Loss of the de novo DNA methyltransferases Dnmt3a and Dnmt3b in embryonic stem cells obstructs differentiation; however, the role of these enzymes in somatic stem cells is largely unknown. Using conditional ablation, we show that Dnmt3a loss progressively impairs hematopoietic stem cell (HSC) differentiation over serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Dnmt3a-null HSCs show both increased and decreased methylation at distinct loci, including substantial CpG island hypermethylation. Dnmt3a-null HSCs upregulate HSC multipotency genes and downregulate differentiation factors, and their progeny exhibit global hypomethylation and incomplete repression of HSC-specific genes. These data establish Dnmt3a as a critical participant in the epigenetic silencing of HSC regulatory genes, thereby enabling efficient differentiation.

Summary The traditional view of hematopoiesis has been that all the cells of the peripheral blood are the progeny of a unitary homogeneous pool of hematopoietic stem cells (HSCs). Recent evidence suggests that the hematopoietic system is actually maintained by a consortium of HSC subtypes with distinct functional characteristics. We show here that myeloid-biased HSCs (My-HSCs) and lymphoid-biased (Ly-HSCs) can be purified according to their capacity for Hoechst dye efflux in combination with canonical HSC markers. These phenotypes are stable under natural (aging) or artificial (serial transplantation) stress and are exacerbated in the presence of competing HSCs. My- and Ly-HSCs respond differently to TGFβ1, presenting a possible mechanism for differential regulation of HSC subtype activation. This study demonstrates definitive isolation of lineage-biased HSC subtypes and contributes to the fundamental change in view that the hematopoietic system is maintained by a continuum of HSC subtypes, rather than a functionally uniform pool.

Clonal haematopoiesis is thought to be a rare condition that increases in frequency with age and predisposes individuals to haematological malignancy. Recent studies, utilizing next-generation sequencing (NGS), observed haematopoietic clones in 10% of 70-year olds and rarely in younger individuals. However, these studies could only detect common haematopoietic clones—>0.02 variant allele fraction (VAF)—due to the error rate of NGS. To identify and characterize clonal mutations below this threshold, here we develop methods for targeted error-corrected sequencing, which enable the accurate detection of clonal mutations as rare as 0.0003 VAF. We apply these methods to study serially banked peripheral blood samples from healthy 50–60-year-old participants in the Nurses' Health Study. We observe clonal haematopoiesis, frequently harbouring mutations in DNMT3A and TET2, in 95% of individuals studied. These clonal mutations are often stable longitudinally and present in multiple haematopoietic compartments, suggesting a long-lived haematopoietic stem and progenitor cell of origin.

Gains and losses in DNA methylation are prominent features of mammalian cell types. To gain insight into mechanisms that could promote shifts in DNA methylation and contribute to cell fate changes, including malignant transformation, we performed genome-wide mapping of 5-methylcytosine and 5-hydroxymethylcytosine in purified murine hematopoietic stem cells. We discovered extended regions of low methylation (Canyons) that span conserved domains frequently containing transcription factors and are distinct from CpG islands and shores. The genes in about half of these methylation Canyons are coated with repressive histone marks while the remainder are covered by activating histone marks and are highly expressed in HSCs. Canyon borders are demarked by 5-hydroxymethylcytosine and become eroded in the absence of DNA methyltransferase 3a (Dnmt3a). Genes dysregulated in human leukemias are enriched for Canyon-associated genes. The novel epigenetic landscape we describe may provide a mechanism for the regulation of hematopoiesis and may contribute to leukemia development.

