TET2 loss-of-function mutations are recurrent events in a wide range of hematological malignancies and a physiologic occurrence in blood cells of healthy older adults. It is currently unknown what ...determines if a person harboring a somatic TET2 mutation will progress to myelodysplastic syndrome or acute myeloid leukemia. Here we develop a zebrafish tet2 mutant through which we show that tet2 loss leads to restricted hematopoietic differentiation combined with a modest upregulation of p53, which is also characteristic of many inherited bone marrow failure syndromes. Uniquely in the context of emergency hematopoiesis by external stimuli, such as infection or cytokine stimulation, lack of tet2 leads hematopoietic stem cells to undergo excessive proliferation, resulting in an accumulation of immature cells, which are poised to become leukemogenic following additional genetic/epigenetic perturbations. This same phenomenon observed in zebrafish extends to human hematopoietic stem cells, identifying TET2 as a critical relay switch in the context of stress hematopoiesis.
Most human breast cancers have diversified genomically and biologically by the time they become clinically evident. Early events involved in their genesis and the cellular context in which these ...events occur have thus been difficult to characterize. Here we present the first formal evidence of the shared and independent ability of basal cells and luminal progenitors, isolated from normal human mammary tissue and transduced with a single oncogene (KRAS(G12D)), to produce serially transplantable, polyclonal, invasive ductal carcinomas within 8 weeks of being introduced either subrenally or subcutaneously into immunodeficient mice. DNA barcoding of the initial cells revealed a dramatic change in the numbers and sizes of clones generated from them within 2 weeks, and the first appearance of many 'new' clones in tumours passaged into secondary recipients. Both primary and secondary tumours were phenotypically heterogeneous and primary tumours were categorized transcriptionally as 'normal-like'. This system challenges previous concepts that carcinogenesis in normal human epithelia is necessarily a slow process requiring the acquisition of multiple driver mutations. It also presents the first description of initial events that accompany the genesis and evolution of malignant human mammary cell populations, thereby contributing new understanding of the rapidity with which heterogeneity in their properties can develop.
The somatic missense point mutation c.402C>G (p.C134W) in the FOXL2 transcription factor is pathognomonic for adult-type granulosa cell tumors (AGCT) and a diagnostic marker for this tumor type. ...However, the molecular consequences of this mutation and its contribution to the mechanisms of AGCT pathogenesis remain unclear. To explore these mechanisms, we engineered V5-FOXL2
- and V5-FOXL2
-inducible isogenic cell lines and performed chromatin immunoprecipitation sequencing and transcriptome profiling. FOXL2
associated with the majority of the FOXL2 wild-type DNA elements as well as a large collection of unique elements genome wide. This model enabled confirmation of altered DNA-binding specificity for FOXL2
and identification of unique targets of FOXL2
including
, whose expression increased sensitivity to YM155. Our results suggest FOXL2
drives AGCT by altering the binding affinity of FOXL2-containing complexes to engage an oncogenic transcriptional program. SIGNIFICANCE: A mechanistic understanding of FOXL2
-induced regulatory state alterations drives discovery of a rationally designed therapeutic strategy.
The normal adult human mammary gland is a continuous bilayered epithelial system. Bipotent and myoepithelial progenitors are prominent and unique components of the outer (basal) layer. The inner ...(luminal) layer includes both luminal-restricted progenitors and a phenotypically separable fraction that lacks progenitor activity. We now report an epigenomic comparison of these three subsets with one another, with their associated stromal cells, and with three immortalized, non-tumorigenic human mammary cell lines. Each genome-wide analysis contains profiles for six histone marks, methylated DNA, and RNA transcripts. Analysis of these datasets shows that each cell type has unique features, primarily within genomic regulatory regions, and that the cell lines group together. Analyses of the promoter and enhancer profiles place the luminal progenitors in between the basal cells and the non-progenitor luminal subset. Integrative analysis reveals networks of subset-specific transcription factors.
