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1.
Cell Stem Cell ; 31(2): 244-259.e10, 2024 02 01.
Article in English | MEDLINE | ID: mdl-38183977

ABSTRACT

The paradigmatic hematopoietic tree model is increasingly recognized to be limited, as it is based on heterogeneous populations largely defined by non-homeostatic assays testing cell fate potentials. Here, we combine persistent labeling with time-series single-cell RNA sequencing to build a real-time, quantitative model of in vivo tissue dynamics for murine bone marrow hematopoiesis. We couple cascading single-cell expression patterns with dynamic changes in differentiation and growth speeds. The resulting explicit linkage between molecular states and cellular behavior reveals widely varying self-renewal and differentiation properties across distinct lineages. Transplanted stem cells show strong acceleration of differentiation at specific stages of erythroid and neutrophil production, illustrating how the model can quantify the impact of perturbations. Our reconstruction of dynamic behavior from snapshot measurements is akin to how a kinetoscope allows sequential images to merge into a movie. We posit that this approach is generally applicable to understanding tissue-scale dynamics at high resolution.


Subject(s)
Bone Marrow , Hematopoietic Stem Cells , Animals , Mice , Hematopoietic Stem Cells/metabolism , Hematopoiesis/genetics , Cell Differentiation
2.
Science ; 381(6659): eadd7564, 2023 08 18.
Article in English | MEDLINE | ID: mdl-37590359

ABSTRACT

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.


Subject(s)
Embryonic Development , Yolk Sac , Female , Humans , Pregnancy , Blood Coagulation/genetics , Macrophages , Yolk Sac/cytology , Yolk Sac/metabolism , Embryonic Development/genetics , Atlases as Topic , Gene Expression , Gene Expression Profiling , Hematopoiesis/genetics , Liver/embryology
3.
Mol Cell ; 83(14): 2417-2433.e7, 2023 07 20.
Article in English | MEDLINE | ID: mdl-37348497

ABSTRACT

Aged hematopoietic stem cells (HSCs) display diminished self-renewal and a myeloid differentiation bias. However, the drivers and mechanisms that underpin this fundamental switch are not understood. HSCs produce genotoxic formaldehyde that requires protection by the detoxification enzymes ALDH2 and ADH5 and the Fanconi anemia (FA) DNA repair pathway. We find that the HSCs in young Aldh2-/-Fancd2-/- mice harbor a transcriptomic signature equivalent to aged wild-type HSCs, along with increased epigenetic age, telomere attrition, and myeloid-biased differentiation quantified by single HSC transplantation. In addition, the p53 response is vigorously activated in Aldh2-/-Fancd2-/- HSCs, while p53 deletion rescued this aged HSC phenotype. To further define the origins of the myeloid differentiation bias, we use a GFP genetic reporter to find a striking enrichment of Vwf+ myeloid and megakaryocyte-lineage-biased HSCs. These results indicate that metabolism-derived formaldehyde-DNA damage stimulates the p53 response in HSCs to drive accelerated aging.


Subject(s)
Aging , Aldehydes , DNA Damage , Hematopoiesis , Tumor Suppressor Protein p53 , Animals , Mice , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Aldehydes/metabolism , Transcriptome , Single-Cell Gene Expression Analysis , Hematopoietic Stem Cells/cytology , Myeloid Cells/cytology , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology
4.
Mol Metab ; 66: 101604, 2022 12.
Article in English | MEDLINE | ID: mdl-36184065

