Mira Jeong

Dnmt3a is essential for hematopoietic stem cell differentiation

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.

Epigenomic profiling of young and aged HSCs reveals concerted changes during aging that reinforce self-renewal

To investigate the cell-intrinsic aging mechanisms that erode the function of somatic stem cells during aging, we have conducted a comprehensive integrated genomic analysis of young and aged cells. We profiled the transcriptome, DNA methylome, and histone modifications of young and old murine hematopoietic stem cells (HSCs). Transcriptome analysis indicated reduced TGF-β signaling and perturbation of genes involved in HSC proliferation and differentiation. Aged HSCs exhibited broader H3K4me3 peaks across HSC identity and self-renewal genes and showed increased DNA methylation at transcription factor binding sites associated with differentiation-promoting genes combined with a reduction at genes associated with HSC maintenance. Altogether, these changes reinforce HSC self-renewal and diminish differentiation, paralleling phenotypic HSC aging behavior. Ribosomal biogenesis emerged as a particular target of aging with increased transcription of ribosomal protein and RNA genes and hypomethylation of rRNA genes. This data set will serve as a reference for future epigenomic analysis of stem cell aging.

Large conserved domains of low DNA methylation maintained by Dnmt3a

Gains and losses in DNA methylation are prominent features of mammalian cell types. To gain insight into the mechanisms that promote shifts in DNA methylation and contribute to changes in cell fate, including malignant transformation, we performed genome-wide mapping of 5-methylcytosine and 5-hydroxymethylcytosine in purified mouse 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. About half of the genes in these methylation canyons are coated with repressive histone marks, whereas the remainder are covered by activating histone marks and are highly expressed in hematopoietic stem cells (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 new epigenetic landscape we describe may provide a mechanism for the regulation of hematopoiesis and may contribute to leukemia development.

Less Is More: Unveiling the Functional Core of Hematopoietic Stem Cells through Knockout Mice

Hematopoietic stem cells (HSCs) represent one of the first recognized somatic stem cell types. As such, nearly 200 genes have been examined for roles in HSC function in knockout mice. In this review, we compile the majority of these reports to provide a broad overview of the functional modules revealed by these genetic analyses and highlight some key regulatory pathways involved, including cell cycle control, Tgf-β signaling, Pten/Akt signaling, Wnt signaling, and cytokine signaling. Finally, we propose recommendations for characterization of HSC function in knockout mice to facilitate cross-study comparisons that would generate a more cohesive picture of HSC biology.

Genomic Hypomethylation in the Human Germline Associates with Selective Structural Mutability in the Human Genome

The hotspots of structural polymorphisms and structural mutability in the human genome remain to be explained mechanistically. We examine associations of structural mutability with germline DNA methylation and with non-allelic homologous recombination (NAHR) mediated by low-copy repeats (LCRs). Combined evidence from four human sperm methylome maps, human genome evolution, structural polymorphisms in the human population, and previous genomic and disease studies consistently points to a strong association of germline hypomethylation and genomic instability. Specifically, methylation deserts, the ∼1% fraction of the human genome with the lowest methylation in the germline, show a tenfold enrichment for structural rearrangements that occurred in the human genome since the branching of chimpanzee and are highly enriched for fast-evolving loci that regulate tissue-specific gene expression. Analysis of copy number variants (CNVs) from 400 human samples identified using a custom-designed array comparative genomic hybridization (aCGH) chip, combined with publicly available structural variation data, indicates that association of structural mutability with germline hypomethylation is comparable in magnitude to the association of structural mutability with LCR–mediated NAHR. Moreover, rare CNVs occurring in the genomes of individuals diagnosed with schizophrenia, bipolar disorder, and developmental delay and de novo CNVs occurring in those diagnosed with autism are significantly more concentrated within hypomethylated regions. These findings suggest a new connection between the epigenome, selective mutability, evolution, and human disease.

