RESUMEN
As embryonic stem cells (ESCs) transition from naive to primed pluripotency during early mammalian development, they acquire high DNA methylation levels. During this transition, the germline is specified and undergoes genome-wide DNA demethylation, while emergence of the three somatic germ layers is preceded by acquisition of somatic DNA methylation levels in the primed epiblast. DNA methylation is essential for embryogenesis, but the point at which it becomes critical during differentiation and whether all lineages equally depend on it is unclear. Here, using culture modeling of cellular transitions, we found that DNA methylation-free mouse ESCs with triple DNA methyltransferase knockout (TKO) progressed through the continuum of pluripotency states but demonstrated skewed differentiation abilities toward neural versus other somatic lineages. More saliently, TKO ESCs were fully competent for establishing primordial germ cell-like cells, even showing temporally extended and self-sustained capacity for the germline fate. By mapping chromatin states, we found that neural and germline lineages are linked by a similar enhancer dynamic upon exit from the naive state, defined by common sets of transcription factors, including methyl-sensitive ones, that fail to be decommissioned in the absence of DNA methylation. We propose that DNA methylation controls the temporality of a coordinated neural-germline axis of the preferred differentiation route during early development.
Asunto(s)
Metilación de ADN , Células Madre Embrionarias , Animales , Ratones , Diferenciación Celular/genética , Células Madre Embrionarias/metabolismo , Factores de Transcripción/metabolismo , Células Madre Embrionarias de Ratones , Células Germinativas/metabolismo , Estratos Germinativos/metabolismo , Mamíferos/metabolismoRESUMEN
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.
Asunto(s)
Médula Ósea , Células Madre Hematopoyéticas , Animales , Ratones , Células Madre Hematopoyéticas/metabolismo , Hematopoyesis/genética , Diferenciación CelularRESUMEN
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.
Asunto(s)
Desarrollo Embrionario , Saco Vitelino , Femenino , Humanos , Embarazo , Coagulación Sanguínea/genética , Macrófagos , Saco Vitelino/citología , Saco Vitelino/metabolismo , Desarrollo Embrionario/genética , Atlas como Asunto , Expresión Génica , Perfilación de la Expresión Génica , Hematopoyesis/genética , Hígado/embriologíaRESUMEN
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.
Asunto(s)
Envejecimiento , Aldehídos , Daño del ADN , Hematopoyesis , Proteína p53 Supresora de Tumor , Animales , Ratones , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Aldehídos/metabolismo , Transcriptoma , Análisis de Expresión Génica de una Sola Célula , Células Madre Hematopoyéticas/citología , Células Mieloides/citología , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologíaRESUMEN
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.
Asunto(s)
Ingestión de Alimentos , Neuronas , Receptores Acoplados a Proteínas G , Animales , Ratones , Hipotálamo/citología , Hipotálamo/metabolismo , Neuronas/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
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.
Asunto(s)
Células Madre Hematopoyéticas , Megacariocitos , Factor de Transcripción STAT1 , Animales , Proliferación Celular , Fluorouracilo/farmacología , Células Madre Hematopoyéticas/metabolismo , Humanos , Interferones , Megacariocitos/metabolismo , Ratones , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismoRESUMEN
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.
Asunto(s)
Linaje de la Célula/fisiología , Sistema Hematopoyético/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula/genética , Epigenómica , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Ratones , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/metabolismo , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/metabolismo , Células Precursoras de Linfocitos B , Factores de Transcripción/genéticaRESUMEN
During mouse embryonic development, pluripotent cells rapidly divide and diversify, yet the regulatory programs that define the cell repertoire for each organ remain ill-defined. To delineate comprehensive chromatin landscapes during early organogenesis, we mapped chromatin accessibility in 19,453 single nuclei from mouse embryos at 8.25 days post-fertilization. Identification of cell-type-specific regions of open chromatin pinpointed two TAL1-bound endothelial enhancers, which we validated using transgenic mouse assays. Integrated gene expression and transcription factor motif enrichment analyses highlighted cell-type-specific transcriptional regulators. Subsequent in vivo experiments in zebrafish revealed a role for the ETS factor FEV in endothelial identity downstream of ETV2 (Etsrp in zebrafish). Concerted in vivo validation experiments in mouse and zebrafish thus illustrate how single-cell open chromatin maps, representative of a mammalian embryo, provide access to the regulatory blueprint for mammalian organogenesis.
Asunto(s)
Cromatina/química , Células Endoteliales/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Organogénesis/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Animales , Linaje de la Célula/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Embrión de Mamíferos , Embrión no Mamífero , Desarrollo Embrionario , Células Endoteliales/citología , Perfilación de la Expresión Génica , Ratones , Ratones Transgénicos , Especificidad de Órganos , Unión Proteica , Análisis de la Célula Individual , Proteína 1 de la Leucemia Linfocítica T Aguda/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
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.
Asunto(s)
Transformación Celular Neoplásica , N-Metiltransferasa de Histona-Lisina/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Células Madre Hematopoyéticas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/fisiopatología , Ratones , Ratones Endogámicos C57BL , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Aorta/embriología , Proteínas de Unión al Calcio/genética , División Celular , Células Progenitoras Endoteliales/fisiología , Femenino , Hemangioblastos/fisiología , Células Madre Hematopoyéticas/fisiología , Ratones , Ratones Endogámicos C57BL , Modelos TeóricosRESUMEN
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.
