Non-catalytic Roles of Tet2 Are Essential to Regulate Hematopoietic Stem and Progenitor Cell Homeostasis

The Ten-eleven translocation (TET) enzymes regulate gene expression by promoting DNA demethylation and partnering with chromatin modifiers. TET2, a member of this family, is frequently mutated in hematological disorders. The contributions of TET2 in hematopoiesis have been attributed to its DNA demethylase activity, and the significance of its nonenzymatic functions has remained undefined. To dissect the catalytic and non-catalytic requirements of Tet2, we engineered catalytically inactive Tet2 mutant mice and conducted comparative analyses of Tet2 mutant and Tet2 knockout animals. Tet2 knockout mice exhibited expansion of hematopoietic stem and progenitor cells (HSPCs) and developed myeloid and lymphoid disorders, while Tet2 mutant mice predominantly developed myeloid malignancies reminiscent of human myelodysplastic syndromes. HSPCs from Tet2 knockout mice exhibited distinct gene expression profiles, including downregulation of Gata2. Overexpression of Gata2 in Tet2 knockout bone marrow cells ameliorated disease phenotypes. Our results reveal the non-catalytic roles of TET2 in HSPC homeostasis

Tet2 disruption leads to enhanced self-renewal and altered differentiation of fetal liver hematopoietic stem cells

Somatic mutation of ten-eleven translocation 2 (TET2) gene is frequently found in human myeloid malignancies. Recent reports showed that loss of Tet2 led to pleiotropic hematopoietic abnormalities including increased competitive repopulating capacity of bone marrow (BM) HSCs and myeloid transformation. However, precise impact of Tet2 loss on the function of fetal liver (FL) HSCs has not been examined. Here we show that disruption of Tet2 results in the expansion of Lin−Sca-1+c-Kit+ (LSK) cells in FL. Furthermore, Tet2 loss led to enhanced self-renewal and long-term repopulating capacity of FL-HSCs in in vivo serial transplantation assay. Disruption of Tet2 in FL also led to altered differentiation of mature blood cells, expansion of common myeloid progenitors and increased resistance for hematopoietic progenitor cells (HPCs) to differentiation stimuli in vitro. These results demonstrate that Tet2 plays a critical role in homeostasis of HSCs and HPCs not only in the BM, but also in FL

Genetic Variation and Population Substructure in Outbred CD-1 Mice: Implications for Genome-Wide Association Studies

Outbred laboratory mouse populations are widely used in biomedical research. Since little is known about the degree of genetic variation present in these populations, they are not widely used for genetic studies. Commercially available outbred CD-1 mice are drawn from an extremely large breeding population that has accumulated many recombination events, which is desirable for genome-wide association studies. We therefore examined the degree of genome-wide variation within CD-1 mice to investigate their suitability for genetic studies. The CD-1 mouse genome displays patterns of linkage disequilibrium and heterogeneity similar to wild-caught mice. Population substructure and phenotypic differences were observed among CD-1 mice obtained from different breeding facilities. Differences in genetic variation among CD-1 mice from distinct facilities were similar to genetic differences detected between closely related human populations, consistent with a founder effect. This first large-scale genetic analysis of the outbred CD-1 mouse strain provides important considerations for the design and analysis of genetic studies in CD-1 mice

The hypomethylating agent Decitabine causes a paradoxical increase in 5-hydroxymethylcytosine in human leukemia cells

The USFDA approved "epigenetic drug", Decitabine, exerts its effect by hypomethylating DNA, demonstrating the pivotal role aberrant genome-wide DNA methylation patterns play in cancer ontology. Using sensitive technologies in a cellular model of Acute Myeloid Leukemia, we demonstrate that while Decitabine reduces the global levels of 5-methylcytosine (5mC), it results in paradoxical increase of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) levels. Hitherto, the only biological mechanism known to generate 5hmC, 5fC and 5caC, involving oxidation of 5mC by members of Ten-Eleven-Translocation (TET) dioxygenase family, was not observed to undergo any alteration during DAC treatment. Using a multi-compartmental model of DNA methylation, we show that partial selectivity of TET enzymes for hemi-methylated CpG dinucleotides could lead to such alterations in 5hmC content. Furthermore, we investigated the binding of TET1-catalytic domain (CD)-GFP to DNA by Fluorescent Correlation Spectroscopy in live cells and detected the gradual increase of the DNA bound fraction of TET1-CD-GFP after treatment with Decitabine. Our study provides novel insights on the therapeutic activity of DAC in the backdrop of the newly discovered derivatives of 5mC and suggests that 5hmC has the potential to serve as a biomarker for monitoring the clinical success of patients receiving DAC

