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The crucial molecular mechanisms that control stem cell fate have received widespread attention because these mechanisms could potentially be manipulated to engineer stem cell biology for thera-peutic interventions or tissue repair. Moreover, increasing evidence indicates that many tumors are sustained by cancer stem cells (CSCs) whose self-renewal may be controlled by mechanisms similar to those that control normal stem cells1,2.

The hematopoietic system provides a paradigm for studying molec-ular mechanisms controlling stem cell function3,4. Lifelong replen-ishment of all hematopoietic cells is maintained by HSCs, which in a tightly controlled process give rise to a hierarchy of multipotent and lineage-committed progenitors5. Regulation of the diverse func-tional repertoire of HSCs requires the coordinated action of tran-scription factors6. The activity of most transcription factors relies on the recruitment of cofactors, many of which control gene expression by catalyzing epigenetic modifications of chromatin7. However, the functional impact of epigenetic modification mechanisms on coordi-nation of stem cell fate programs is still poorly understood.

Methylation of CpG dinucleotides within the DNA is a major epi-genetic modification, which in mammals is controlled by at least three different DNA methyltransferases (DNMTs): DNMT3a and DNMT3b for de novo methylation, and DNMT1 for methylation maintenance8. The impact of methylation on stem cell features has been studied in embryonic stem cells, but little is known about its function in somatic stem cells in vivo9,10. Recent advances in the genome-wide mapping

of DNA methylation revealed that methylated CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation11. However, whether and how these changes are required for cell fate choices, particularly with respect to stem cells, remains unknown. Moreover, altered DNA methylation is a hallmark of cancer, and drugs targeting methylating enzymes are used in cancer therapy. However, the relationship between tumor-associated altera-tions in methylation and CSC properties is still elusive.

Here we address this issue using mice with gradually diminished Dnmt1 expression. We show that distinct methylation threshold levels are required for alternative fate decisions of both HSCs and CSCs. The data suggest that competing stem cell programs require differ-ent methylation dosage–dependent control mechanisms and identify CpG methylation as a shared epigenetic program in the control of normal and neoplastic stem cells.

RESULTSDNMT1 is indispensable for cell-autonomous survival of HSCsHSCs express high levels of Dnmt1, the major methyltransferase of postnatal mammalian cells10. To investigate the role of DNA methyla-tion in HSCs, we bred mice in which exons 4 and 5 of Dnmt1 were flanked by loxP sites12 with mice expressing Cre recombinase under the control of the type I interferon–inducible Mx1 promoter13 (trans-gene officially named Tg(Mx1-cre); referred to here as MxCre). This strategy allowed inducible deletion of the catalytic Dnmt1 domain

DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restrictionAnn-Marie Bröske1,7, Lena Vockentanz1,7, Shabnam Kharazi2, Matthew R Huska1, Elena Mancini3, Marina Scheller1, Christiane Kuhl1, Andreas Enns1, Marco Prinz4, Rudolf Jaenisch5, Claus Nerlov3, Achim Leutz1, Miguel A Andrade-Navarro1, Sten Eirik W Jacobsen2,6 & Frank Rosenbauer1

DNA methylation is a dynamic epigenetic mark that undergoes extensive changes during differentiation of self-renewing stem cells. However, whether these changes are the cause or consequence of stem cell fate remains unknown. Here, we show that alternative functional programs of hematopoietic stem cells (HSCs) are governed by gradual differences in methylation levels. Constitutive methylation is essential for HSC self-renewal but dispensable for homing, cell cycle control and suppression of apoptosis. Notably, HSCs from mice with reduced DNA methyltransferase 1 activity cannot suppress key myeloerythroid regulators and thus can differentiate into myeloerythroid, but not lymphoid, progeny. A similar methylation dosage effect controls stem cell function in leukemia. These data identify DNA methylation as an essential epigenetic mechanism to protect stem cells from premature activation of predominant differentiation programs and suggest that methylation dynamics determine stem cell functions in tissue homeostasis and cancer.

1Max Delbrück Center for Molecular Medicine, Berlin, Germany. 2Hematopoietic Stem Cell Laboratory, Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden. 3European Molecular Biology Laboratory, Mouse Biology Unit, Monterotondo, Italy. 4Department of Neuropathology, University of Freiburg, Freiburg, Germany. 5The Whitehead Institute, Cambridge, Massachusetts, USA. 6Haematopoietic Stem Cell Laboratory, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, England. 7These authors contributed equally to this work. Correspondence should be addressed to F.R. (f.rosenbauer@mdc-berlin.de).

Received 3 April; accepted 25 August; published online 4 October 2009; doi:10.1038/ng.463

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upon administration of the interferon-α inducer polyinosinic- polycytidylic acid (poly(I:C)), thus creating a functional knockout of the gene in hematopoietic cells (Supplementary Fig. 1a,b).

Poly(I:C) injection of 6-week-old Dnmt1lox/lox; MxCre mice (here-after designated Dnmt1∆/∆ when treated with poly(I:C)) resulted in rapid death of all mice (Fig. 1a). In contrast, all similarly treated Dnmt1lox/lox control mice (without the MxCre transgene) survived this procedure. Peripheral blood samples of moribund Dnmt1∆/∆ mice showed anemia (hemoglobin levels: Dnmt1lox/lox, 12.6 ± 1.7 g/dl; Dnmt1∆/∆, 7.9 ± 2.5 g/dl; n = 7 per group; P = 0.001) and cytopenia (white blood cell counts: Dnmt1lox/lox, 11.2 ± 3.6 × 103 cells/µl, Dnmt1∆/∆, 2.9 ± 1.4 × 103 cells/µl; n = 7 per group; P < 0.001). Histological analysis of sternum sections revealed profound bone marrow cytopenia (Fig. 1b), which was caused by depletion of cells from all major bone marrow lineages (Fig. 1c). Cell counts from bone marrow and spleen suspensions confirmed hemat-opoietic pancytopenia (Supplementary Fig. 1c). Further analysis of the bone marrow lineage-negative (Lin−) compartment revealed a near-complete absence of phenotypic HSCs and early progenitors in Dnmt1∆/∆ mice (Fig. 1d). Moreover, mutant bone marrow cells were unable to generate myeloid and B-lymphoid colonies in vitro, indi-cating that Dnmt1 ablation completely disrupted the differentiation potential of hematopoietic stem and progenitor cells (Fig. 1e).

We next tested whether the severe hematopoietic phenotype was caused by cell-intrinsic DNMT1 effects in vivo. We generated chimeric mice by transplanting untreated bone marrow cells of Dnmt1lox/lox; MxCre or Dnmt1lox/lox control mice into lethally irradiated, congenic wild-type recipients. We confirmed stable engraftment of the donor cells 8 weeks after transplantation (Supplementary Fig. 1d) and subsequently administered poly(I:C) to selectively delete Dnmt1 in

hematopoietic cells. As in primary Dnmt1∆/∆ mutants, Dnmt1 loss in chimeric mice led to profound hematopoietic cell depletion, followed by rapid death of all mice (Fig. 1f and data not shown). Finally, staining of bone marrow cells from poly(I:C)-injected Dnmt1∆/∆ mice with antibody to cell-surface annexin V revealed that rapid bone mar-row failure was caused by apoptosis (Fig. 1g). Collectively, these data indicate that complete Dnmt1 deletion eliminates HSCs and bone marrow progenitors by induction of cell-autonomous apoptosis.