ABSTRACT

OBJECTIVE: Insulin-like peptide 5 (INSL5) signalling, through its cognate receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), has been reported to be orexigenic, and the high fat diet (HFD) preference observed in wildtype mice is altered in Rxfp4 knock-out mice. In this study, we used a new Rxfp4-Cre mouse model to investigate the mechanisms underlying these observations. METHODS: We generated transgenic Rxfp4-Cre mice and investigated central expression of Rxfp4 by RT-qPCR, RNAscope and intraparenchymal infusion of INSL5. Rxfp4-expressing cells were chemogenetically manipulated in global Cre-reporter mice using designer receptors exclusively activated by designer drugs (DREADDs) or after stereotactic injection of a Cre-dependent AAV-DIO-Dq-DREADD targeting a population located in the ventromedial hypothalamus (RXFP4VMH). Food intake and feeding motivation were assessed in the presence and absence of a DREADD agonist. Rxfp4-expressing cells in the hypothalamus were characterised by single-cell RNA-sequencing (scRNAseq) and the connectivity of RXFP4VMH cells was investigated using viral tracing. RESULTS: Rxfp4-Cre mice displayed Cre-reporter expression in the hypothalamus. Active expression of Rxfp4 in the adult mouse brain was confirmed by RT-qPCR and RNAscope. Functional receptor expression was supported by cyclic AMP-responses to INSL5 application in ex vivo brain slices and increased HFD and highly palatable liquid meal (HPM), but not chow, intake after intra-VMH INSL5 infusion. scRNAseq of hypothalamic RXFP4 neurons defined a cluster expressing VMH markers, alongside known appetite-modulating neuropeptide receptors (Mc4r, Cckar and Nmur2). Viral tracing demonstrated RXFP4VMH neural projections to nuclei implicated in hedonic feeding behaviour. Whole body chemogenetic inhibition (Di-DREADD) of Rxfp4-expressing cells, mimicking physiological INSL5-RXFP4 Gi-signalling, increased intake of the HFD and HPM, but not chow, whilst activation (Dq-DREADD), either at whole body level or specifically within the VMH, reduced HFD and HPM intake and motivation to work for the HPM. CONCLUSION: These findings identify RXFP4VMH neurons as regulators of food intake and preference, and hypothalamic RXFP4 signalling as a target for feeding behaviour manipulation.


Subject(s)
Eating , Neurons , Receptors, G-Protein-Coupled , Animals , Mice , Hypothalamus/cytology , Hypothalamus/metabolism , Neurons/metabolism , Receptors, G-Protein-Coupled/metabolism
5.
Blood ; 140(14): 1592-1606, 2022 10 06.
Article in English | MEDLINE | ID: mdl-35767701

ABSTRACT

Adult hematopoietic stem cells (HSCs) are predominantly quiescent and can be activated in response to acute stress such as infection or cytotoxic insults. STAT1 is a pivotal downstream mediator of interferon (IFN) signaling and is required for IFN-induced HSC proliferation, but little is known about the role of STAT1 in regulating homeostatic hematopoietic stem/progenitor cells (HSPCs). Here, we show that loss of STAT1 altered the steady state HSPC landscape, impaired HSC function in transplantation assays, delayed blood cell regeneration following myeloablation, and disrupted molecular programs that protect HSCs, including control of quiescence. Our results also reveal STAT1-dependent functional HSC heterogeneity. A previously unrecognized subset of homeostatic HSCs with elevated major histocompatibility complex class II (MHCII) expression (MHCIIhi) displayed molecular features of reduced cycling and apoptosis and was refractory to 5-fluorouracil-induced myeloablation. Conversely, MHCIIlo HSCs displayed increased megakaryocytic potential and were preferentially expanded in CALR mutant mice with thrombocytosis. Similar to mice, high MHCII expression is a feature of human HSCs residing in a deeper quiescent state. Our results therefore position STAT1 at the interface of stem cell heterogeneity and the interplay between stem cells and the adaptive immune system, areas of broad interest in the wider stem cell field.