Asunto(s)
Leucemia Mieloide Aguda/patología , Proteínas de Fusión Oncogénica/genética , Adolescente , Factores de Edad , Animales , Niño , Preescolar , Femenino , Humanos , Lactante , Leucemia Mieloide Aguda/genética , Ratones , Trasplante de Neoplasias , Factores de Transcripción , Células Tumorales CultivadasRESUMEN
Mouse heart development arises from Mesp1-expressing cardiovascular progenitors (CPs) that are specified during gastrulation. The molecular processes that control early regional and lineage segregation of CPs have been unclear. We performed single-cell RNA sequencing of wild-type and Mesp1-null CPs in mice. We showed that populations of Mesp1 CPs are molecularly distinct and span the continuum between epiblast and later mesodermal cells, including hematopoietic progenitors. Single-cell transcriptome analysis of Mesp1-deficient CPs showed that Mesp1 is required for the exit from the pluripotent state and the induction of the cardiovascular gene expression program. We identified distinct populations of Mesp1 CPs that correspond to progenitors committed to different cell lineages and regions of the heart, identifying the molecular features associated with early lineage restriction and regional segregation of the heart at the early stage of mouse gastrulation.
Asunto(s)
Corazón/embriología , Células Madre/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , División Celular , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Mesodermo/citología , Ratones , Ratones Mutantes , ARN/genética , Análisis de Secuencia de ARN , Células Madre/metabolismoRESUMEN
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.
Asunto(s)
Redes Reguladoras de Genes , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Algoritmos , Animales , Diferenciación Celular , Ratones Endogámicos C57BL , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
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.
Asunto(s)
Redes Reguladoras de Genes , Hematopoyesis , Células Madre Hematopoyéticas/fisiología , Factores de Transcripción/metabolismo , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Simulación por Computador , Perfilación de la Expresión Génica , Ratones , Modelos Teóricos , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Biología Computacional/métodos , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos/genética , Regiones Promotoras Genéticas/genética , Alineación de Secuencia/métodos , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Regulación de la Expresión Génica/genética , Humanos , Análisis de Secuencia de ADNRESUMEN
Comprehensive study of transcriptional control processes will be required to enhance our understanding of both normal and malignant hematopoiesis. Modern sequencing technologies have revolutionized our ability to generate genome-scale expression and histone modification profiles, transcription factor (TF)-binding maps, and also comprehensive chromatin-looping information. Many of these technologies, however, require large numbers of cells, and therefore cannot be applied to rare hematopoietic stem/progenitor cell (HSPC) populations. The stem cell factor-dependent multipotent progenitor cell line HPC-7 represents a well-recognized cell line model for HSPCs. Here we report genome-wide maps for 17 TFs, 3 histone modifications, DNase I hypersensitive sites, and high-resolution promoter-enhancer interactomes in HPC-7 cells. Integrated analysis of these complementary data sets revealed TF occupancy patterns of genomic regions involved in promoter-anchored loops. Moreover, preferential associations between pairs of TFs bound at either ends of chromatin loops led to the identification of 4 previously unrecognized protein-protein interactions between key blood stem cell regulators. All HPC-7 data sets are freely available both through standard repositories and a user-friendly Web interface. Together with previously generated genome-wide data sets, this study integrates HPC-7 data into a genomic resource on par with ENCODE tier 1 cell lines and, importantly, is the only current model with comprehensive genome-scale data that is relevant to HSPC biology.
Asunto(s)
Regulación de la Expresión Génica , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismo , Animales , Sitios de Unión/genética , Células Cultivadas , Inmunoprecipitación de Cromatina , Embrión de Mamíferos , Genoma , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Unión Proteica/genética , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Hematopoyesis/genética , Elementos Reguladores de la Transcripción , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Sitios de Unión , Células Sanguíneas/metabolismo , Inmunoprecipitación de Cromatina , ADN/química , ADN/metabolismo , Ratones , Motivos de Nucleótidos , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Estudio de Asociación del Genoma Completo , Hematopoyesis/genética , Células Madre Hematopoyéticas/fisiología , Factores de Transcripción/genética , Diferenciación Celular/genética , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Humanos , ARN no Traducido , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Regulación Leucémica de la Expresión Génica/fisiología , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Transactivadores/genética , Factores de Transcripción/metabolismo , Animales , Antineoplásicos , Elementos de Facilitación Genéticos/genética , Genómica , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Células Progenitoras Mieloides/fisiología , Trasplante de Neoplasias , Transcripción Genética/fisiología , Regulador Transcripcional ERGRESUMEN
MOTIVATION: Combinatorial interactions of transcription factors with cis-regulatory elements control the dynamic progression through successive cellular states and thus underpin all metazoan development. The construction of network models of cis-regulatory elements, therefore, has the potential to generate fundamental insights into cellular fate and differentiation. Haematopoiesis has long served as a model system to study mammalian differentiation, yet modelling based on experimentally informed cis-regulatory interactions has so far been restricted to pairs of interacting factors. Here, we have generated a Boolean network model based on detailed cis-regulatory functional data connecting 11 haematopoietic stem/progenitor cell (HSPC) regulator genes. RESULTS: Despite its apparent simplicity, the model exhibits surprisingly complex behaviour that we charted using strongly connected components and shortest-path analysis in its Boolean state space. This analysis of our model predicts that HSPCs display heterogeneous expression patterns and possess many intermediate states that can act as 'stepping stones' for the HSPC to achieve a final differentiated state. Importantly, an external perturbation or 'trigger' is required to exit the stem cell state, with distinct triggers characterizing maturation into the various different lineages. By focusing on intermediate states occurring during erythrocyte differentiation, from our model we predicted a novel negative regulation of Fli1 by Gata1, which we confirmed experimentally thus validating our model. In conclusion, we demonstrate that an advanced mammalian regulatory network model based on experimentally validated cis-regulatory interactions has allowed us to make novel, experimentally testable hypotheses about transcriptional mechanisms that control differentiation of mammalian stem cells. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.