DNA methylation maintains homeostasis within the HSC poolGiven the short life span and complete HSC loss in Dnmt1∆/∆ mice, we next combined a hypomorphic Dnmt1 allele (Dnmt1chip) with a null allele14. Dnmt1−/chip mice were viable, expressed low amounts of Dnmt1 and showed DNA hypomethylation in multiple tissues at levels substantially milder than those in Dnmt1-null cells14 (Supplementary Fig. 2a,b).

Dnmt1−/chip mice had normal frequencies of LSK (Lin−Sca-1+ c-Kit+) cells, indicating that hypomorphic Dnmt1 expression is suf-ficient to rescue HSC formation (Fig. 2a). However, further analysis revealed a markedly skewed distribution of mutant HSC subpopu-lations. Frequencies of short-term HSCs (ST-HSCs) were normal, whereas long-term HSCs (LT-HSCs) were enriched by 2.7-fold and lymphoid-primed multipotential progenitors (LMPPs)—representing the earliest stage of lymphoid transcriptional priming in the hemato-poietic hierachy15,16—were virtually absent (Fig. 2b). Because Flt3 transcript numbers were normal in Dnmt1−/chip HSCs, the lack of LMPPs was not likely to have resulted from reduced Flt3 expres-sion (Supplementary Fig. 3a). Notably, Dnmt1+/+ and Dnmt1−/chip LT-HSCs expressed CD150 (a SLAM family protein that is the most stringent marker for HSCs17) at similar levels, suggesting that both subsets are uniform populations of phenotypic stem cells (Supplementary Fig. 3b).

We detected no significant alterations in cell cycle or apoptosis rates of Dnmt1−/chip HSCs (Supplementary Fig. 4a,b). Furthermore, breeding of the antiapoptotic Tg(H2-K-BCL2) transgene18 (referred to here as H2K-Bcl2) into the Dnmt1−/chip line did not correct the

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Figure 1 DNMT1 is indispensable for early hematopoiesis. (a) Cumulative survival of poly(I:C)-injected Dnmt1lox/lox; MxCre (Dnmt1∆/∆; n = 21) and Dnmt1lox/lox control (n = 19) mice. Arrowheads indicate time points of poly(I:C) administration. (b) Massive bone marrow hypoplasia in moribund Dnmt1∆/∆ mice 7 d after first poly(I:C) injection (hematoxylin and eosin stain; original magnification, ×400). (c) Numbers of B-lymphoid (B220+), myeloid (Mac1+Gr1+) and erythroid (Ter119+) cells in bone marrow of Dnmt1∆/∆ and Dnmt1lox/lox mice, as determined by FACS. Values in c and d are mean ± s.d. *P ≤ 0.05; **P ≤ 0.001; n = 4 per group. (d) FACS results showing near-complete absence of phenotypic HSCs (Lin−Sca-1+c-Kit+) and progenitors (Lin−Sca-1−c-Kit+) from bone marrow of Dnmt1∆/∆ mice 7 d after poly(I:C) administration. (e) DNMT1 loss blocks myeloid and B-lymphoid differentiation in vitro. Bone marrow cells from Dnmt1lox/lox or Dnmt1∆/∆ mice were plated in methylcellulose containing either 10 ng/ml interleukin (IL)-3, 10 ng/ml IL-6 and 50 ng/ml stem cell factor (SCF) to promote myeloid differentiation, or 10 ng/ml IL-7 to promote differentiation of pre-B cells. Each dot represents average colony number of one mouse from triplicate platings. Bars indicate mean values. P ≤ 0.001 each. (f) Rapid lethality by hematopoietic cell–intrinsic Dnmt1 loss. Bone marrow cells (1–2 × 106 total) from untreated Dnmt1lox/lox; MxCre and control mice were injected into lethally irradiated wild-type mice. Stably engrafted mice were injected five times with poly(I:C) (arrowheads) 6 weeks after transplantation. n = 8–9 per group. (g) Rates of apoptosis were determined in bone marrow cells of Dnmt1∆/∆ and Dnmt1lox/lox mice by annexin V and 4′,6-diamidino-2-phenylindole (DAPI) co-staining. Values are mean percentages (n = 2). Mean fluorescence intensities (MFI) for annexin are indicated above.

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mutant HSC phenotype (Supplementary Fig. 4c). Collectively, these results indicated that gradually reduced Dnmt1 alters HSC homeostasis by a mechanism independent of effects on cell cycle or apoptosis.

HSC self-renewal requires constitutive DNA methylationWe next tested whether Dnmt1−/chip HSCs have altered stem cell func-tion, using a series of adoptive transplantation assays. Competitive repopulation analysis revealed that Dnmt1−/chip donor cells have a >99.5% (1:1 competition) lower ability than do control cells to reconstitute hematopoietic cells (Fig. 2c and Supplementary Fig. 5a). Furthermore, the repopulating efficiency of Dnmt1−/chip cells progres-sively decreased with each generation in a serial transplantation assay, whereas control donor-derived hematopoiesis remained constant (Fig. 2d). This revealed that low Dnmt1−/chip HSC activity is caused by impaired self-renewal.

In a competitive short-term (24 h) engraftment assay, bone marrow cells from Dnmt1−/chip and control mice showed equal contribution to the bone marrow progenitor chimerism in recipients, indicating that HSC homing is unaffected by hypomethylation (Supplementary Fig. 5b). We used a conditional genetic approach to formally exclude the possibility that altered homing causes impaired repopulation ability. We generated Dnmt1lox/chip; MxCre and Dnmt1lox/chip control mice and used their untreated bone marrow cells for repopulation, thus permitting normal homing of the stem cells (Supplementary Fig. 5c). We confirmed stable donor cell engraftment and injected poly(I:C) to induce hypomethylation after Cre-mediated excision

of the Dnmt1lox allele (hereafter termed Dnmt1∆). After poly(I:C) administration, numbers of Dnmt1∆/chip donor-derived peripheral blood cells decreased (Fig. 2e). Analysis of the donor-derived bone marrow compartment at the experimental endpoint confirmed excised donor cells and revealed a 90% decline in Dnmt1∆/chip LSK cell numbers, indicating that constitutive methylation is essential for HSC self-renewal, even in the absence of transplantation stress (Fig. 2f and Supplementary Fig. 5d).

Methylation regulates myeloerythroid versus lymphoid fateRecent evidence has indicated that the transition from multipotent HSCs to LMPPs is accompanied by loss of megakaryocyte-erythroid potential and a corresponding shift from megakaryocyte-erythroid to lymphoid transcriptional priming16. Consequently, the absence of LMPPs in Dnmt1−/chip mice suggested an unanticipated effect of HSC methylation status on the choice between myeloerythroid and lymphoid lineages. Indeed, although frequencies of differentiated myeloid and erythroid cells were not affected in Dnmt1−/chip mice, B cell frequencies were lower than in control mice (Fig. 3a). Similarly, whereas myeloerythroid progenitor frequencies were comparable in Dnmt1−/chip and control mice, common lymphoid progenitors as well as B- and T-lineage–restricted progenitors were diminished in Dnmt1−/chip mice (Fig. 3b,c). Dnmt1−/chip Lin− bone marrow progeni-tors had reduced expression of genes encoding lymphocyte progenitor markers, whereas genes encoding myeloid progenitor markers were expressed at normal levels (Fig. 3d). Notably, Dnmt1−/chip progenitors lacked expression of essential B-cell factors (such as Pax5; ref. 19)