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•Epigenomes of four normal human breast cell fractions and three cell lines are reported•Luminal progenitor and mature luminal cell epigenomes differ greatly•Enhancers define luminal progenitors as intermediate between basal and luminal cells•Transcription factor binding sites in active enhancers point to distinct regulators
Pellacani et al. present comprehensive histone and DNA modification profiles for four cell types in normal human breast tissue and three immortalized human mammary epithelial cell lines. Analysis of activated enhancers place luminal progenitors in between bipotent progenitor-containing basal cells and nonproliferative luminal cells. Explore consortium data at the Cell Press IHEC webportal at http://www.cell.com/consortium/IHEC.
Human cord blood (CB) offers an attractive source of cells for clinical transplants because of its rich content of cells with sustained repopulating ability in spite of an apparent deficiency of ...cells with rapid reconstituting ability. Nevertheless, the clonal dynamics of nonlimiting CB transplants remain poorly understood. To begin to address this question, we exposed CD34+ CB cells to a library of barcoded lentiviruses and used massively parallel sequencing to quantify the clonal distributions of lymphoid and myeloid cells subsequently detected in sequential marrow aspirates obtained from 2 primary NOD/SCID-IL2Rγ−/− mice, each transplanted with ∼105 of these cells, and for another 6 months in 2 secondary recipients. Of the 196 clones identified, 68 were detected at 4 weeks posttransplant and were often lympho-myeloid. The rest were detected later, after variable periods up to 13 months posttransplant, but with generally increasing stability throughout time, and they included clones in which different lineages were detected. However, definitive evidence of individual cells capable of generating T-, B-, and myeloid cells, for over a year, and self-renewal of this potential was also obtained. These findings highlight the caveats and utility of this model to analyze human hematopoietic stem cell control in vivo.
Key Points
The leukemia inhibitory factor (Lif) signaling pathway is a crucial determinant for mouse embryonic stem (mES) cell self-renewal and pluripotency. One of the hallmarks of mES cells, their compact ...growth morphology, results from tight cell adhesion mediated through E-cadherin, β-catenin (Ctnnb1) and α-catenin with the actin cytoskeleton. β-catenin is also involved in canonical Wnt signaling, which has also been suggested to control mES cell stemness. Here, we analyze Ctnnb1(-/-) mES cells in which cell adhesion is preserved by an E-cadherin-α-catenin (Eα) fusion protein (Ctnnb1(-/-)Eα mES cells), and show that mimicking only the adhesive function of β-catenin is necessary and sufficient to maintain the mES cell state, making β-catenin/Wnt signaling obsolete in this process. Furthermore, we propose a role for E-cadherin in promoting the Lif signaling cascade, showing an association of E-cadherin with the Lifr-Gp130 receptor complex, which is most likely facilitated by the extracellular domain of E-cadherin. Without Eα, and thus without maintained cell adhesion, Ctnnb1(-/-) mES cells downregulate components of the Lif signaling pathway, such as Lifr, Gp130 and activated Stat3, as well as pluripotency-associated markers. From these observations, we hypothesize that the changes in gene expression accompanying the loss of pluripotency are a direct consequence of dysfunctional cell adhesion. Supporting this view, we find that the requirement for intact adhesion can be circumvented by the forced expression of constitutively active Stat3. In summary, we put forward a model in which mES cells can be propagated in culture in the absence of Ctnnb1, as long as E-cadherin-mediated cell adhesion is preserved.
Hematopoietic clones with leukemogenic mutations arise in healthy people as they age, but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may ...play an important role in modulating human AML population dynamics. To investigate this concept further, we examined the combined and separate effects of an oncogene (c-MYC) and exposure to interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized, rapidly fatal, and serially transplantable blast population, phenotypically and transcriptionally similar to human AML cells, but only in mice producing IL-3, GM-CSF, and SCF transgenically or in regular mice in which the cells were exposed to IL-3 or GM-CSF delivered using a cotransduction strategy. In their absence, the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months, but, on transfer to secondary mice producing the human cytokines, the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38-) and late granulocyte-macrophage progenitor (GMP) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them.