Loss of Function of TET2 Cooperates with Constitutively Active KIT in Murine and Human Models of Mastocytosis

Systemic Mastocytosis (SM) is a clonal disease characterized by abnormal accumulation of mast cells in multiple organs. Clinical presentations of the disease vary widely from indolent to aggressive forms, and to the exceedingly rare mast cell leukemia. Current treatment of aggressive SM and mast cell leukemia is unsatisfactory. An imatinib-resistant activating mutation of the receptor tyrosine kinase KIT (KIT D816V) is most frequently present in transformed mast cells and is associated with all clinical forms of the disease. Thus the etiology of the variable clinical aggressiveness of abnormal mast cells in SM is unclear. TET2 appears to be mutated in primary human samples in aggressive types of SM, suggesting a possible role in disease modification. In this report, we demonstrate the cooperation between KIT D816V and loss of function of TET2 in mast cell transformation and demonstrate a more aggressive phenotype in a murine model of SM when both mutations are present in progenitor cells. We exploit these findings to validate a combination treatment strategy targeting the epigenetic deregulation caused by loss of TET2 and the constitutively active KIT receptor for the treatment of patients with aggressive SM

Developmental functions of the dynamic DNA methylome and hydroxymethylome in the mouse and zebrafish: similarities and differences

5-methylcytosine (5mC) is the best understood DNA modification and is generally believed to be associated with repression of gene expression. Over the last decade, sequentially oxidized forms of 5mC (oxi-mCs) have been discovered within the genomes of vertebrates. Their discovery was accompanied by that of the ten-eleven translocation (TET) methylcytosine dioxygenases, the enzymes that catalyse the formation of the oxi-mCs. Although a number of studies performed on different vertebrate models and embryonic stem cells demonstrated that both TET enzymes and oxi-mCs are likely to be important for several developmental processes it is currently unclear whether their developmental roles are conserved among vertebrates. Here, we summarise recent developments in this field suggesting that biological roles of TETs/oxi-mCs may significantly differ between mice and zebrafish. Thus, although the role of TET proteins in late organogenesis has been documented for both these systems; unlike in mice the enzymatic oxidation of 5mC does not seem to be involved in zygotic reprogramming or gastrulation in zebrafish. Our analysis may provide an insight into the general principles of epigenetic regulation of animal development and cellular differentiation

Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity

The TET family of FE(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) promote DNA demethylation by converting 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), which they further oxidize into 5-formylcytosine and 5-carboxylcytosine. Tet1 is robustly expressed in mouse embryonic stem cells (mESCs) and has been implicated in mESC maintenance. Here we demonstrate that, unlike genetic deletion, RNAi-mediated depletion of Tet1 in mESCs led to a significant reduction in 5hmC and loss of mESC identity. The differentiation phenotype due to Tet1 depletion positively correlated with the extent of 5hmC loss. Meta-analyses of genomic data sets suggested interaction between Tet1 and leukemia inhibitory factor (LIF) signaling. LIF signaling is known to promote self-renewal and pluripotency in mESCs partly by opposing MAPK/ERK-mediated differentiation. Withdrawal of LIF leads to differentiation of mESCs. We discovered that Tet1 depletion impaired LIF-dependent Stat3-mediated gene activation by affecting Stat3's ability to bind to its target sites on chromatin. Nanog overexpression or inhibition of MAPK/ERK signaling, both known to maintain mESCs in the absence of LIF, rescued Tet1 depletion, further supporting the dependence of LIF/Stat3 signaling on Tet1. These data support the conclusion that analysis of mESCs in the hours/days immediately following efficient Tet1 depletion reveals Tet1's normal physiological role in maintaining the pluripotent state that may be subject to homeostatic compensation in genetic models

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 integrityopen0

Cell cycle regulation in hematopoietic stem cells

Hematopoietic stem cells (HSCs) give rise to all lineages of blood cells. Because HSCs must persist for a lifetime, the balance between their proliferation and quiescence is carefully regulated to ensure blood homeostasis while limiting cellular damage. Cell cycle regulation therefore plays a critical role in controlling HSC function during both fetal life and in the adult. The cell cycle activity of HSCs is carefully modulated by a complex interplay between cell-intrinsic mechanisms and cell-extrinsic factors produced by the microenvironment. This fine-tuned regulatory network may become altered with age, leading to aberrant HSC cell cycle regulation, degraded HSC function, and hematological malignancy

TET1 is a tumor suppressor of hematopoietic malignancy

The methylcytosine dioxygenase TET1 (‘ten-eleven translocation 1’) is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.National Institutes of Health (U.S.) (5RO1HD045022)National Institutes of Health (U.S.) (5R37CA084198