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Figure 2 DNA methylation is required for HSC homeostasis and self-renewal. (a,b) Total HSCs (LSK, Lin−Sca-1+c-Kit+) from 8- to 12-week-old Dnmt1+/+ and Dnmt1−/chip mice (a) were separated into LT-HSCs (LSK CD34−/lowFlt3−), ST-HSCs (LSK CD34+Flt3−) and LMPPs (LSK CD34+Flt3hi; b). Numbers represent mean ± s.d. of three mice each. Three independent experiments were done. (c) Bone marrow cells from Dnmt1−/chip mice are outcompeted by age-matched wild-type cells in competitive transplantation assays. Either 8 × 105 (5:1), 4 × 106 (1:1) or 1.6 × 107 (1:4) B6SJL CD45.1+ wild-type competitor cells were mixed with 4 × 106 CD45.2+ test cells from Dnmt1−/chip or Dnmt1+/chip mice and injected intravenously into lethally irradiated (10.5 Gy) F1 129ola/B6.SJL CD45.1+CD45.2+ recipient mice. Data show frequencies of CD45.2+ donor cells in peripheral blood of recipients 12 weeks after transplantation. n = 3–5 per group. Data in c–f are mean ± s.d.; *P ≤ 0.05, **P ≤ 0.001. (d) Dnmt1−/chip HSCs show impaired long-term reconstitution capacity in serial transplantation assays. Four rounds of transplantation with 5 × 106 bone marrow cells each were done in 12- to 16-week intervals. Bar graphs reflect donor chimerism in peripheral blood 4 weeks after transplantation. n = 5–7 per group. (e) Inducible bone marrow chimeras confirm an intrinsic and proliferation-independent deficiency in long-term maintenance of hypomethylated HSCs. Bone marrow cells (1 × 106) from uninduced Dnmt1lox/chip; MxCre or Dnmt1lox/chip mice (CD45.2+) were transplanted along with 1 × 106 wild-type bone marrow cells (CD45.1+) in lethally irradiated recipients. After 6 weeks, mice were injected five times with poly(I:C) (arrowheads), and CD45.2+ cells were monitored in peripheral blood. Mean value of CD45.2+ control cells was set to 100% at each time point. n = 4 per group. (f) Twenty weeks after poly(I:C) induction, FACS analysis of bone marrow of the chimeras described in e revealed a marked decline in hypomethylated donor HSCs (LSK). n = 4 each.

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and early B-cell factor 1 (Ebf1; ref. 20), indicating that methylation is indispensable for establishing the transcriptional B-cell program.

Analysis of the bone marrow chimeras described in Figure 2d revealed that the selective inefficiency of Dnmt1−/chip precursors in generating lymphocytes was cell intrinsic in vivo. Transplanted Dnmt1−/chip donor cells reconstituted myeloid cells with a 50% decrease, reminiscent of the impaired HSC function, but were totally incompetent to generate B cells (Fig. 3e). Because mature T cells are known to undergo substantial homeostatic proliferation, we analyzed the ability of Dnmt1−/chip cells to reconstitute T cells after removing the CD3ε +, CD4+ and CD8α+ frac-tions from injected donor bone marrow cells. This approach revealed that Dnmt1−/chip progenitors have impaired capacity to regenerate T cells (Fig. 3f). The H2K-Bcl2 transgene line did not rescue disrupted lymphopoiesis in Dnmt1−/chip mice, suggesting that lymphopenia was not caused by enhanced hypomethylation-induced apoptosis of lymphocytes (Supplementary Fig. 6a).

We directly quantified the lineage potential of purified HSCs using clonal single-cell in vitro differentiation assays. Both Dnmt1−/chip LT-HSCs and ST-HSCs showed normal potential to differentiate into granulomonocytic and megakaryocytic lineages (Fig. 3g and

Supplementary Fig. 6b). In contrast, the two groups showed reduced capacity to generate T and B cells.

Finally, both LMPP and B-cell frequencies were reduced in poly (I:C)-treated Dnmt1lox/chip; MxCre mice, indicating that methylation is required not only for establishing, but also for maintaining, lymphoid potential in HSCs (Supplementary Fig. 6c,d).

Committed B cells no longer require sustained DNA methylationTo determine whether constitutive methylation is continuously required throughout lymphopoiesis, we crossed Dnmt1lox/chip mice with mice carrying the Tg(CD19-cre) transgene21 (referred to here as CD19Cre), thus inducing DNA hypomethylation after commitment to the B-cell lineage had been established. Addition of a Gt(ROSA) allele carrying an inducible loxP-STOP-loxP-EYFP (enhanced yellow fluorescent protein) reporter cassette knock-in22 allowed us to trace cells in which Cre was active. In contrast to the B-cell depletion in Dnmt1−/chip mice, we detected normal numbers and undisturbed homeostasis of EYFP+ B cells in Dnmt1∆/chip; CD19Cre mice (Fig. 3h,i and Supplementary Fig. 7a,b). Consequently, whereas B-cell com-mitment from HSCs requires a high methylation threshold, a lower

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thymuses. DN1, CD3ε−CD4−CD8α−CD44+CD25−; DN2, CD3ε−CD4−CD8α−CD44+CD25+; DN3, CD3ε−CD4−CD8α−CD44−CD25+. n = 3–4 mice per group. (d) Semiquantitative RT-PCR from Lin− bone marrow of marker genes for B cells, early lymphocytes and myeloid cells. Actb was used as a positive control for cDNA input. Samples were taken at four-cycle intervals. One representative of three independent experiments is shown. (e) Loss of B-lymphoid potential is cell intrinsic. Bone marrow of F1 chimeras described in Figure 2d was analyzed for CD45.2+ donor-derived B (B220+) and myeloid (Mac1+ and/or Gr1+) cells 12 weeks after transplantation. n = 5–7 mice per group. (f) DNA hypomethylation impairs T-cell reconstitution. CD3ε−CD4−CD8α− bone marrow cells (1.5 × 106) were transplanted into lethally irradiated wild-type recipients. Analysis of CD45.2+ donor cells 8 weeks after transplantation showed fewer T cells (CD3ε+, CD4+ and/or CD8α+) in mice reconstituted with Dnmt1 mutant cells. n = 4 per group. n.a., not analyzed. (g) LT-HSCs were subjected to in vitro single-cell differentiation assays (see Online Methods). Cloning frequencies with indicated lineage potential were scored at weeks 3–4 for T and B and days 8–10 for granulomonocytic (GM) and megakaryocytic (Mk) cells. Lineage affiliations of individual clones were determined by morphology (GM and Mk) or FACS (T cell, NK1.1−Thy1.2hiCD25hi; B cell, B220+CD19+; data not shown). One of at least two independent experiments is shown. (h) FACS analysis of Dnmt1∆/chip; CD19Cre and Dnmt1∆/+; CD19Cre spleen cells carrying an additional loxP-STOP-loxP-EYFP knock-in Gt(ROSA) allele revealed equal frequencies of EYFP+ cells. Numbers indicate percentage of cells from one representative mouse (n = 4). (i) Unchanged numbers of EYFP+B220+IgM+ and EYFP+B220+IgM− B cells in spleens of Dnmt1∆/chip; CD19Cre and Dnmt1∆/+; CD19Cre mice (n = 4 per group).