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386 Aberrant genomic DNA methylation patterns are widely reported in human cancers but the prognostic value and pathological consequences of these marks remain uncertain. CpG methylation is catalyzed by a family of DNA methyltransferase enzymes comprised of three members – Dnmt1, Dnmt3a and Dnmt3b. Mutations in the de novo DNA methyltransferase enzyme DNMT3A have now been reported in over 20% of adult acute myeloid leukemia (AML) and 10–15% of myelodysplastic syndrome (MDS) patients. However, analysis of promoter methylation and gene expression in these patients has thus far failed to yield any mechanistic insight into the pathology of DNMT3A mutation-driven leukemia. In this study, we have used a conditional knockout mouse model to study the role of Dnmt3a in normal hematopoiesis. Hematopoietic stem cells (HSCs) from Mx1-Cre:Dnmt3afl/fl mice were serially transplanted into lethally irradiated recipient mice to study the effect of loss of Dnmt3a on HSC self-renewal and differentiation. We show that loss of Dnmt3a progressively impedes HSC differentiation over four-rounds of serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Examination of the bone marrow post-transplant revealed that control HSCs showed a gradual decline in their ability to regenerate the HSC pool at each successive round of transplantation, while in contrast Dnmt3a-KO HSCs show a remarkably robust capacity for amplification, generating 40,000 – 100,000 HSCs per mouse. Quantification of peripheral blood differentiation on a per HSC basis demonstrated in the absence of Dnmt3a, a cell division is more likely to result in a self-renewal rather than differentiation fate (Figure 1). Using semi-global reduced representation bisulfite sequencing (RRBS), we show that Dnmt3a-KO HSCs manifest both increased and decreased methylation at distinct loci, including dramatic CpG island hypermethylation. Global transcriptional analysis by microarray revealed that Dnmt3a-KO HSCs show upregulation of HSC multipotency genes coupled with simultaneous downregulation of early differentiation factors (e.g. Flt3, PU.1, Mef2c), likely inhibiting the initial stages of HSC differentiation. Upregulation of key HSC regulators including Runx1, Gata3 and Nr4a2 was associated with gene-body hypomethylation and activated chromatin marks (H3K4me3) in Dnmt3a-KO HSCs. Finally, we show that Dnmt3a-KO HSCs are unable to methylate and transcriptionally repress these key HSC multipotency genes in response to chemotherapeutic ablation of the hematopoietic system, leading to inefficient differentiation and manifesting hypomethylation and incomplete repression of HSC-specific genes in their limited differentiated progeny. In conclusion, we show that Dnmt3a plays a specific role in permitting HSC differentiation, as in its absence, phenotypically normal but impotent stem cells accumulate and differentiation capacity is progressively lost. This differentiation-deficit phenotype is reminiscent of Dnmt3a/Dnmt3b-null embryonic stem (ES) cells while markedly distinct from that of Dnmt1-KO HSCs which show premature HSC exhaustion and lymphoid-deficient differentiation, demonstrating distinct roles for the different DNA methyltransferase enzymes in HSCs. In light of the recently-identified DNMT3A mutations in AML and MDS patients, these studies are the first biological models linking mutation of Dnmt3a with inhibition of HSC differentiation which may be one of the first pathogenic steps occuring in such patients.Figure 1Dnmt3a-KO HSCs become biased towards self-renewal as opposed to differentiation. At each transplant round, the self-renewal quotient was calculated as the number of donor-derived HSCs recovered at the end of the transplant divided by 250 (the number of HSC initially transplanted). The differentiation quotient was calculated as (the white blood cell count per μl of blood at 16 weeks) X (percentage of donor-cell chimerism)/number of donor HSC at the end of the transplant. Over serial transfer, Dnmt3a-KO HSCs more rapidly lose their differentiation capacity compared to control HSCs, while sustaining robust self-renewal.Figure 1. Dnmt3a-KO HSCs become biased towards self-renewal as opposed to differentiation. At each transplant round, the self-renewal quotient was calculated as the number of donor-derived HSCs recovered at the end of the transplant divided by 250 (the number of HSC initially transplanted). The differentiation quotient was calculated as (the white blood cell count per μl of blood at 16 weeks) X (percentage of donor-cell chimerism)/number of donor HSC at the end of the transplant. Over serial transfer, Dnmt3a-KO HSCs more rapidly lose their differentiation capacity compared to control HSCs, while sustaining robust self-renewal. Disclosures: Issa: Novartis: Honoraria; GSK: Consultancy; SYNDAX: Consultancy; Merck: Research Funding; Eisai: Research Funding; Celgene: Research Funding; Celgene: Honoraria; J&J: Honoraria.