The thyroid gland, necessary for normal human growth and development, functions as an essential regulator of metabolism by the production and secretion of appropriate levels of thyroid hormone. ...However, assessment of abnormal thyroid function may be challenging suggesting a more fundamental understanding of normal function is needed. One way to characterize normal gland function is to study the epigenome and resulting transcriptome within its constituent cells. This study generates the first published reference epigenomes for human thyroid from four individuals using ChIP-seq and RNA-seq. We profiled six histone modifications (H3K4me1, H3K4me3, H3K27ac, H3K36me3, H3K9me3, H3K27me3), identified chromatin states using a hidden Markov model, produced a novel quantitative metric for model selection and established epigenomic maps of 19 chromatin states. We found that epigenetic features characterizing promoters and transcription elongation tend to be more consistent than regions characterizing enhancers or Polycomb-repressed regions and that epigenetically active genes consistent across all epigenomes tend to have higher expression than those not marked as epigenetically active in all epigenomes. We also identified a set of 18 genes epigenetically active and consistently expressed in the thyroid that are likely highly relevant to thyroid function. Altogether, these epigenomes represent a powerful resource to develop a deeper understanding of the underlying molecular biology of thyroid function and provide contextual information of thyroid and human epigenomic data for comparison and integration into future studies.
The life of a plant is characterized by major phase transitions. This includes the agriculturally important transitions from seed to seedling (germination) and from vegetative to generative growth ...(flowering induction). In many plant species, including Arabidopsis thaliana, freshly harvested seeds are dormant and incapable of germinating. Germination can occur after the release of dormancy and the occurrence of favourable environmental conditions. Although the hormonal control of seed dormancy is well studied, the molecular mechanisms underlying the induction and release of dormancy are not yet understood.In this study, we report the cloning and characterization of the mutant reduced dormancy 2-1 (rdo2-1). We found that RDO2 is allelic to the recently identified dormancy gene TFIIS, which is a transcription elongation factor. HUB1, which was previously called RDO4, was identified in the same mutagenesis screen for reduced dormancy as rdo2-1 and was also shown to be involved in transcription elongation. The human homologues of RDO2 and HUB1 interact with the RNA Polymerase II Associated Factor 1 Complex (PAF1C). Therefore, we investigated the effect of other Arabidopsis PAF1C related factors; VIP4, VIP5, ELF7, ELF8 and ATXR7 on seed dormancy. Mutations in these genes resulted in reduced dormancy, similar to hub1-2 and rdo2-1. Consistent with a role at the end of seed maturation, we found that HUB1, RDO2 and VIP5 are upregulated during this developmental phase. Since mutants in PAF1C related factors are also described to be early flowering, we conclude that these components are involved in the regulation of both major developmental transitions in the plant.
Cellular barcoding offers a powerful approach to characterize the growth and differentiation activity of large numbers of cotransplanted stem cells. Here, we describe a lentiviral genomic-barcoding ...and analysis strategy and its use to compare the clonal outputs of transplants of purified mouse and human basal mammary epithelial cells. We found that both sources of transplanted cells produced many bilineage mammary epithelial clones in primary recipients, although primary clones containing only one detectable mammary lineage were also common. Interestingly, regardless of the species of origin, many clones evident in secondary recipients were not detected in the primary hosts, and others that were changed from appearing luminal-restricted to appearing bilineage. This barcoding methodology has thus revealed conservation between mice and humans of a previously unknown diversity in the growth and differentiation activities of their basal mammary epithelial cells stimulated to grow in transplanted hosts.
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•A method for analyzing clones in regenerating epithelial populations is shown•Transplanted mouse and human basal mammary cells show similar growth patterns•In serial transplants of mammary cells, some clones show very delayed growth•Mammary clones may switch their differentiation behavior when serially transplanted
Hirst et al. use barcoding to analyze clonal growth of transplanted mammary stem cells. They find that lineage phenotypes change and that many clones are only detected in secondary transplants.
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