Figure 3 DNA methylation controls myeloerythroid versus lymphoid differentiation of HSCs. (a) Frequencies of B cells (CD19+) and myeloid cells (Mac1+) in bone marrow and erythroid cells (Ter119+) in spleens were analyzed in 8- to 12-week-old Dnmt1−/chip and Dnmt1+/chip mice by FACS. n = 3–7 mice per group. Data in a–c,e–g,i are mean ± s.d.; *P ≤ 0.05, **P ≤ 0.001. (b,c) Multiple-color FACS shows reduced lymphoid but normal myeloid and erythroid progenitor frequencies in 2-month-old Dnmt1−/chip mice compared with age-matched controls. In b, common myeloid progenitors (CMP), Lin−Sca-1−c-Kit+CD34+Fc-γRII/IIIlo; granulocyte monocyte progenitors (GMP), Lin−Sca- 1−c-Kit+CD34+Fc-γRII/III+; megakaryocyte erythrocyte progenitors (MEP), Lin−Sca-1−c-Kit+CD34−Fc-γRII/III−; pre-B cells, Lin−IgM−B220+CD43−; proB cells, Lin−IgM−B220+CD43+; common lymphoid progenitors (CLP), Lin−Sca-1 loc-KitloIL7R-α+. Cells were analyzed from bone marrow. In c, double-negative (DN) T cells were analyzed from

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threshold is sufficient to maintain B-cell identity and maturation once the B-cell program has initially been established.

DNA methylation controls leukemia stem cell functionTo address the effect of hypomethylation on CSC function, we trans-formed Dnmt1−/chip cells by retroviral coexpression of Myc and Bcl2 (referred to here as Myc-Bcl2) in a mouse transplantation approach (Supplementary Fig. 8a). The Myc-Bcl2 combination was chosen as a model to study lineage choice in cancer because it induces a bilinear myeloid–B lymphoid leukemia in wild-type mice23. To avoid initial transduction biases of different progenitors, we used sorted LSK cells for retroviral infection and transplantation into sublethally irradiated recipients. LSK cells from Dnmt1+/+ and Dnmt1−/chip mice could be infected with similar efficiencies and expressed the Myc-Bcl2 tran-script in equal quantities (Supplementary Fig. 8b,c).

All recipient mice that received Myc-Bcl2; Dnmt1+/+ cells rapidly succumbed to lethal leukemias consisting mainly of Mac1+B220− myeloblasts and B220+CD19+IgM−Mac1− B lymphoblasts (Fig. 4a, Supplementary Fig. 8d and data not shown). Recipient mice trans-planted with Myc-Bcl2; Dnmt1−/chip cells developed leukemia with substantially prolonged latency. Notably, whereas all mutant mice

developed myeloid leukemia, none succumbed to a B-lymphoid disease (Fig. 4b–d). Myc-Bcl2; Dnmt1−/chip cells gave rise to bilinear myeloid and T-lymphoid leukemias in two of the transplanted mice, indicating that hypomethylation completely abolished leukemic B cell potential but did not disrupt malignant T-cell outgrowth (data not shown). We confirmed the transformed nature of Myc-Bcl2; Dnmt1−/chip and Dnmt1+/+ cells by transplantation into secondary recipients (data not shown).

The increased latency of Myc-Bcl2; Dnmt1−/chip leukemias suggested that constitutive methylation is required for proper renewal of leukemia stem cells (LSCs). To test this hypothesis, we transduced Lin− bone marrow cells with a retrovirus expressing MLL-AF9, a prototype human oncogene in which the MLL and AF9 (offi-cially known as MLLT3) genes are fused by a t(9;11)(p22;q23) translocation24,25, and conducted serial replating assays26 (Supplementary Fig. 9a). Dnmt1−/chip cells transduced with empty vector formed as many colonies as did the control cells, confirming that hypomethylation does not impair response to myeloid growth factors (Fig. 4e). In contrast, Dnmt1−/chip cells transduced with MLL-AF9 showed a severe replating deficiency, indicating that they had impaired self-renewal capacity in vitro (Fig. 4f).

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Figure 4 DNA methylation controls LSC functions. (a) Delayed leukemia latency in mice that received Myc-Bcl2–transduced Dnmt1−/chip LSK cells compared with those that received the same number of Myc-Bcl2; Dnmt1+/+ LSK cells. Mice were autopsied when visibly ill. Median survival was 27.70 ± 3.5 d for Dnmt1+/+ (n = 11) and 93.25 ± 28.05 d for Dnmt1−/chip (n = 8); P < 0.0001. (b) Representative FACS plots of bone marrow (BM) and lymph node (LN) suspensions from diseased mice. Neoplastic B cell population was lost in leukemic mice reconstituted with Myc-Bcl2; Dnmt1−/chip cells. n = 7–11 mice per group. Plots show cells within donor (CD45.2+) gates. Some mice had an additional, rarer population with abnormal surface marker combinations, such as coexpression of myeloid, B and occasionally T-lymphoid antigens (data not shown). However, because these cells had an early myeloid morphology and neither B- nor T-cell receptor loci were rearranged, they represented abnormal myeloblasts (data not shown). (c) Bone marrow cytospins confirming absence of B-cell blasts (red arrowheads) and presence of myeloblasts (black arrowheads) in Dnmt1−/chip leukemic mice (May-Gruenwald stain, ×1,000 magnification). (d) Genes indicative of myeloid cells (Mpo, myeloperoxidase; Csf2ra, granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor-α) were expressed normally in leukemic bone marrow samples of moribund Myc-Bcl2; Dnmt1−/chip LSK cell–reconstituted mice, whereas expression of genes indicative of B cells (Pax5, Ebf1) was not detectable. Shown is one of two independent RT-PCR experiments with similar outcomes. Donor-derived blast cell infiltration in recipient bone marrow was >95% in all cases. (e) Dnmt1+/+ and Dnmt1−/chip bone marrow cells transduced with green fluorescent protein (GFP)-expressing control virus gave rise to equal numbers of colonies in methylcellulose in the presence of IL3, IL6, SCF and GM-CSF. Experiment was done in triplicate; data show mean ± s.d. (f) Serial replating assay of MLL-AF9–transduced Dnmt1+/+ and Dnmt1−/chip bone marrow cells. GFP+ cells were sorted, and for each round, 5,000 cells were plated in methylcellulose containing IL3, IL6, SCF and GM-CSF. After 5 d, colonies were counted. Data show mean ± s.d. Experiment was done twice in triplicate. (g) Survival of secondary recipient mice injected with limiting dilutions (continuous line, 10,000 cells; dashed line, 1,000 cells; dotted line, 100 cells) of MLL-AF9; Dnmt1lox/chip and MLL-AF9; Dnmt1∆/chip GFP+ bone marrow cells (containing >95% leukemic cells) from diseased mice. Detailed numbers are shown in supplementary table 1.

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We next injected equal numbers of MLL-AF9–transduced bone marrow cells from 5-fluorouracil–treated conditional Dnmt1lox/chip; MxCre and Dnmt1lox/chip mice into sublethally irradiated recipients and administered poly(I:C) 3 weeks after the transplantation (Supplementary Fig. 9b). Similar to the Myc-Bcl2 model, MLL-AF9; Dnmt1∆/chip cell transplantation led to leukemia with prolonged latency (median survival after poly(I:C): MLL-AF9; Dnmt1lox/chip, 48.2 ± 11.8 d; MLL-AF9; Dnmt1∆/chip, 61.95 ± 13.5 d; P = 0.0005). The leukemic cells had near-complete Dnmt1lox excision and were polyclonal, and the major-ity of them coexpressed c-Kit and Mac1 (Supplementary Fig. 9c–e). To quantify LSCs in this model, we conducted limited-dilution transplan-tation in secondary recipients27 (Fig. 4g and Supplementary Table 1). This approach confirmed the increased latency of hypomethylated

leukemias and revealed a LSC frequency of 1 in 91 MLL-AF9; Dnmt1lox/chip cells and 1 in 1,072 MLL-AF9; Dnmt1∆/chip cells. Thus, hypometh-ylation reduced MLL-AF9–transduced LSCs by 91.5%. Notably, the proliferation rates of cultured MLL-AF9-transduced cells from Dnmt1∆/chip and Dnmt1lox/chip leukemias were comparable, suggesting that hypomethylation has a limited effect on cancer bulk cell growth (Supplementary Fig. 9f).