Subject(s)
Hematopoietic Stem Cells , Megakaryocytes , STAT1 Transcription Factor , Animals , Cell Proliferation , Fluorouracil/pharmacology , Hematopoietic Stem Cells/metabolism , Humans , Interferons , Megakaryocytes/metabolism , Mice , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism
6.
EMBO J ; 39(24): e104983, 2020 12 15.
Article in English | MEDLINE | ID: mdl-33103827

ABSTRACT

Recent advances in molecular profiling provide descriptive datasets of complex differentiation landscapes including the haematopoietic system, but the molecular mechanisms defining progenitor states and lineage choice remain ill-defined. Here, we employed a cellular model of murine multipotent haematopoietic progenitors (Hoxb8-FL) to knock out 39 transcription factors (TFs) followed by RNA-Seq analysis, to functionally define a regulatory network of 16,992 regulator/target gene links. Focussed analysis of the subnetworks regulated by the B-lymphoid TF Ebf1 and T-lymphoid TF Gata3 revealed a surprising role in common activation of an early myeloid programme. Moreover, Gata3-mediated repression of Pax5 emerges as a mechanism to prevent precocious B-lymphoid differentiation, while Hox-mediated activation of Meis1 suppresses myeloid differentiation. To aid interpretation of large transcriptomics datasets, we also report a new method that visualises likely transitions that a progenitor will undergo following regulatory network perturbations. Taken together, this study reveals how molecular network wiring helps to establish a multipotent progenitor state, with experimental approaches and analysis tools applicable to dissecting a broad range of both normal and perturbed cellular differentiation landscapes.


Subject(s)
Cell Lineage/physiology , Hematopoietic System/metabolism , Transcription Factors/metabolism , Animals , Cell Differentiation , Cell Lineage/genetics , Epigenomics , GATA3 Transcription Factor/genetics , GATA3 Transcription Factor/metabolism , Hematopoiesis , Hematopoietic Stem Cell Transplantation , Mice , Myeloid Ecotropic Viral Integration Site 1 Protein/metabolism , PAX5 Transcription Factor/genetics , PAX5 Transcription Factor/metabolism , Precursor Cells, B-Lymphoid , Transcription Factors/genetics
7.
Nat Commun ; 11(1): 1407, 2020 03 16.
Article in English | MEDLINE | ID: mdl-32179751

ABSTRACT

Leukaemogenic mutations commonly disrupt cellular differentiation and/or enhance proliferation, thus perturbing the regulatory programs that control self-renewal and differentiation of stem and progenitor cells. Translocations involving the Mll1 (Kmt2a) gene generate powerful oncogenic fusion proteins, predominantly affecting infant and paediatric AML and ALL patients. The early stages of leukaemogenic transformation are typically inaccessible from human patients and conventional mouse models. Here, we take advantage of cells conditionally blocked at the multipotent haematopoietic progenitor stage to develop a MLL-r model capturing early cellular and molecular consequences of MLL-ENL expression based on a clear clonal relationship between parental and leukaemic cells. Through a combination of scRNA-seq, ATAC-seq and genome-scale CRISPR-Cas9 screening, we identify pathways and genes likely to drive the early phases of leukaemogenesis. Finally, we demonstrate the broad utility of using matched parental and transformed cells for small molecule inhibitor studies by validating both previously known and other potential therapeutic targets.


Subject(s)
Cell Transformation, Neoplastic , Histone-Lysine N-Methyltransferase/metabolism , Leukemia, Myeloid, Acute/metabolism , Myeloid-Lymphoid Leukemia Protein/metabolism , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Disease Models, Animal , Female , Hematopoietic Stem Cells/metabolism , Histone-Lysine N-Methyltransferase/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/physiopathology , Mice , Mice, Inbred C57BL , Myeloid-Lymphoid Leukemia Protein/genetics , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
8.
EMBO J ; 39(8): e104270, 2020 04 15.
Article in English | MEDLINE | ID: mdl-32149421

ABSTRACT

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three-dimensional organization of the niche. Here, we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modeling to show the two-step process of intra-aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modeling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Calcium-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Aorta/embryology , Calcium-Binding Proteins/genetics , Cell Division , Endothelial Progenitor Cells/physiology , Female , Hemangioblasts/physiology , Hematopoietic Stem Cells/physiology , Mice , Mice, Inbred C57BL , Models, Theoretical
9.
Cancer Discov ; 9(12): 1736-1753, 2019 12.
Article in English | MEDLINE | ID: mdl-31662298

ABSTRACT

Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as ETO2-GLIS2, are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2-GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2-GLIS2-induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. SIGNIFICANCE: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state.See related commentary by Cruz Hernandez and Vyas, p. 1653.This article is highlighted in the In This Issue feature, p. 1631.