Flow cytometry analysis of murine hematopoietic stem cells

Hematopoietic stem cells (HSCs) remain 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 challenging because of the relative abundance (or lack thereof) of these cells in the bone marrow. The frequency of HSCs in murine 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. 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. In this updated version of our review from 2009, we review different strategies for hematopoietic stem and progenitor cell identification and purification. 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 updating this review is to provide insight into some of the recent experimental and technical advances in mouse hematopoietic stem cell biology.

Long Non-Coding RNAs Control Hematopoietic Stem Cell Function

Hematopoietic stem cells (HSCs) possess unique gene expression programs that enforce their identity and regulate lineage commitment. Long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression and cell fate decisions, although their functions in HSCs are unclear. Here we profiled the transcriptome of purified HSCs by deep sequencing and identified 323 unannotated lncRNAs. Comparing their expression in differentiated lineages revealed 159 lncRNAs enriched in HSCs, some of which are likely HSC specific (LncHSCs). These lncRNA genes share epigenetic features with protein-coding genes, including regulated expression via DNA methylation, and knocking down two LncHSCs revealed distinct effects on HSC self-renewal and lineage commitment. We mapped the genomic binding sites of one of these candidates and found enrichment for key hematopoietic transcription factor binding sites, especially E2A. Together, these results demonstrate that lncRNAs play important roles in regulating HSCs, providing an additional layer to the genetic circuitry controlling HSC function.

Acute loss of TET function results in aggressive myeloid cancer in mice

TET-family dioxygenases oxidize 5-methylcytosine (5mC) in DNA, and exert tumour suppressor activity in many types of cancers. Even in the absence of TET coding region mutations, TET loss-of-function is strongly associated with cancer. Here we show that acute elimination of TET function induces the rapid development of an aggressive, fully-penetrant and cell-autonomous myeloid leukaemia in mice, pointing to a causative role for TET loss-of-function in this myeloid malignancy. Phenotypic and transcriptional profiling shows aberrant differentiation of haematopoietic stem/progenitor cells, impaired erythroid and lymphoid differentiation and strong skewing to the myeloid lineage, with only a mild relation to changes in DNA modification. We also observe progressive accumulation of phospho-H2AX and strong impairment of DNA damage repair pathways, suggesting a key role for TET proteins in maintaining genome integrity.

DNMT3A and TET2 compete and cooperate to repress lineage-specific transcription factors in hematopoietic stem cells

Mutations in the epigenetic modifiers DNMT3A and TET2 non-randomly co-occur in lymphoma and leukemia despite their epistasis in the methylation–hydroxymethylation pathway. Using Dnmt3a and Tet2 double-knockout mice in which the development of malignancy is accelerated, we show that the double-knockout methylome reflects regions of independent, competitive and cooperative activity. Expression of lineage-specific transcription factors, including the erythroid regulators Klf1 and Epor, is upregulated in double-knockout hematopoietic stem cells (HSCs). DNMT3A and TET2 both repress Klf1, suggesting a model of cooperative inhibition by epigenetic modifiers. These data demonstrate a dual role for TET2 in promoting and inhibiting HSC differentiation, the loss of which, along with DNMT3A, obstructs differentiation, leading to transformation.

DNMT3A Loss Drives Enhancer Hypomethylation in FLT3-ITD-Associated Leukemias

DNMT3A, the gene encoding the de novo DNA methyltransferase 3A, is among the most frequently mutated genes in hematologic malignancies. However, the mechanisms through which DNMT3A normally suppresses malignancy development are unknown. Here, we show that DNMT3A loss synergizes with the FLT3 internal tandem duplication in a dose-influenced fashion to generate rapid lethal lymphoid or myeloid leukemias similar to their human counterparts. Loss of DNMT3A leads to reduced DNA methylation, predominantly at hematopoietic enhancer regions in both mouse and human samples. Myeloid and lymphoid diseases arise from transformed murine hematopoietic stem cells. Broadly, our findings support a role for DNMT3A as a guardian of the epigenetic state at enhancer regions, critical for inhibition of leukemic transformation.