Myeloerythroid gene activation in hypomethylated HSCsTo gain insight into the molecular basis of the defects in Dnmt1−/chip stem cells, we generated genome-wide mRNA expression profiles of Lin−Sca-1+c-Kit+Flt3− HSCs (which were all CD150+) and, for comparison, Lin−Sca-1−c-Kit+CD34+ (which were CD150−)

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Figure 5 DNA hypomethylation leads to derepression of myeloerythroid genes in HSCs. (a) Genome-wide mRNA expression profiles of HSCs (Flt3− LSK) and myeloerythroid progenitors (MP; Lin−Sca-1−c-Kit+CD34+) were generated using Affymetrix mouse genome 430 2.0 arrays. Numbers of up- and downregulated genes in HSCs and MPs from 8- to 12-week-old Dnmt1+/+ and Dnmt1−/chip mice were compared. Numbers below circles indicate total unique transcripts expressed in HSCs or MPs. (b) Expression of profiled populations was compared using principal component analysis (PCA). When vectors of gene expression were projected in the plane defined by their two main eigenvectors, Dnmt1−/chip HSCs resided at a midpoint between Dnmt1+/+ HSCs and both Dnmt1+/+ and Dnmt1−/chip MPs, revealing increased transcriptional similarity of Dnmt1−/chip HSCs to myeloerythroid cells. For comparison, expression profiles of CD19+IgM− B-cell progenitors (BP) of Dnmt1+/+ mice were generated. Both wild-type and mutant HSCs were distant from B-cell progenitors. (c) HSC and MP signature probe sets were defined as probe sets that were differentially expressed between Dnmt1+/+ HSCs and MPs, as calculated by a false discovery rate <0.001 and a log fold change >2.0. Absolute expression values were transformed to probe set Z scores before visualization. (d,e) Expression of representative genes with known functions in stem cells and myeloerythroid cells (d) or lymphoid cells (e) are shown for five profiled populations. (f) Representative FACS showing increased surface expression of CD48 and Fc-γRII/III on LT-HSCs from Dnmt1−/chip mice compared with their counterparts from Dnmt1+/+ mice. MPs of Dnmt1+/+ mice served as high-expression control. MFI values for CD48 are 4,290.24 (LT-HSC +/+), 16,955.89 (LT-HSC −/chip) and 24,708.68 (MP +/+). MFI values for Fc-γRII/III are 921.85 (LT-HSC +/+), 1,514.31 (LT-HSC −/chip) and 1,538.74 (MP +/+). (g) MassARRAY profiles showing methylation statuses of Gata1 and Cd48 promoters in HSCs and MPs from Dnmt1+/+ and Dnmt1−/chip mice. Profiles of T cells (CD3+) and erythrocytes (Ter119+) of Dnmt1+/+ mice are shown as controls. Percentages of methylated CpGs are indicated. Genomic localizations of analyzed promoter regions: Gata1, chr. X 7545431–7545769 and chr. X 7553755–7554238; Cd48, chr. 1 173612289–173612577. (h) Rescue of Dnmt1−/chip B-cell differentiation by ectopic expression of Ebf1. c-Kit+ bone marrow cells were purified from Dnmt1+/+ and Dnmt1−/chip mice, infected with mouse stem cell virus (MSCV) expressing Ebf1-IRES-GFP or IRES-GFP, and cultured on OP9 stromal cells in presence of IL-7, Flt3 and SCF. Representative FACS plots show GFP-gated cells after 19 d of coculture. Numbers indicate frequencies of B220+Mac1− B cells. Experiment was done twice independently.

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myeloerythroid progenitors. We found a marked difference between the numbers of deregulated transcripts in hypomethylated HSCs and myeloerythroid progenitors. The expression levels of 1,186 unique transcripts (11.4% of all expressed) were different between Dnmt1+/+ and Dnmt1−/chip HSCs, but only 44 (0.4% of all expressed) were dif-ferent between myeloerythroid progenitor populations (Fig. 5a and Supplementary Tables 2 and 3). Notably, the array data confirmed that Dnmt1 mRNA levels in Dnmt1−/chip HSCs and myeloerythroid progenitors decreased by a similar degree (Supplementary Fig. 10). In contrast, Dnmt3a and Dnmt3b levels were not significantly changed in either population, suggesting that neither enzyme compensates for the diminished Dnmt1 activity.

We used principal component analysis to explore relationships between wild-type and mutant HSCs and myeloerythroid progeni-tors based on comparisons of their transcriptomes28. This approach revealed a clear separation of Dnmt1−/chip HSCs from their wild-type counterparts, whereas myeloerythroid progenitors remained closely related (Fig. 5b). Furthermore, Dnmt1−/chip HSCs had an increased transcriptional similarity to myeloerythroid progenitors, but not to CD19+IgM− B-cell progenitors, a finding that was confirmed by an unbiased cluster analysis (Supplementary Fig. 11a).

We generated gene signatures specific for HSCs or myeloerythroid progenitors by subtracting the genes expressed in wild-type myelo-erythroid progenitors from those expressed in wild-type HSCs or vice versa. Of the HSC signature genes, 96.57% showed lower expression, and 3.43% showed higher expression, in Dnmt1−/chip HSCs compared to wild-type HSCs (Fig. 5c). In contrast, only 10.30% of myeloerythroid progenitor signature genes showed lower expression in Dnmt1−/chip cells, whereas 89.71% showed higher expression. Density plots further

showed reciprocal expression shifts of HSC and myeloerythroid progenitor signature genes in Dnmt1−/chip HSCs (Supplementary Fig. 11b). Many genes with known functions in myeloerythroid differentiation were among the most highly upregulated genes in Dnmt1−/chip HSCs (Fig. 5d). These included the genes encoding the potent transcription factors Gata1, Id2 and Cebpa (Supplementary Fig. 11c,d). Notably, Gata1 was expressed by a greater frequency of Dnmt1−/chip HSCs, as shown by single-cell RT-PCR (Supplementary Fig. 11e). In contrast to the greater expression of myeloerythroid progenitor genes, most genes known to control self-renewal and/or lymphoid differentiation showed lower expression in mutant HSCs (Fig. 5d,e).

Several surface receptors expressed on differentiated cells, but normally not on HSCs, were upregulated in Dnmt1−/chip stem cells. Analysis of surface expression of two of these receptors, Fc-γRII/III (ref. 29) and CD48 (ref. 17), revealed that both are expressed on Dnmt1−/chip HSCs at levels comparable to those on myeloerythroid progenitors (Fig. 5f), confirming that increased transcription of myeloerythroid progenitor genes leads to higher protein expression.

We quantified the CpG methylation status of the promoters of Gata1, Id2, Cebpa and Cd48 in purified HSCs using MassARRAY tech-nology (Sequenom). Of these, the Gata1 and Cd48 promoters showed methylation in wild-type HSCs (Fig. 5g and data not shown). Notably, methylation of both promoters was reduced in Dnmt1−/chip HSCs.