Subject(s)
Leukemia, Myeloid, Acute/pathology , Oncogene Proteins, Fusion/genetics , Adolescent , Age Factors , Animals , Child , Child, Preschool , Female , Humans , Infant , Leukemia, Myeloid, Acute/genetics , Mice , Neoplasm Transplantation , Transcription Factors , Tumor Cells, Cultured
10.
Proc Natl Acad Sci U S A ; 114(23): 5822-5829, 2017 06 06.
Article in English | MEDLINE | ID: mdl-28584094

ABSTRACT

Adult blood contains a mixture of mature cell types, each with specialized functions. Single hematopoietic stem cells (HSCs) have been functionally shown to generate all mature cell types for the lifetime of the organism. Differentiation of HSCs toward alternative lineages must be balanced at the population level by the fate decisions made by individual cells. Transcription factors play a key role in regulating these decisions and operate within organized regulatory programs that can be modeled as transcriptional regulatory networks. As dysregulation of single HSC fate decisions is linked to fatal malignancies such as leukemia, it is important to understand how these decisions are controlled on a cell-by-cell basis. Here we developed and applied a network inference method, exploiting the ability to infer dynamic information from single-cell snapshot expression data based on expression profiles of 48 genes in 2,167 blood stem and progenitor cells. This approach allowed us to infer transcriptional regulatory network models that recapitulated differentiation of HSCs into progenitor cell types, focusing on trajectories toward megakaryocyte-erythrocyte progenitors and lymphoid-primed multipotent progenitors. By comparing these two models, we identified and subsequently experimentally validated a difference in the regulation of nuclear factor, erythroid 2 (Nfe2) and core-binding factor, runt domain, alpha subunit 2, translocated to, 3 homolog (Cbfa2t3h) by the transcription factor Gata2. Our approach confirms known aspects of hematopoiesis, provides hypotheses about regulation of HSC differentiation, and is widely applicable to other hierarchical biological systems to uncover regulatory relationships.


Subject(s)
Gene Regulatory Networks , Hematopoiesis/genetics , Hematopoietic Stem Cells/cytology , Algorithms , Animals , Cell Differentiation , Mice, Inbred C57BL , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription Factors/physiology
11.
Elife ; 5: e11469, 2016 Feb 22.
Article in English | MEDLINE | ID: mdl-26901438

ABSTRACT

Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes.


Subject(s)
Gene Regulatory Networks , Hematopoiesis , Hematopoietic Stem Cells/physiology , Transcription Factors/metabolism , Animals , Cell Line , Chromatin Immunoprecipitation , Computer Simulation , Gene Expression Profiling , Mice , Models, Theoretical , Sequence Analysis, DNA
12.
Nucleic Acids Res ; 44(8): e72, 2016 05 05.
Article in English | MEDLINE | ID: mdl-26721389

ABSTRACT

Eukaryotic gene expression is regulated by transcription factors (TFs) binding to promoter as well as distal enhancers. TFs recognize short, but specific binding sites (TFBSs) that are located within the promoter and enhancer regions. Functionally relevant TFBSs are often highly conserved during evolution leaving a strong phylogenetic signal. While multiple sequence alignment (MSA) is a potent tool to detect the phylogenetic signal, the current MSA implementations are optimized to align the maximum number of identical nucleotides. This approach might result in the omission of conserved motifs that contain interchangeable nucleotides such as the ETS motif (IUPAC code: GGAW). Here, we introduce ConBind, a novel method to enhance alignment of short motifs, even if their mutual sequence similarity is only partial. ConBind improves the identification of conserved TFBSs by improving the alignment accuracy of TFBS families within orthologous DNA sequences. Functional validation of the Gfi1b + 13 enhancer reveals that ConBind identifies additional functionally important ETS binding sites that were missed by all other tested alignment tools. In addition to the analysis of known regulatory regions, our web tool is useful for the analysis of TFBSs on so far unknown DNA regions identified through ChIP-sequencing.


