RESUMO
In vitro generation and expansion of hematopoietic stem cells (HSCs) holds great promise for the treatment of any ailment that relies on bone marrow or blood transplantation. To achieve this, it is essential to resolve the molecular and cellular pathways that govern HSC formation in the embryo. HSCs first emerge in the aorta-gonad-mesonephros (AGM) region, where a rare subset of endothelial cells, hemogenic endothelium (HE), undergoes an endothelial-to-hematopoietic transition (EHT). Here, we present full-length single-cell RNA sequencing (scRNA-seq) of the EHT process with a focus on HE and dorsal aorta niche cells. By using Runx1b and Gfi1/1b transgenic reporter mouse models to isolate HE, we uncovered that the pre-HE to HE continuum is specifically marked by angiotensin-I converting enzyme (ACE) expression. We established that HE cells begin to enter the cell cycle near the time of EHT initiation when their morphology still resembles endothelial cells. We further demonstrated that RUNX1 AGM niche cells consist of vascular smooth muscle cells and PDGFRa+ mesenchymal cells and can functionally support hematopoiesis. Overall, our study provides new insights into HE differentiation toward HSC and the role of AGM RUNX1+ niche cells in this process. Our expansive scRNA-seq datasets represents a powerful resource to investigate these processes further.
Assuntos
Embrião de Mamíferos/embriologia , Hemangioblastos/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Animais , Diferenciação Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Hemangioblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Mesonefro/citologia , Mesonefro/embriologia , Mesonefro/metabolismo , Camundongos , Análise de Célula Única , Transcriptoma , Peixe-ZebraRESUMO
The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2LRR1 is required for ubiquitylation of the CMG-MCM7 subunit during S-phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis-regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.
Assuntos
DNA Helicases , Replicação do DNA , Animais , Ciclo Celular/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Camundongos , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Ubiquitinação , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
RUNX transcription factors orchestrate many different aspects of biology, including basic cellular and developmental processes, stem cell biology and tumorigenesis. In this Primer, we introduce the molecular hallmarks of the three mammalian RUNX genes, RUNX1, RUNX2 and RUNX3, and discuss the regulation of their activities and their mechanisms of action. We then review their crucial roles in the specification and maintenance of a wide array of tissues during embryonic development and adult homeostasis.
Assuntos
Subunidades alfa de Fatores de Ligação ao Core/genética , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Mamíferos/embriologia , Mamíferos/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Hematopoese/fisiologia , Homeostase/fisiologia , Humanos , Camundongos , Neoplasias/metabolismoRESUMO
During ontogeny, hematopoietic stem and progenitor cells arise from hemogenic endothelium through an endothelial-to-hematopoietic transition that is strictly dependent on the transcription factor RUNX1. Although it is well established that RUNX1 is essential for the onset of hematopoiesis, little is known about the role of RUNX1 dosage specifically in hemogenic endothelium and during the endothelial-to-hematopoietic transition. Here, we used the mouse embryonic stem cell differentiation system to determine if and how RUNX1 dosage affects hemogenic endothelium differentiation. The use of inducible Runx1 expression combined with alterations in the expression of the RUNX1 co-factor CBFß allowed us to evaluate a wide range of RUNX1 levels. We demonstrate that low RUNX1 levels are sufficient and necessary to initiate an effective endothelial-to-hematopoietic transition. Subsequently, RUNX1 is also required to complete the endothelial-to-hematopoietic transition and to generate functional hematopoietic precursors. In contrast, elevated levels of RUNX1 are able to drive an accelerated endothelial-to-hematopoietic transition, but the resulting cells are unable to generate mature hematopoietic cells. Together, our results suggest that RUNX1 dosage plays a pivotal role in hemogenic endothelium maturation and the establishment of the hematopoietic system.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Endotélio Vascular/fisiologia , Dosagem de Genes/fisiologia , Hemangioblastos/fisiologia , Hematopoese/genética , Células-Tronco Hematopoéticas/fisiologia , Animais , Diferenciação Celular/genética , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Regulação da Expressão Gênica , Camundongos , Camundongos KnockoutRESUMO
Gene expression governs cell fate, and is regulated via a complex interplay of transcription factors and molecules that change chromatin structure. Advances in sequencing-based assays have enabled investigation of these processes genome-wide, leading to large datasets that combine information on the dynamics of gene expression, transcription factor binding and chromatin structure as cells differentiate. While numerous studies focus on the effects of these features on broader gene regulation, less work has been done on the mechanisms of gene-specific transcriptional control. In this study, we have focussed on the latter by integrating gene expression data for the in vitro differentiation of murine ES cells to macrophages and cardiomyocytes, with dynamic data on chromatin structure, epigenetics and transcription factor binding. Combining a novel strategy to identify communities of related control elements with a penalized regression approach, we developed individual models to identify the potential control elements predictive of the expression of each gene. Our models were compared to an existing method and evaluated using the existing literature and new experimental data from embryonic stem cell differentiation reporter assays. Our method is able to identify transcriptional control elements in a gene specific manner that reflect known regulatory relationships and to generate useful hypotheses for further testing.