Restoring B-cell potential of Dnmt1−/chip HSCsThe myeloerythroid transcription factors that were more highly expressed in Dnmt1−/chip HSCs block lymphoid differentiation by repressing key lymphoid genes30,31. Consequently, the defect in

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Figure 6 Epigenetic Gata1 and Cd48 activation in renewal of myeloid leukemia cells. (a) Real time RT-PCR of Gata1 in two myeloid cell lines, 416B and PU1null, and primary leukemia cells derived from moribund MLL-AF9 wild-type mouse after 72 h of treatment with 5-Aza-dC (5 µM for 416B and MLL-AF9 cells; 1 µM for PU1null cells). Data were normalized to expression of Actb. Experiment was done three times independently, with similar outcomes. (b) FACS analysis showing enhanced expression of CD48 in 416B cells after treatment with 5 µM 5-Aza-dC. Data in a,b,d,e show mean ± s.d. *P ≤ 0.05. (c) Methylation of Gata1 and Cd48 promoters was reversed by 5-Aza-dC in 416B and PU1null cells, as assessed by quantitative MassARRAY technology. Percentages of methylated CpGs are indicated. Genomic localization of analyzed regions: Gata1, chr. X 7545431–7545769 and chr. X 7553755–7554238; CD48, chr. 1 173612289–173612577. (d) Luciferase assay of K562 cells transfected with in vitro methylated or unmethylated pCpGL-Gata1 promoter–firefly luciferase construct. Firefly luciferase activity was normalized using Renilla luciferase activity as internal transfection control. (e) Enforced Gata1 expression reduces growth of myeloid leukemia cells. PU1null cells were transduced with retrovirus expressing Gata1 and GFP, or GFP only, and subjected to liquid culture in presence of IL-3. Percentage of GFP+ cells on day 0 was equalized to 1. (f) Model of DNA dosage effects on stem cell multipotency. MP, myeloerythroid progeny; LP, lymphoid progeny; †, apoptosis; methyl +++, high methylation level required; methyl +, low methylation level required.

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lymphoid development might be a direct result of the incompetence of Dnmt1−/chip HSCs to upregulate crucial lymphoid genes, owing to their inability to epigenetically silence predominant myeloeryth-roid factors. To test this hypothesis, we assessed whether the block in lymphoid differentiation could be overcome by forced expression of a potent lymphoid transcription factor. We ectopically expressed Ebf1 in c-Kit–enriched Dnmt1−/chip precursors with a retrovirus, and we conducted B-cell differentiation assays on OP9 mouse stromal cell cultures. We chose Ebf1 because it is a key transcription factor for early B-cell development32 and was repressed in Dnmt1−/chip bone marrow precursors (Fig. 3d). Control virus–transduced Dnmt1−/chip precursors were unable to generate B cells, whereas Ebf1 virus– transduced mutant cells generated B cells at numbers comparable to those of wild-type precursors (Fig. 5h and Supplementary Fig. 12). Thus, ectopic activation of Ebf1 rescues the lymphoid potential of Dnmt1−/chip cells.

Pharmacological demethylation activates differentiation genesTo evaluate whether activation of myeloerythroid genes represents a mechanism by which demethylating chemicals can inhibit the renewal of cancer cells, we treated mouse myeloid leukemia lines and primary myeloid leukemia cells with 5-aza-2′-deoxycytidine (5- Aza-dC) and measured expression of Gata1, Cebpa and Cd48. Whereas Cebpa expression remained unchanged, Gata1 and Cd48 expression was induced by 5-Aza-dC (Fig. 6a,b and data not shown). Moreover, methylation of the promoter regions of Gata1 and Cd48 in the leuke-mia cells was reduced after 5-Aza-dC treatment (Fig. 6c).

We next inserted the Gata1 promoter into a CpG-free luciferase plasmid33, incubated the plasmid with the CpG methyltransferase SSSI to methylate the promoter, and compared it to its unmethylated version in transient reporter assays. The unmethylated promoter had higher reporter activity than did the methylated version, suggesting that demethylation increases Gata1 promoter activity (Fig. 6d). Finally, we analyzed the functional significance of Gata1 activation in leukemia cells using retroviral gene transfer. We found that ectopic Gata1 expression impaired leukemic cell growth, suggesting that demethylating agents suppress self-renewal of cancer cells through derepression of differentiation factors (Fig. 6e).

DISCUSSIONRecent data have suggested that the transition of stem cells from self-renewal to differentiation is accompanied by extensive changes in their methylation pattern9,11,34. However, this observation left unanswered the important question of whether changes in methyla-tion are etiologically relevant for changes in stem cell fate. Here, we addressed this question and found that alternative functional stem cell programs require distinct threshold levels in their methylation status (Fig. 6f).

HSCs that were devoid of Dnmt1 underwent rapid apoptosis, which was fully blocked by reintroduction of a hypomorphic Dnmt1chip allele. Notably, Dnmt1-hypomorphic HSCs showed specific defects: although they had normal homing capacity, their repopulation poten-tial was severely impaired, revealing that the self-renewal program of HSCs requires constitutive maintenance of a critical threshold of methylation. A recent analysis of Dnmt3a−/−; Dnmt3b−/− HSCs showed that de novo methylation is also important for HSC renewal10. These findings, combined with our results, suggest that de novo meth-ylation represents a fail-safe mechanism to replace lost methylation marks during stem cell renewal.

Dnmt3a−/−; Dnmt3b−/− HSCs give rise to normal numbers of cells of all hematopoietic lineages, indicating that de novo methylation is

dispensable for differentiation. In contrast, the preexisting methyla-tion pattern in HSCs seems to be crucial for this process. Dnmt1−/chip HSCs had a normal ability to form myeloid and erythroid progeny, but they were impaired in their ability to commit to lymphoid dif-ferentiation. This finding indicated that methylation-dependent control mechanisms differentially regulate myeloerythroid versus lymphoid lineage choice, thus uncovering a previously unrecog-nized gradual methylation sensitivity of opposing lineage programs. Because Dnmt1−/chip mice lacked LMPPs, hypomethylation blocked the lymphoid lineage choice before lymphoid transcriptional prim-ing was initiated. Notably, conditional induction of hypomethylation in B-cell progenitors led to normal numbers of mature B cells. We conclude that a high methylation threshold is required for lymphoid commitment in HSCs, but a lower threshold is sufficient to maintain the lymphoid program once it has been established. Together, these data strongly argue for a fundamental difference in the role of DNA methylation in stem cells and lineage-specified progenitors.

Although aberrant methylation had been found in most, if not all, cancers, its role in controlling CSC functions remained elusive35. We addressed this question by transforming Dnmt1−/chip cells with the oncogenes Myc, Bcl2 and MLL-AF9. We found that hypometh-ylation impairs malignant self-renewal of AML cells and completely blocks initiation of acute B-lymphoid leukemia (B-ALL) from transformed stem cells. Thus, demethylating chemicals may impair CSC function in cancer therapy. However, because initiation, but not maintenance, of normal B cells requires high Dnmt1 activity, it remains possible that fully developed B-ALL is not a relevant target for demethylation therapy. Our future experiments will address this issue by assessing the functional role of Dnmt1 in B-ALL maintenance, as opposed to initiation.