Subject(s)
Computational Biology/methods , DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic/genetics , Promoter Regions, Genetic/genetics , Sequence Alignment/methods , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites/genetics , Gene Expression Regulation/genetics , Humans , Sequence Analysis, DNA
13.
Nucleic Acids Res ; 42(22): 13513-24, 2014 Dec 16.
Article in English | MEDLINE | ID: mdl-25428352

ABSTRACT

Combinatorial transcription factor (TF) binding is essential for cell-type-specific gene regulation. However, much remains to be learned about the mechanisms of TF interactions, including to what extent constrained spacing and orientation of interacting TFs are critical for regulatory element activity. To examine the relative prevalence of the 'enhanceosome' versus the 'TF collective' model of combinatorial TF binding, a comprehensive analysis of TF binding site sequences in large scale datasets is necessary. We developed a motif-pair discovery pipeline to identify motif co-occurrences with preferential distance(s) between motifs in TF-bound regions. Utilizing a compendium of 289 mouse haematopoietic TF ChIP-seq datasets, we demonstrate that haematopoietic-related motif-pairs commonly occur with highly conserved constrained spacing and orientation between motifs. Furthermore, motif clustering revealed specific associations for both heterotypic and homotypic motif-pairs with particular haematopoietic cell types. We also showed that disrupting the spacing between motif-pairs significantly affects transcriptional activity in a well-known motif-pair-E-box and GATA, and in two previously unknown motif-pairs with constrained spacing-Ets and Homeobox as well as Ets and E-box. In this study, we provide evidence for widespread sequence-specific TF pair interaction with DNA that conforms to the 'enhanceosome' model, and furthermore identify associations between specific haematopoietic cell-types and motif-pairs.


Subject(s)
Hematopoiesis/genetics , Regulatory Elements, Transcriptional , Transcription Factors/metabolism , Transcription, Genetic , Animals , Binding Sites , Blood Cells/metabolism , Chromatin Immunoprecipitation , DNA/chemistry , DNA/metabolism , Mice , Nucleotide Motifs , Sequence Analysis, DNA
14.
Blood ; 122(14): e12-22, 2013 Oct 03.
Article in English | MEDLINE | ID: mdl-23974199

ABSTRACT

Genome-wide combinatorial binding patterns for key transcription factors (TFs) have not been reported for primary human hematopoietic stem and progenitor cells (HSPCs), and have constrained analysis of the global architecture of molecular circuits controlling these cells. Here we provide high-resolution genome-wide binding maps for a heptad of key TFs (FLI1, ERG, GATA2, RUNX1, SCL, LYL1, and LMO2) in human CD34(+) HSPCs, together with quantitative RNA and microRNA expression profiles. We catalog binding of TFs at coding genes and microRNA promoters, and report that combinatorial binding of all 7 TFs is favored and associated with differential expression of genes and microRNA in HSPCs. We also uncover a previously unrecognized association between FLI1 and RUNX1 pairing in HSPCs, we establish a correlation between the density of histone modifications that mark active enhancers and the number of overlapping TFs at a peak, we demonstrate bivalent histone marks at promoters of heptad target genes in CD34(+) cells that are poised for later expression, and we identify complex relationships between specific microRNAs and coding genes regulated by the heptad. Taken together, these data reveal the power of integrating multifactor sequencing of chromatin immunoprecipitates with coding and noncoding gene expression to identify regulatory circuits controlling cell identity.