SUMMARY Hematopoietic stem cells (HSCs) are the precursors of the hematopoietic system responsible for the lifelong production of blood and bone marrow. Given the emerging importance of epigenetic regulation in HSC fate decisions and malignant transformation, we investigated the role of the DNA methyltransferase Dnmt3b through genetic ablation in HSCs – either alone or in combinatorial deletion with its paralog Dnmt3a. While conditional inactivation of Dnmt3b alone in adult HSCs had minor functional impact, simultaneous deletion of Dnmt3a and Dnmt3b was synergistic resulting in a severe block in differentiation and enhanced HSC self-renewal. Dnmt3a/b-null HSCs displayed activated β-catenin signaling, partly accounting for the differentiation block. Loss of Dnmt3a in HSCs resulted in global DNA hypomethylation, but a paradoxical hypermethylation of CpG islands, most of which was eliminated in Dnmt3a/b-null HSCs. These data demonstrate distinct roles for Dnmt3b in HSC differentiation and provide unprecedented resolution into the epigenetic regulation of HSC fate decisions.

Hematopoietic stem cells (HSCs) remain by far the most well-characterized adult stem cell population both in terms of markers for purification and assays to assess functional potential. However, despite over 40 years of research, working with HSCs in the mouse remains difficult because of the relative abundance (or lack thereof) of these cells in the bone marrow. The frequency of HSCs in bone marrow is about 0.01% of total nucleated cells and $5,000 can be isolated from an individual mouse depending on the age, sex, and strain of mice as well as purification scheme utilized. This prohibits the study of processes in HSCs, which require large amounts of starting material. Adding to the challenge is the continual reporting of new markers for HSC purification, which makes it difficult for the uninitiated in the field to know which purification strategies yield the highest proportion of long-term, multilineage HSCs. This report will review different hematopoietic stem and progenitor purification strategies and compare flow cytometry profiles for HSC sorting and analysis on different instruments. We will also discuss methods for rapid flow cytometric analysis of peripheral blood cell types, and novel strategies for working with rare cell populations such as HSCs in the analysis of cell cycle status by BrdU, Ki-67, and Pyronin Y staining. The purpose of this review is to provide insight into some of the recent experimental and technical advances in mouse hematopoietic stem cell biology. ' International Society for Advancement of CytometryKey terms hematopoietic stem cell; flow cytometry; peripheral blood; cell cycle HEMATOPOIETIC stem cells have tremendous therapeutic potential and have been harnessed in the clinic for more than 40 years in the context of bone marrow transplantation. Multipotent long-term HSCs (LT-HSCs) reside in the bone marrow and through a process of asymmetric cell division, can self-renew to sustain the stem cell pool or differentiate into short-term HSCs (ST-HSCs) or lineage-restricted progenitors that undergo extensive proliferation and differentiation to produce terminally differentiated, functional hematopoietic cells. ST-HSCs or multipotent progenitors (MPPs) are only able to sustain hematopoiesis in the short term, whereas the LTHSCs must persist for the lifespan of the organism to perpetually replenish the hematopoietic system. HSCs can be isolated from bone marrow or peripheral blood using enrichment (magnetic cell separation-MACS) and/or single-cell sorting (fluorescence-activated cell sorting-FACS) based on cell surface markers and/or vital dye staining. The HSC has served as the paradigm for adult stem cell populations by virtue of a well-defined differentiation cascade with distinct intermediaries connecting the differentiation of LT-HSCs into mature, functional hematopoietic cells. Each of the cell stages of HSC differentiation can be purified from the bone marrow or peripheral blood using characteristic cell surface markers, which has greatly facilitated the study of hemato...