We also found that DNA hypomethylation caused widespread tran-scriptional deregulation in HSCs. In sharp contrast, transcription was only mildly disturbed in myeloerythroid progenitors, supporting the idea that methylation is most relevant at the stem cell level and loses importance after lineage commitment. The hypomethylation-induced transcriptional changes were surprisingly specific, in that Dnmt1−/chip HSCs showed an increase in the expression of many myeloerythroid genes, including the transcription factors Gata1, Id2 and Cebpa, but a decrease in the expression of ‘stemness’ and lymphoid genes. Forced expression of Ebf1 corrected the myeloerythroid bias and restored the potential of Dnmt1−/chip HSCs to differentiate into B cells. Ebf1 is a crucial transcription factor for early B-cell development that is tran-scriptionally repressed by Gata1 and C/epb-α30,31 and was repressed in Dnmt1−/chip bone marrow precursors. Together, these data pro-vide a molecular explanation for the hematopoietic phenotype of Dnmt1−/chip mice by showing that the inability to silence myeloeryth-roid genes directly inhibits lymphoid potential by blocking the ability of Dnmt1−/chip HSCs to switch on crucial lymphoid genes.

We found that promoters of Gata1 and Cd48 were strongly methylated in normal HSCs and that this methylation was lower in Dnmt1−/chip HSCs. Although we did not detect promoter methylation of Id2 and Cebpa, it is possible that these were activated by demethylation of more distally located regulatory elements. Both Id2 and Cebpa were silenced by methylation in cancer cells, supporting the idea that their expression is controlled by methylation-dependent mechanisms (refs. 36–38 and our unpublished observations). Furthermore, Gata1 expression was silenced by promoter methylation in myeloid leukemia cells, which could be reversed by 5-Aza-dC treatment. Together, these data raise the possibility that pharmacological Dnmt1 repression suppresses growth of leukemia cells by activating a differentiation program that is silenced to allow CSC renewal.

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Nature GeNetics  volume 41 | number 11 | november 2009 1215

A rt i c l e s

In summary, DNA methylation is an important epigenetic mecha-nism to silence predominant differentiation programs in stem cells as a prerequisite to maintaining self-renewal and multipotency. Moreover, the data suggest that retention or re-establishment of stem cell–specific methylation patterns is an important step in the develop-ment and function of CSCs.

METHODSMethods and any associated references are available in the online version of the paper at http://www.nature.com/naturegenetics/.

Accession codes. GEO: array data, GSE17765

Note: Supplementary information is available on the Nature Genetics website.

ACKNoWLEdgMENtSWe thank V. Malchin, C. Graubmann and J.F. Zinke for their excellent technical assistance; J. Schoenheit for his help with real-time PCR; H.P. Rahn and Z. Ma for assistance with high-speed cell sorting; M.L. Cleary (Stanford University School of Medicine), S. Fillatreau (Deutsches Rheuma-Forschungszentrum), A. Müller (University of Würzburg), K. Rajewsky (Harvard Medical School), M. Rehli (University of Regensburg), M. Tomasson (Washington University School of Medicine), B.L. Kee (University of Chicago) and T. Somervaille (Stanford University School of Medicine) for reagents; members of the Rosenbauer lab for discussions; D.G. Tenen for support during the initial phase of this study; and C. Plass, C. Müller-Tidow, C. Bonifer and D.G. Tenen for advice on the manuscript. This work was supported by grants from the German Research Foundation, the German Cancer Aid (Mildred Scheel) and the Helmholtz Association of German Research Centers to F.R. S.E.W.J. is the recipient of a strategic appointment grant from the UK Medical Research Council.

AUtHoR CoNtRIBUtIoNSA.-M.B., L.V., S.K., E.M., C.K., M.S. and A.E. conducted experiments. M.R.H. and M.A.A.-N. analyzed the microarray data. A.-M.B., L.V. and F.R. wrote the manuscript. R.J. provided key mouse strains and gave advice on the manuscript. M.P. conducted the pathological analysis. C.N., A.L. and S.E.W.J. provided essential ideas, experimental support and suggestions on the manuscript. F.R. designed and supervised the project and provided financial support.

Published online at http://www.nature.com/naturegenetics/. Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/

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ONLINE METHODSMice and cell lines. Mice carrying targeted Dnmt1−, Dnmt1chip, Dnmt1lox or Gt(ROSA)loxP-STOP-loxP-EYFP alleles, or MxCre, CD19Cre or H2K-Bcl2 trans-genes, were described previously12–14,18,21,22. All mice were kept in specific pathogen–free animal facilities at the Max Delbrück Center. B6.SJL-Ptprca (CD45.1+) mice were purchased from Taconic and were crossed with 129ola mice to obtain 129ola/B6.SJL (CD45.1+CD45.2+) F1 mice. To induce excision of loxP-flanked (floxed) alleles, we intraperitoneally injected mice with 300 µg of poly(I:C) (GE Healthcare) every other day for a total of five injec-tions. All mouse experiments were approved by the local authorities according to the German Federal Animal Protection Act.

PLAT-E and 416B cells were cultured in DMEM supplemented with 10% or 20% FBS, respectively. PU1null cells39 were kept in RPMI supplemented with 10% FBS and 10 ng/ml IL-3.

Transplantation experiments. For serial and competitive transplantation assays, 8-week-old CD45.1+CD45.2+ recipients were irradiated with a lethal dose of 10.5 Gy total-body irradiation with the 18-MeV photon beam of a linear electron accelerator at a dose rate of 0.18 Gy/min. The mice were recon-stituted with 5 × 106 freshly isolated bone marrow cells within 24 h of irra-diation. For short-term engraftment assays, 1 × 107 CD45.2+ Dnmt1−/chip or Dnmt1+/+ bone marrow cells were mixed with 1 × 107 CD45.1+ SJL bone mar-row cells and transplanted into lethally irradiated CD45.1+CD45.2+ recipients. For transplantation of Dnmt1 conditional mutant cells, 1 × 106 Dnmt1lox/chip; MxCre or Dnmt1lox/chip whole bone marrow cells were mixed with 1 × 106 CD45.1+ SJL whole bone marrow cells and transplanted into lethally irradiated CD45.1+CD45.2+ recipient mice. Eight weeks after transplantation, poly(I:C) was administered as described above. For transplantation of Myc-Bcl2 cells, 2 × 104 transduced LSK cells were transplanted into CD45.1+CD45.2+ recipients irradiated with a sublethal dose of 6.0 Gy.

Histology. Mouse sterna were fixed in 4% buffered formalin, dissected, paraffin embedded, and stained with hematoxylin and eosin as described previously40. Cytocentrifuge preparations were fixed in methanol and stained with May-Gruenwald-Giemsa (Sigma-Aldrich).

Retroviral constructs, viral supernatant production and cell transduction. MSCV retroviral constructs expressing c-Myc-IRES-Bcl2, MLL-AF9-IRES-GFP or Ebf1-IRES-GFP have been described23,24,41. The Gata1-expressing virus was generated by inserting the mouse Gata1 cDNA into the XhoI site of MSCV expressing IRES-eGFP. Retroviral supernatants were produced as described42. For retroviral transduction, LSK or c-Kit–enriched cells were cultured for 24 h at 37 °C in Iscove’s modified Dulbecco medium (20% FCS, 100 µg/ml penicillin and 2 mM L-glutamine) containing 50 ng/ml mouse SCF (Tebu-Bio), 20 ng/ml mouse IL-6 (Tebu-Bio) and 20 ng/ml mouse leukemia inhibitory factor (Santa Cruz Biotechnology). Cells were then mixed with retroviral supernatants in the presence of 50 ng/ml mouse SCF, 20 ng/ml mouse IL-6, 20 ng/ml mouse leukemia inhibitory factor and 8 µg/ml polybrene (hexadimethrine bromide; Sigma-Aldrich) and cultured for 24 h (LSK) to 48 h (c-Kit+) at 37 °C. Lin− bone marrow or total bone marrow cells from 5-fluorouracil–treated mice were transduced in a cytokine cocktail containing 50 ng/ml mouse SCF, 20 ng/ml mouse IL-6 and 10 ng/ml IL-3 (all from Tebu-Bio).