Subject(s)
Genome-Wide Association Study , Hematopoiesis/genetics , Hematopoietic Stem Cells/physiology , Transcription Factors/genetics , Cell Differentiation/genetics , Chromatin Immunoprecipitation , Cluster Analysis , Flow Cytometry , Hematopoietic Stem Cells/cytology , Humans , RNA, Untranslated , Transcription, Genetic
15.
Blood ; 122(15): 2694-703, 2013 Oct 10.
Article in English | MEDLINE | ID: mdl-23974202

ABSTRACT

The ETS transcription factor ERG plays a central role in definitive hematopoiesis, and its overexpression in acute myeloid leukemia (AML) is associated with a stem cell signature and poor prognosis. Yet how ERG causes leukemia is unclear. Here we show that pan-hematopoietic ERG expression induces an early progenitor myeloid leukemia in transgenic mice. Integrated genome-scale analysis of gene expression and ERG binding profiles revealed that ERG activates a transcriptional program similar to human AML stem/progenitor cells and to human AML with high ERG expression. This transcriptional program was associated with activation of RAS that was required for leukemia cells growth in vitro and in vivo. We further show that ERG induces expression of the Pim1 kinase oncogene through a novel hematopoietic enhancer validated in transgenic mice and human CD34(+) normal and leukemic cells. Pim1 inhibition disrupts growth and induces apoptosis of ERG-expressing leukemic cells. The importance of the ERG/PIM1 axis is further underscored by the poorer prognosis of AML highly expressing ERG and PIM1. Thus, integrative genomic analysis demonstrates that ERG causes myeloid progenitor leukemia characterized by an induction of leukemia stem cell transcriptional programs. Pim1 and the RAS pathway are potential therapeutic targets of these high-risk leukemias.


Subject(s)
Gene Expression Regulation, Leukemic/physiology , Leukemia, Myeloid, Acute/genetics , Proto-Oncogene Proteins c-pim-1/metabolism , Trans-Activators/genetics , Transcription Factors/metabolism , Animals , Antineoplastic Agents , Enhancer Elements, Genetic/genetics , Genomics , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Myeloid Progenitor Cells/physiology , Neoplasm Transplantation , Transcription, Genetic/physiology , Transcriptional Regulator ERG
16.
PLoS One ; 7(3): e31484, 2012.
Article in English | MEDLINE | ID: mdl-22396734

ABSTRACT

The Scl gene encodes a transcription factor essential for haematopoietic development. Scl transcription is regulated by a panel of cis-elements spread over 55 kb with the most distal 3' element being located downstream of the neighbouring gene Map17, which is co-regulated with Scl in haematopoietic cells. The Scl/Map17 domain is flanked upstream by the ubiquitously expressed Sil gene and downstream by a cluster of Cyp genes active in liver, but the mechanisms responsible for delineating the domain boundaries remain unclear. Here we report identification of a DNaseI hypersensitive site at the 3' end of the Scl/Map17 domain and 45 kb downstream of the Scl transcription start site. This element is located at the boundary of active and inactive chromatin, does not function as a classical tissue-specific enhancer, binds CTCF and is both necessary and sufficient for insulator function in haematopoietic cells in vitro. Moreover, in a transgenic reporter assay, tissue-specific expression of the Scl promoter in brain was increased by incorporation of 350 bp flanking fragments from the +45 element. Our data suggests that the +45 region functions as a boundary element that separates the Scl/Map17 and Cyp transcriptional domains, and raise the possibility that this element may be useful for improving tissue-specific expression of transgenic constructs.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/physiology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/physiology , Repressor Proteins/genetics , Transcription, Genetic , Animals , Binding Sites , CCCTC-Binding Factor , Chromatin Immunoprecipitation , Chromosome Mapping/methods , Deoxyribonuclease I/metabolism , Enhancer Elements, Genetic , Genes, Reporter , Hematopoietic Stem Cells/cytology , Humans , Liver/metabolism , Mice , Multigene Family , Oligonucleotide Array Sequence Analysis , Protein Structure, Tertiary , T-Cell Acute Lymphocytic Leukemia Protein 1 , Transgenes
17.
Blood ; 117(26): 7079-89, 2011 Jun 30.
Article in English | MEDLINE | ID: mdl-21536859

