The mechanisms underlying haematopoietic lineage decisions remain disputed. Lineage-affiliated transcription factors with the capacity for lineage reprogramming, positive auto-regulation and mutual inhibition have been described as being expressed in uncommitted cell populations. This led to the assumption that lineage choice is cell-intrinsically initiated and determined by stochastic switches of randomly fluctuating cross-antagonistic transcription factors. However, this hypothesis was developed on the basis of RNA expression data from snapshot and/or population-averaged analyses. Alternative models of lineage choice therefore cannot be excluded. Here we use novel reporter mouse lines and live imaging for continuous single-cell long-term quantification of the transcription factors GATA1 and PU.1 (also known as SPI1). We analyse individual haematopoietic stem cells throughout differentiation into megakaryocytic-erythroid and granulocytic-monocytic lineages. The observed expression dynamics are incompatible with the assumption that stochastic switching between PU.1 and GATA1 precedes and initiates megakaryocytic-erythroid versus granulocytic-monocytic lineage decision-making. Rather, our findings suggest that these transcription factors are only executing and reinforcing lineage choice once made. These results challenge the current prevailing model of early myeloid lineage choice.
Mice with an impaired Type I interferon (IFN) signaling (IFNAR1- and IFNβ-deficient mice) display an increased susceptibility toward v-ABL-induced B-cell leukemia/lymphoma. The enhanced leukemogenesis in the absence of an intact Type I IFN signaling is caused by alterations within the tumor environment. Deletion of Ifnar1 in tumor cells (as obtained in Ifnar1f/f CD19-Cre mice) failed to impact on disease latency or type. In line with this observation, the initial transformation and proliferative capacity of tumor cells were unaltered irrespective of whether the cells expressed IFNAR1 or not. v-ABL-induced leukemogenesis is mainly subjected to natural killer (NK) cell-mediated tumor surveillance. Thus, we concentrated on NK cell functions in IFNAR1 deficient animals. Ifnar1-/- NK cells displayed maturation defects as well as an impaired cytolytic activity. When we deleted Ifnar1 selectively in mature NK cells (by crossing Ncr1-iCre mice to Ifnar1f/f animals), maturation was not altered. However, NK cells derived from Ifnar1f/f Ncr1-iCre mice showed a significant cytolytic defect in vitro against the hematopoietic cell lines YAC-1 and RMA-S, but not against the melanoma cell line B16F10. Interestingly, this defect was not related to an in vivo phenotype as v-ABL-induced leukemogenesis was unaltered in Ifnar1f/f Ncr1-iCre compared with Ifnar1f/f control mice. Moreover, the ability of Ifnar1f/f Ncr1-iCre NK cells to kill B16F10 melanoma cells was unaltered, both in vitro and in vivo. Our data reveal that despite the necessity for Type I IFN in NK cell maturation the expression of IFNAR1 on mature murine NK cells is not required for efficient tumor surveillance.
The ten-eleven translocation 2 gene (TET2) encodes a member of the TET family of DNA methylcytosine oxidases that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) to initiate the demethylation of DNA within genomic CpG islands. Somatic loss-of-function mutations of TET2 are frequently observed in human myelodysplastic syndrome (MDS), which is a clonal malignancy characterized by dysplastic changes of developing blood cell progenitors, leading to ineffective hematopoiesis. We used genome-editing technology to disrupt the zebrafish Tet2 catalytic domain. T ET2 belongs to the TET (ten-eleven translocation) family of methylcytosine oxidases, which require 2-oxoglutarate, oxygen, and Fe(II) for their activity. TET2, like TET1 and TET3, modifies the methylation status of the genome, regulating the transcription of specific genes by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and then to 5-formylcytosine (5fC) and finally to 5-carboxylcytosine (5caC). Each of the last 3 products is recognized and excised by thymine DNA glycosylase (TDG), completing the removal of the 5-methyl group and regenerating unmodified cytosine (1). Hydroxylation of 5mC by the TET enzymes, returning cytosine to its unmethylated state, has been shown to be crucial to many aspects of embryonic development, including embryonic stem cell (ESC) renewal, epigenetic programming of zygotic cells, and meiosis of primordial germ cells (PGCs) (reviewed in references 2 and 3).A variety of alterations, including deletions and missense, nonsense, and frameshift mutations, inactivate the TET2 enzyme in different types of human myeloid malignancies, such as myelodysplastic syndromes (MDS) (25 to 35%) (4-7), myeloproliferative neoplasms (MPN) (2 to 20%) (8, 9), de novo acute myeloid leukemia (AML) (12 to 17%) (10-14), secondary AML (24 to 32%) (11,12), and chronic myelomonocytic leukemia (CMML) (50 to 60%) (5). In these diseases, TET2 gene alterations lead to a marked reduction in global levels of 5hmC (15). TET2 mutations have also been identified in the hematopoietic cells of otherwise healthy adults over 50 years of age who have "clonal skewing" of their bone marrow cells (16), indicating that TET2 mutations may represent one of the first mutations leading to clonal expansion and the eventual development of myeloid malignancies.The role of TET2 mutations in myeloid malignancies has been studied in a number of mouse models (17-20). The hematopoietic stem cells (HSCs) in these models have low 5hmC content and exhibit increased self-renewal ability and a competitive advantage over wild-type HSCs for repopulating hematopoietic lineages. Tet2 knockout mice are viable and fertile and appear to develop normally. However, as they age, Tet2-deficient mice are prone to develop myeloid malignancies, predominantly CMML, with 20 to 30% developing disease after 8 months of age, clearly suggesting that additional genetic lesions are needed to initiate myeloid malignancy.Thus, the essential role of TET2 in maintaining the normal growt...
In E-myc transgenic animals lymphoma formation requires additional genetic alterations, which frequently comprise loss of p53 or overexpression of BCL-2. We describe that the nature of the "second hit" affects the ability of the immune system to contain lymphoma development. Tumors with disrupted p53 signaling killed the host more rapidly than BCL-2 overexpressing ones. Relaxing immunologic control, using Tyk2 ؊/؊ mice or by Ab-mediated depletion of CD8 ؉ T or natural killer (
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