Assuntos
Diferenciação Celular/genética , Ensaios de Triagem em Larga Escala/métodos , Elementos Reguladores de Transcrição/genética , Animais , Diferenciação Celular/fisiologia , Cromatina/metabolismo , Bases de Dados Genéticas , Epigênese Genética , Epigenômica , Regulação da Expressão Gênica/genética , Genoma , Macrófagos/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/metabolismo , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismoRESUMO
Endothelial to hematopoietic transition (EHT) is a dynamic process involving the shutting down of endothelial gene expression and switching on of hematopoietic gene transcription. Although the factors regulating EHT in hemogenic endothelium (HE) of the dorsal aorta have been relatively well studied, the molecular regulation of yolk sac HE remains poorly understood. Here, we show that SOX7 inhibits the expression of RUNX1 target genes in HE, while having no effect on RUNX1 expression itself. We establish that SOX7 directly interacts with RUNX1 and inhibits its transcriptional activity. Through this interaction we demonstrate that SOX7 hinders RUNX1 DNA binding as well as the interaction between RUNX1 and its co-factor CBFß. Finally, we show by single-cell expression profiling and immunofluorescence that SOX7 is broadly expressed across the RUNX1+ yolk sac HE population compared with SOX17. Collectively, these data demonstrate for the first time how direct protein-protein interactions between endothelial and hematopoietic transcription factors regulate contrasting transcriptional programs during HE differentiation and EHT.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Endotélio/citologia , Hemangioblastos/citologia , Fatores de Transcrição SOXF/metabolismo , Saco Vitelino/citologia , Animais , Diferenciação Celular , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Feminino , Proteínas HMGB/metabolismo , Células-Tronco Hematopoéticas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição SOXF/genética , Transcrição Gênica/fisiologiaRESUMO
The transmission of extracellular signals into the nucleus involves inducible transcription factors, but how different signalling pathways act in a cell type-specific fashion is poorly understood. Here, we studied the regulatory role of the AP-1 transcription factor family in blood development using embryonic stem cell differentiation coupled with genome-wide transcription factor binding and gene expression analyses. AP-1 factors respond to MAP kinase signalling and comprise dimers of FOS, ATF and JUN proteins. To examine genes regulated by AP-1 and to examine how it interacts with other inducible transcription factors, we abrogated its global DNA-binding activity using a dominant-negative FOS peptide. We show that FOS and JUN bind to and activate a specific set of vascular genes and that AP-1 inhibition shifts the balance between smooth muscle and hematopoietic differentiation towards blood. Furthermore, AP-1 is required for de novo binding of TEAD4, a transcription factor connected to Hippo signalling. Our bottom-up approach demonstrates that AP-1- and TEAD4-associated cis-regulatory elements form hubs for multiple signalling-responsive transcription factors and define the cistrome that regulates vascular and hematopoietic development by extrinsic signals.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Proteínas Musculares/metabolismo , Músculo Liso Vascular/citologia , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Fatores Ativadores da Transcrição/metabolismo , Animais , Sítios de Ligação/genética , Linhagem Celular , Proteínas de Ligação a DNA/genética , Expressão Gênica/genética , Perfilação da Expressão Gênica , Camundongos , Músculo Liso Vascular/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição de Domínio TEA , Fator de Transcrição AP-1/antagonistas & inibidoresRESUMO
Cell fate decisions during haematopoiesis are governed by lineage-specific transcription factors, such as RUNX1, SCL/TAL1, FLI1 and C/EBP family members. To gain insight into how these transcription factors regulate the activation of haematopoietic genes during embryonic development, we measured the genome-wide dynamics of transcription factor assembly on their target genes during the RUNX1-dependent transition from haemogenic endothelium (HE) to haematopoietic progenitors. Using a Runx1-/- embryonic stem cell differentiation model expressing an inducible Runx1 gene, we show that in the absence of RUNX1, haematopoietic genes bind SCL/TAL1, FLI1 and C/EBPß and that this early priming is required for correct temporal expression of the myeloid master regulator PU.1 and its downstream targets. After induction, RUNX1 binds to numerous de novo sites, initiating a local increase in histone acetylation and rapid global alterations in the binding patterns of SCL/TAL1 and FLI1. The acquisition of haematopoietic fate controlled by Runx1 therefore does not represent the establishment of a new regulatory layer on top of a pre-existing HE program but instead entails global reorganization of lineage-specific transcription factor assemblies.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Epigênese Genética/fisiologia , Hematopoese/fisiologia , Acetilação , Animais , Sequência de Bases , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Células-Tronco Embrionárias/fisiologia , Epigênese Genética/genética , Hematopoese/genética , Histonas/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Fatores de Transcrição/fisiologiaRESUMO