Real-time RT-PCR. We extracted RNA using a TRIzol or RNeasy kit (Qiagen), reverse-transcribed it and then amplified it using a 7300 Real-Time PCR System (Applied Biosystems) using exon-spanning primer-probe sets. For semiquan-titative PCR, aliquots were taken at five-cycle intervals to ensure that PCR remained within the exponential range of amplifications. Primer sequences and TaqMan assay ID numbers are provided in Supplementary Table 4.

RT-PCR analysis of single cells. Single-cell RT-PCR analysis was done as previously described15.

Southern blot analysis. Southern blot analysis of genomic methylation was done using an intracisternal A particle cDNA probe as described previously43. To analyze retroviral integration sites, genomic DNA was digested with EcoRI and probed with a radiolabeled EGFP probe that was derived as a 1-kb NcoI-HindIII fragment from the pMIG vector.

Detection of DNA methylation by PCR. Genomic DNA was digested with HpaII or MspI overnight. The digestion products were purified, ligated to an adaptor, PCR amplified and electrophoresed as described44.

Flow cytometry and cell sorting. We analyzed single-cell suspensions from various organs by flow cytometry using antibodies conjugated with phyco-erythrin, phycoerythrin-Cy7, FITC, Tricolor, allophycocyanin, allophycocy-anin-Cy7, Pacific blue or biotin specific for the following cell surface molecules: Mac-1/CD11b (M1/70), CD3ε (145-2C11), CD4 (GK1.5), CD8α (53-6.7), B220 (RA3-6B2), Gr-1 (RB6-8C5), TER119 (TER-119), CD19 (1D3), IgM (R6-60.2), IL-7Rα chain (SB/199), Sca1 (E13-161-7), c-Kit (2B8), Flt3 (A2F10.1) Fc-γRII/III (2.4G2), CD34 (RAM34), CD48 (HM48-1) and CD150 (TC15-12F12.2). We obtained antibodies from BD Biosciences, eBioscience, BioLegend and Caltag Laboratories. Nonspecific binding was reduced by preincubation with unconjugated antibody to Fc-γRII/III (2.4G2). Analysis and sorting of HSCs and intermediate progenitors was done as described16,17,29. Before sorting progenitor populations, lineage depletion of bone marrow cells was achieved with a lineage ‘cocktail’ of antibodies to CD3ε, CD4, CD8α, B220, CD19 and Gr-1 and immunomagnetic beads conjugated to anti-rat IgG (Invitrogen). Alternatively, c-Kit enrichment was done with CD117 microbeads (Miltenyi). For B-cell progenitor analysis, the lineage cocktail contained antibodies to CD3ε, CD4, CD8α and Gr-1. For annexin V staining, freshly isolated bone marrow cells were stained with the appropriate antibodies, washed in annexin binding buffer (BD Biosciences) and incubated for 20 min at 4 °C in the dark with allophycocyanin-conjugated antibody to annexin V (BD Biosciences). We analyzed all samples on a FACSCalibur or LSR II cytometer (BD Biosciences) and sorted them on a FACSAria (BD Biosciences) using standard protocols. Gates on viable cells were set according to the exclusion of propidium iodide staining.

Cell cycle analysis. The cell cycle status of LSK cells was determined by staining at 37 °C for 30 min with Hoechst 33342 (Invitrogen) as described45. Staining with DAPI was used for exclusion of dead cells. Cells were analyzed on an LSR II cytometer equipped with a violet laser (407 nm).

In vitro clonogenic stem cell differentiation assays. Bone marrow cells were collected, and LSKCD34−Flt3− and LSKCD34+Flt3− populations were FACS purified and cultured under appropriate conditions to quantify megakaryo-cytic, granulomonocytic, B-cell and T-cell growth as previously described16. Megakaryocytic and granulomonocytic colonies were scored with an inverted light microscope after 8–10 d of culture46. B-cell clones (B220+CD19+) and T-cell clones (CD4+CD8α+ and/or Nk1.1−Thy1.2hiCD25hi) were analyzed on days 21 and 28 by FACS. Clones were required to have ≥20 gated events (of indicated cell surface phenotypes) with appropriate scatter profile to be scored as positive. Small clones were cultured an additional week before being analyzed as described16.

MassARRAY. Quantitative DNA methylation analysis at single CpG units was done by MassARRAY (Sequenom) as previously described47. Briefly, genomic DNA (500 ng from sorted primary cells or 1 µg from cell lines) was treated with sodium bisulfite, PCR amplified, in vitro transcribed, cleaved with RNase A and subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Methylation standards (0%, 20%, 40%, 60%, 80% and 100% methylated genomic DNA) and correction algorithms based on the R statistical computing environment were used for data normalization.

Linear amplification of RNA and microarray hybridization. We sorted 35,000 HSCs (Lin−Sca-1+c-Kit+Flt3−) and myeloerythroid progenitors (Lin−Sca-1−c-Kit+CD34+) from three independent pools of Dnmt1+/+ and Dnmt1−/chip mice, with three or four mice per pool. We sorted 35,000 B-cell progenitors (CD19+IgM−) from bone marrow of Dnmt1+/+ mice. RNA extraction, linear amplification and microarray procedure were done by ImaGenes. Briefly, RNA was extracted according to the RNeasy Micro method (Qiagen) optimized for small amounts of RNA. For linear amplification of RNA, a strategy of two rounds of reverse transcription followed by T7 promoter-dependent in vitro transcription was used. Amplified RNA (10 µg) was hybridized to an Affymetrix mouse genome 430 2.0 array.

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Microarray analysis. All microarray analysis was done using tools contained in the Bioconductor project. Raw expression values were normalized using robust multichip average (RMA), and differential expression was determined using the LIMMA library. All probe-set annotation information comes from the mouse4302.db library, version 2.2.0. To determine the total number of genes up- and downregulated in HSCs and myeloerythroid progenitors (Fig. 4a), we selected one representative probe set for each gene. Genes with a false discovery rate <0.05 were considered differentially expressed (controlled using the procedure of Benjamini and Hochberg48). Presence and absence of each gene was determined using the MAS 5.0 calls algorithm. When defin-ing stem and myeloid signature probe sets, probe sets that were differen-tially expressed with a false discovery rate ≤0.001 and log2 fold change >2.0 were chosen, with HSC signature probe sets being more highly expressed in HSCs and myeloerythroid progenitor signature probe sets being more highly expressed in myeloerythroid progenitors.

Luciferase assay and in vitro methylation. A ~800-bp fragment (−856 bp to −31 bp) of the mouse Gata1 promoter was amplified by PCR and cloned into the CpG-free pCpGL-basic luciferase vector33 using HindIII and NcoI. In vitro methylation was done as in ref. 33. K562 cells were transfected by electroporation as described in ref. 49, in the presence of 10 µg of methyl-ated or unmethylated vector DNA and 1 µg of Renilla luciferase. At 24 h after transfection, cells were washed and lysed in passive lysis buffer (Promega). Luciferase activity was measured using a Centro LB960 luminometer (Berthold Technologies). Firefly luciferase activity was normalized to Renilla luciferase activity. Experiments were done in duplicate.

Statistical analysis. We used Student t tests to determine the statistical significance of experimental results. P ≤ 0.05 was considered significant.

URLs. Bioconductor project, http://www.bioconductor.org.

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42. Rosenbauer, F. et al. Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1. Nat. Genet. 36, 624–630 (2004).

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