Dnmt3a Is Essential for Hematopoietic Stem Cell Differentiation

Abstract

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.

PIAS1 SUMO ligase regulates the self-renewal and differentiation of hematopoietic stem cells

“…It has been documented that DNA methylation plays an important role in the regulation of HSC self-renewal and differentiation (Tadokoro et al, 2007;Broske et al, 2009;Trowbridge et al, 2009;Challen et al, 2011), but how DNMTs are regulated to control methylation dynamics in hematopoiesis is not known. PIAS1 interacts with DNMTs in vivo (Liu et al, 2010) (Fig 7D).…”

“…The EMBO Journal PIAS1 regulates hematopoietic stem cells Bin Liu et al Studies from several groups have shown that DNMTs play important roles in regulating the self-renewal and differentiation of HSCs (Tadokoro et al, 2007;Broske et al, 2009;Trowbridge et al, 2009;Challen et al, 2011). The conditional knockout of Dnmt3a impaired HSC differentiation, and Dnmt3a-null HSCs upregulated HSC multipotency genes and downregulated differentiation factors (Challen et al, 2011). The phenotype of Pias1 À/À mice in regulating HSC lineage differentiation resembles that of Dnmt1 knockout mice (Broske et al, 2009).…”

“…DNA methylation is mediated mainly by three DNA methyltransferases (DNMTs), including the de novo methyltransferases DNMT3A and DNMT3B, and the maintenance methyltransferase DNMT1 (Jaenisch & Bird, 2003). Recent studies have shown that DNMTs are critical for regulating the self-renewal and differentiation of HSCs (Tadokoro et al, 2007;Broske et al, 2009;Trowbridge et al, 2009;Challen et al, 2011). Although DNMTs do not possess sequence-specific DNA binding properties, gene-specific and temporal epigenetic changes are associated with the proper differentiation of self-renewing HSCs.…”

Genetic profiling in acute myeloid leukaemia ─ where are we and what is its role in patient management

“…The DNA methyltransferase 3A (DNMT3A) gene was recently recognized to play an important role in haematopoietic stem cell self-renewal and differentiation (Challen et al, 2012;Trowbridge & Orkin, 2012). Mutations in DNMT3A are associated with an adverse outcome.…”

PIAS adds methyl-bias to HSC-differentiation

“…DNA methylation is critical for the maintenance of hematopoietic stem cells and for their lineage commitment and stepwise differentiation (Broske et al, 2009;Challen et al, 2012;Tadokoro et al, 2007;Trowbridge et al, 2009). Specifically, DNMT1 is essential for hematopoietic stem cell selfrenewal and homeostasis as well as for myeloid versus lymphoid fate choice (Broske et al, 2009;Trowbridge et al, 2009).…”