ABSTRACT

The Ets-related gene (ERG) is an Ets-transcription factor required for normal blood stem cell development. ERG expression is down-regulated during early T-lymphopoiesis but maintained in T-acute lymphoblastic leukemia (T-ALL), where it is recognized as an independent risk factor for adverse outcome. However, it is unclear whether ERG is directly involved in the pathogenesis of T-ALL and how its expression is regulated. Here we demonstrate that transgenic expression of ERG causes T-ALL in mice and that its knockdown reduces the proliferation of human MOLT4 T-ALL cells. We further demonstrate that ERG expression in primary human T-ALL cells is mediated by the binding of other T-cell oncogenes SCL/TAL1, LMO2, and LYL1 in concert with ERG, FLI1, and GATA3 to the ERG +85 enhancer. This enhancer is not active in normal T cells but in transgenic mice targets expression to fetal liver c-kit(+) cells, adult bone marrow stem/progenitors and early CD4(-)CD8(-) double-negative thymic progenitors. Taken together, these data illustrate that ERG promotes T-ALL and that failure to extinguish activity of stem cell enhancers associated with regulatory transcription factors such as ERG can contribute to the development of leukemia.


Subject(s)
Gene Expression Regulation, Leukemic , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , T-Lymphocytes/metabolism , Trans-Activators/metabolism , Adaptor Proteins, Signal Transducing , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Line, Tumor , Cell Proliferation , Cells, Cultured , DNA-Binding Proteins/metabolism , Gene Knockdown Techniques , Humans , LIM Domain Proteins , Metalloproteins/metabolism , Mice , Mice, Transgenic , Molecular Sequence Data , Neoplasm Proteins/metabolism , Neoplasm Transplantation , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Promoter Regions, Genetic , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-vav/genetics , Proto-Oncogene Proteins c-vav/metabolism , RNA, Messenger/metabolism , Sequence Alignment , Survival Analysis , T-Cell Acute Lymphocytic Leukemia Protein 1 , T-Lymphocytes/pathology , Trans-Activators/antagonists & inhibitors , Trans-Activators/chemistry , Trans-Activators/genetics , Transcriptional Regulator ERG
18.
Mol Cell Biol ; 30(15): 3853-63, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20516218

ABSTRACT

The growth factor independence 1 (Gfi1) gene was originally discovered in the hematopoietic system, where it functions as a key regulator of stem cell homeostasis, as well as neutrophil and T-cell development. Outside the blood system, Gfi1 is essential for inner-ear hair and intestinal secretory cell differentiation. To understand the regulatory hierarchies within which Gfi1 operates to control these diverse biological functions, we used a combination of comparative genomics, locus-wide chromatin immunoprecipitation assays, functional validation in cell lines, and extensive transgenic mouse assays to identify and characterize the complete ensemble of Gfi1 regulatory elements. This concerted effort identified five distinct regulatory elements spread over 100kb each driving expression in transgenic mice to a subdomain of endogenous Gfi1. Detailed characterization of an enhancer 35 kb upstream of Gfi1 demonstrated activity in the dorsal aorta region and fetal liver in transgenic mice, which was bound by key stem cell transcription factors Scl/Tal1, PU.1/Sfpi1, Runx1, Erg, Meis1, and Gata2. Taken together, our results reveal the regulatory regions responsible for Gfi1 expression and importantly establish that Gfi1 expression at the sites of hematopoietic stem cell (HSC) emergence is controlled by key HSC regulators, thus integrating Gfi1 into the wider HSC regulatory networks.


Subject(s)
Hematopoiesis/genetics , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors , Cell Differentiation/genetics , Cell Line , Chromatin Immunoprecipitation , Hematopoietic System/metabolism , Homeodomain Proteins , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Transgenic , Myeloid Ecotropic Viral Integration Site 1 Protein , Neoplasm Proteins , Proto-Oncogene Proteins , Regulatory Sequences, Nucleic Acid , T-Cell Acute Lymphocytic Leukemia Protein 1
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