During ontogeny, the transcription factor RUNX1 governs the emergence of definitive hematopoietic cells from specialized endothelial cells called hemogenic endothelium (HE). The ultimate consequence of this endothelial-to-hematopoietic transition is the concomitant activation of the hematopoietic program and downregulation of the endothelial program. However, due to the rare and transient nature of the HE, little is known about the initial role of RUNX1 within this population. We, therefore, developed and implemented a highly sensitive DNA adenine methyltransferase identification-based methodology, including a novel data analysis pipeline, to map early RUNX1 transcriptional targets in HE cells. This novel transcription factor binding site identification protocol should be widely applicable to other low abundance cell types and factors. Integration of the RUNX1 binding profile with gene expression data revealed an unexpected early role for RUNX1 as a positive regulator of cell adhesion- and migration-associated genes within the HE. This suggests that RUNX1 orchestrates HE cell positioning and integration prior to the release of hematopoietic cells. Overall, our genome-wide analysis of the RUNX1 binding and transcriptional profile in the HE provides a novel comprehensive resource of target genes that will facilitate the precise dissection of the role of RUNX1 in early blood development.
Assuntos
Movimento Celular/fisiologia , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Hematopoese/fisiologia , Animais , Adesão Celular/fisiologia , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Células Endoteliais/citologia , Endotélio Vascular/citologia , Camundongos , Camundongos KnockoutRESUMO
Although inhibition of p16(INK4a) expression is critical to preserve the proliferative capacity of stem cells, the molecular mechanisms responsible for silencing p16(INK4a) expression remain poorly characterized. Here, we show that the histone acetyltransferase (HAT) monocytic leukemia zinc finger protein (MOZ) controls the proliferation of both hematopoietic and neural stem cells by modulating the transcriptional repression of p16(INK4a) . In the absence of the HAT activity of MOZ, expression of p16(INK4a) is upregulated in progenitor and stem cells, inducing an early entrance into replicative senescence. Genetic deletion of p16(INK4a) reverses the proliferative defect in both Moz(HAT) (-) (/) (-) hematopoietic and neural progenitors. Our results suggest a critical requirement for MOZ HAT activity to silence p16(INK4a) expression and to protect stem cells from early entrance into replicative senescence.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células-Tronco Hematopoéticas/citologia , Histona Acetiltransferases/metabolismo , Células-Tronco Neurais/citologia , Animais , Antígenos CD34/metabolismo , Ciclo Celular , Proliferação de Células , Separação Celular , Senescência Celular , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Inativação Gênica , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Neurais/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Telencéfalo/citologia , Regulação para Cima/genéticaRESUMO
BACKGROUND: Internationally accepted standards for trustworthy guidelines include the necessity to ground recommendations in values and preferences. Considering values and preferences respects the rights of citizens to participate in health decision-making and ensures that guidelines align with the needs and priorities of the communities they are intended to serve. Early anecdotal reports suggest that COVID-19 public health guidelines did not consider values and preferences. OBJECTIVE: To capture and characterize whether and how COVID-19 public health guidelines considered values and preferences. METHODS: We performed a systematic review of COVID-19 public health guidelines. We searched the eCOVID-19 RecMap platform-a comprehensive international catalog of COVID-19 guidelines-up to July 2023 and the Guidelines International Network (GIN) Library-an international library of guidelines published or endorsed by GIN member organizations-up to May 2024. We included guidelines that made recommendations addressing vaccination, masking, isolation, lockdowns, travel