax5 Blnk (C and D) Quantitative RT-PCR analysis of Ebf1 (C) Irf4 Pax5 Cd79a and Pax5 (D). mRNA levels in indicated progenitor Lef1 Blk Igll1 fractions purified from recipient mouse BM repo- Zfpn1a3 (Aiolos) À/À À/À Vpreb1 pulated by wild-type, Ink4a-Arf , and Bmi1 Ighg À/À Vpreb1/Vpreb2 Ink4a-Arf BM cells were normalized to Hprt1 Bmi1 Prdm1 (Blimp1) expression. Expression levels relative to those Cd79a (mb-1) Il7r Ink4a in the wild-type pro-B cells are shown as the Cd79b (B29) mean ± standard deviation (SD) for triplicate Tcfe2a (E2A) Arf Zfpn1a1 (Ikaros) analyses. **p < 0.01. See also Figure S1. Gapdh Sfpi1 (PU.1) <1/8 1/4 1/2 1 2 4 8< C D to those in wild-type pre-B cells (Figures 1 Wild-type 1 Wild-type 1C and 1D). To confirm these findings at ) ) -/- -/- ** Ink4a-Arf Ink4a-Arf a single-cell level, we compared the dere- -/- -/- -/- -/- Hprt1 Hprt1 Bmi1 Ink4a-Arf ** Bmi1 Ink4a-Arf pressed Pax5 protein levels by immunos- expression expression 0.1 0.1 taning with an ArrayScan, a cell image Ebf1 Pax5 analyzer designed for high-capacity 0.01 0.01 (normalized to (normalized to automated fluorescence imaging and Relative Relative quantitative analysis. Two hundred cells were scanned and the fluorescence - KSL KSL CLP - KSL KSL CLP + pre-B CMP GMP MEP + pre-B CMP GMP MEP Flt3 Flt3 intensity was plotted in Figure S1C. Dere- Flt3 ETP/DN2 Flt3 ETP/DN2 pression of Pax5 protein was evident in HSC/MPPs and LMPPs compared to we compared gene expression profiles of HSC/MPP cells in IL- levels of Pax5 in wild-type cells. Common lymphoid progenitors 7RaÀc-Kit+Sca-1+LineageÀ (IL-7RaÀKSL) fractions from bone (CLPs) that possess both B and T lymphoid potential also ex- marrow (BM) of wild-type, Ink4a-ArfÀ/À, Bmi1À/À, and Bmi1À/À pressed significantly higher levels of Ebf1 and Pax5 in the Ink4a-ArfÀ/À mice. Unexpectedly, a set of genes specific to the absence of Bmi1, whereas Ebf1 and Pax5 were strictly silenced B lymphoid lineage, but not T lymphoid or myeloid lineage, in T cell lineage progenitors. These results suggest that Bmi1 were prematurely derepressed in both Bmi1À/À and Bmi1À/À functions in the silencing of Ebf1 and Pax5 in HSC/MPP/LMPPs Ink4a-ArfÀ/À HSC/MPPs (Figure 1A; Figures S1A and S1B avail- and myeloid progenitors and that Ink4a and Arf are not involved able online). Among these genes, Ebf1 and Pax5 are B cell- in the transcriptional regulation of these genes. specific master transcription factor genes that function in B lineage specification at the earliest stage of B cell differentiation, Direct Repression of Ebf1 and Pax5 Expression and other genes are their downstream targets (Dias et al., 2008; by Bmi1 through Repressive Histone Modifications Laslo et al., 2008). Semiquantitative RT-PCR confirmed that at Their Promoters Ebf1, Pax5, and downstream targets were derepressed in To confirm this, we characterized the Ebf1 and Pax5 promoters Bmi1À/À and Bmi1À/ÀInk4a-ArfÀ/À IL-7RaÀKSL cells, while the by conducting chromatin immunoprecipitation (ChIP) assays. To expression of upstream genes, Sfpi1 (PU.1), Tcfe2a (E2a), and obtain enough cells, we used BM lineage marker-negative (Lin- Il7r, was not altered (Figure 1B). We next quantified the Ebf1 eageÀ) progenitors depleted of cells committed to the lymphoid, and Pax5 expression in each progenitor fraction by quantitative myeloid, and erythroid lineages. Binding of Bmi1 to both of the RT-PCR. Because the BM environment of Bmi1À/À and Bmi1À/À Ebf1 promoters (Roessler et al., 2007) and to the Pax5 promoter Ink4a-ArfÀ/À mice is unable to support HSCs (Oguro et al., 2006), was detected, but not to the promoter of Il7r, a gene upstream of we purified progenitors from wild-type recipient BM reconsti- Ebf1 (Figures 2A and 2B). Bivalent domains in ESCs consist of tuted with Bmi1À/ÀInk4a-ArfÀ/À BM cells to exclude the environ- a repressive mark, trimethylation of histone H3 at lysine 27 mental effect. Ebf1 and Pax5 were derepressed in Flt3ÀIL- (H3K27me3) catalyzed by PRC2, and an active mark, trimethyla- 7RaÀKSL HSC/MPPs and Flt3+IL-7RaÀKSL lymphoid-primed tion of histone H3 at lysine 4 (H3K4me3) catalyzed by trxG multipotent progenitors (LMPPs), and even in myeloid proteins.
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