restrictions, contact tracing, infection surveillance, and school closures. Reviewers worked independently and in duplicate to review guidelines for consideration of values and preferences. RESULTS: Our search yielded 130 eligible guidelines, of which 41 (31.5%) were published by national organizations, 70 (53.8%) by international organizations, and 19 (14.6%) by professional societies and associations. Twenty-eight (21.5%) guidelines considered values and preferences. Among guidelines that considered values and preferences, most did so to assess the acceptability of recommendations (23; 82.1%) and by referencing published research (25; 89.3%). Guidelines only occasionally engaged laypersons as part of the guideline development group (8; 28.6%). None of the guidelines performed systematic reviews of the literature addressing values and preferences. CONCLUSION: Most COVID-19 public health guidelines did not consider values and preferences. When they were considered, it was often suboptimal. Disregard for values and preferences might have partly contributed to divisive and unpopular COVID-19 policies. Given the possibility of future health emergencies, we recommend guideline developers identify efficient and effective methods for considering values and preferences in crisis situations.
RESUMO
The MOnocytic leukaemia Zing finger (MOZ; MYST3 or KAT6A(1)) gene is frequently found translocated in acute myeloid leukaemia. MOZ encodes a large multidomain protein that contains, besides others, a histone acetyl transferase catalytic domain. Several studies have now established the critical function of MOZ in haematopoiesis. In this review we summarize the recent findings that underscore the relevance of the different biological activities of MOZ in the regulation of haematopoiesis.
Assuntos
Epigênese Genética , Hematopoese/genética , Histona Acetiltransferases/genética , Transativadores , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Hematopoese/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Histona Acetiltransferases/imunologia , Histona Acetiltransferases/metabolismo , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Modelos Genéticos , Modelos ImunológicosRESUMO
Although the critical requirement for the transcription factor RUNX1/AML1 at the onset of hematopoietic development is well established, little is known about its transcriptional targets at this pivotal stage of blood development. Using microarrays, we identified the uncharacterized gene AI467606 as a gene whose expression level is dramatically reduced in the absence of RUNX1. We further demonstrated by chromatin immunoprecipitation and promoter assay a direct regulation of its transcription by RUNX1. Using a bacterial artificial chromosome transgenic approach, we established that AI467606 is expressed during the development of the hematopoietic system in vivo and in vitro and that its expression is detected within the CD41(+) population and marks definitive hematopoietic potential. Similarly, in the adult mouse, all hematopoietic cell lineages, except mature erythrocytes, express AI467606. Taken together, these findings indicate that AI467606 is a novel transcriptional target of RUNX1/AML1 at the onset of hematopoietic development that is extensively expressed within the hematopoietic system.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Hematopoese/fisiologia , Proteínas/genética , Proteínas/metabolismo , Transcrição Gênica/fisiologia , Fatores Etários , Animais , Medula Óssea/embriologia , Medula Óssea/fisiologia , Linhagem da Célula/fisiologia , Células Cultivadas , Cromossomos Artificiais Bacterianos/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Membrana , Camundongos , Dados de Sequência Molecular , Glicoproteína IIb da Membrana de Plaquetas/metabolismoRESUMO
Epigenetic dysregulation is a key feature of most acute myeloid leukemia (AML). Recently, it has become clear that long noncoding RNAs (lncRNAs) can play a key role in epigenetic regulation, and consequently also dysregulation. Currently, our understanding of the requirements and roles of lncRNAs in AML is still limited. Here, using CRISPRi screening, we identified the lncRNA SGOL1-AS1 as an essential regulator of survival in THP-1 AML cells. We demonstrated that SGOL1-AS1 interacts with chromatin-modifying proteins involved in gene repression and that SGOL1-AS1 knockdown is associated with increased heterochromatin formation. We also observed that loss of SGOLl-AS1 results in increased apoptosis and the downregulation of pro-inflammatory genes. In AML patients, high expression of SGOL1-AS1 correlates with both pro-inflammatory gene expression and poor survival. Altogether, our data reveal that SGOL1-AS1 is an essential regulator of cell survival in AML cell lines and a possible regulator of pro-inflammatory signaling in AML patients.
RESUMO
The molecular mechanisms that regulate the balance between proliferation and differentiation of precursors at the onset of hematopoiesis specification are poorly understood. By using a global gene expression profiling approach during the course of embryonic stem cell differentiation, we identified Sox7 as a potential candidate gene involved in the regulation of blood lineage formation from the mesoderm germ layer. In the present study, we show that Sox7 is transiently expressed in mesodermal precursors as they undergo specification to the hematopoietic program. Sox7 knockdown in vitro significantly decreases the formation of both primitive erythroid and definitive hematopoietic progenitors as well as endothelial progenitors. In contrast, Sox7-sustained expression in the earliest committed hematopoietic precursors promotes the maintenance of their multipotent and self-renewing status. Removal of this differentiation block driven by Sox7-enforced expression leads to the efficient differentiation of hematopoietic progenitors to all erythroid and myeloid lineages. This study identifies Sox7 as a novel and important player in the molecular regulation of the first committed blood precursors. Furthermore, our data demonstrate that the mere sustained expression of Sox7 is sufficient to completely alter the balance between proliferation and differentiation at the onset of hematopoiesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Fatores de Transcrição SOXF/fisiologia , Animais , Divisão Celular , Linhagem da Célula , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Células Eritroides/citologia , Gástrula/citologia , Gástrula/metabolismo , Técnicas de Silenciamento de Genes , Vetores Genéticos/farmacologia , Hemangioblastos/citologia , Hemangioblastos/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Mesoderma/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/citologia , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/fisiologia , Fatores de Transcrição SOXF/biossíntese , Fatores de Transcrição SOXF/deficiência , Fatores de Transcrição SOXF/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
During ontogeny, the establishment of the hematopoietic system takes place in several phases, separated both in time and location. The process is initiated extra-embryonically in the yolk sac (YS) and concludes in the main arteries of the embryo with the formation of hematopoietic stem cells (HSC). Initially, it was thought that HSC-independent hematopoietic YS cells were transient, and only required to bridge the gap to HSC activity. However, in recent years it has become clear that these cells also contribute to embryonic organogenesis, including the emergence of HSCs. Furthermore, some of these early HSC-independent YS cells persist into adulthood as distinct hematopoietic populations. These previously unrecognized abilities of embryonic HSC-independent hematopoietic cells constitute a new field of interest. Here, we aim to provide a succinct overview of the current knowledge regarding the contribution of YS-derived hematopoietic cells to the development of the embryo and the adult hematopoietic system.
RESUMO
The transcription factor RUNX1 is a master regulator of blood cell specification. During embryogenesis, hematopoietic progenitors are initially generated from hemogenic endothelium through an endothelium-to-hematopoietic transition controlled by RUNX1. Several studies have dissected the expression pattern and role of RUNX1 isoforms at the onset of mouse hematopoiesis, however the precise pattern of RUNX1 isoform expression and biological output of RUNX1-expressing cells at the onset of human hematopoiesis is still not fully understood. Here, we investigated these questions using a RUNX1b:VENUS RUNX1c:TOMATO human embryonic stem cell line which allows multi-parameter single cell resolution via flow cytometry and isolation of RUNX1b-expressing cells for further analysis. Our data reveal the sequential expression of the two RUNX1 isoforms with RUNX1b expressed first in a subset of endothelial cells and during the endothelial to hematopoietic transition while RUNX1c only becomes expressed in fully specified blood cells. Furthermore, our data show that RUNX1b marks endothelial cells endowed with hemogenic potential and that RUNX1b expression level determines hemogenic competency in a dose-dependent manner. Together our data reveal the dynamic of RUNX1 isoforms expression at the onset of human blood specification and establish RUNX1b isoform as the earliest known marker for hemogenic competency.
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Despite absent expression in normal hematopoiesis, the Forkhead factor FOXC1, a critical mesenchymal differentiation regulator, is highly expressed in â¼30% of HOXAhigh acute myeloid leukemia (AML) cases to confer blocked monocyte/macrophage differentiation. Through integrated proteomics and bioinformatics, we find that FOXC1 and RUNX1 interact through Forkhead and Runt domains, respectively, and co-occupy primed and active enhancers distributed close to differentiation genes. FOXC1 stabilizes association of RUNX1, HDAC1, and Groucho repressor TLE3 to limit enhancer activity: FOXC1 knockdown induces loss of repressor proteins, gain of CEBPA binding, enhancer acetylation, and upregulation of nearby genes, including KLF2. Furthermore, it triggers genome-wide redistribution of RUNX1, TLE3, and HDAC1 from enhancers to promoters, leading to repression of self-renewal genes, including MYC and MYB. Our studies highlight RUNX1 and CEBPA transcription factor swapping as a feature of leukemia cell differentiation and reveal that FOXC1 prevents this by stabilizing enhancer binding of a RUNX1/HDAC1/TLE3 transcription repressor complex to oncogenic effect.
Assuntos
Diferenciação Celular , Proteínas Correpressoras/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Proteínas Correpressoras/genética , Subunidade alfa 2 de Fator de Ligação ao Core/química , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Monócitos/citologia , Monócitos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Regulação para CimaRESUMO
BACKGROUND: B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is the most common pediatric cancer. Identifying key players involved in proliferation of BCP-ALL cells is crucial to propose new therapeutic targets. Runt Related Transcription Factor 1 (RUNX1) and Core-Binding Factor Runt Domain Alpha Subunit 2 Translocated To 3 (CBFA2T3, ETO2, MTG16) are master regulators of hematopoiesis and are implicated in leukemia. METHODS: We worked with BCP-ALL mononuclear bone marrow patients' cells and BCP-ALL cell lines, and performed Chromatin Immunoprecipitations followed by Sequencing (ChIP-Seq), co-immunoprecipitations (co-IP), proximity ligation assays (PLA), luciferase reporter assays and mouse xenograft models. RESULTS: We demonstrated that CBFA2T3 transcript levels correlate with RUNX1 expression in the pediatric t(12;21) ETV6-RUNX1 BCP-ALL. By ChIP-Seq in BCP-ALL patients' cells and cell lines, we found that RUNX1 is recruited on its promoter and on an enhancer of CBFA2T3 located - 2 kb upstream CBFA2T3 promoter and that, subsequently, the transcription factor RUNX1 drives both RUNX1 and CBFA2T3 expression. We demonstrated that, mechanistically, RUNX1 and CBFA2T3 can be part of the same complex allowing CBFA2T3 to strongly potentiate the activity of the transcription factor RUNX1. Finally, we characterized a CBFA2T3-mimicking peptide that inhibits the interaction between RUNX1 and CBFA2T3, abrogating the activity of this transcription complex and reducing BCP-ALL lymphoblast proliferation. CONCLUSIONS: Altogether, our findings reveal a novel and important activation loop between the transcription regulator CBFA2T3 and the transcription factor RUNX1 that promotes BCP-ALL proliferation, supporting the development of an innovative therapeutic approach based on the NHR2 subdomain of CBFA2T3 protein.
Assuntos
Antineoplásicos/farmacologia , Subunidade alfa 2 de Fator de Ligação ao Core/antagonistas & inibidores , Peptídeos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Antineoplásicos/química , Linhagem Celular Tumoral , Criança , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos/química , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ativação Transcricional/efeitos dos fármacosRESUMO
BACKGROUND: HIV-1 fails to successfully infect mouse T cells as a result of several blocks in the viral replication cycle. We investigated whether this also impacted on the use of HIV-1 derived lentiviral vectors for stable gene transfer into mouse T cells. METHODS: Freshly isolated primary mouse T cells were immediately mixed with lentiviral vectors encoding an enhanced green fluorescent protein marker gene and transduction frequency was determined after 5 days of culture. RESULTS: Optimal transduction required both mouse T cell activation and cytokine support. Furthermore, transduction was also dependent upon the promoter chosen, with the rank order of potency being PGK > EF1 > SFFV > CMV. HIV-1 lentiviral vectors also efficiently transduced cytokine-stimulated T cells (in the absence of antibody driven T cell activation), albeit with a lower level of transgene expression compared to fully-activated T cells. CONCLUSIONS: The present study demonstrates that primary mouse T cells can be efficiently transduced with HIV-1 lentiviral vectors, opening up prospects for their use in mouse models of gene-modified adoptive cellular therapy.