Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Tratamento Farmacológico da COVID-19 , COVID-19 , Leucemia Promielocítica Aguda , SARS-CoV-2 , Adulto , Trióxido de Arsênio/administração & dosagem , COVID-19/diagnóstico , Humanos , Leucemia Promielocítica Aguda/diagnóstico , Leucemia Promielocítica Aguda/tratamento farmacológico , Masculino , Tretinoína/administração & dosagemRESUMO
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.
Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Linfócitos T/metabolismo , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteínas com Domínio LIM , Metaloproteínas/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sobrevida , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Linfócitos T/patologia , Transativadores/antagonistas & inibidores , Transativadores/química , Transativadores/genética , Regulador Transcricional ERGRESUMO
Hematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors--GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL--in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Genoma Humano/genética , Megacariócitos/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sítios de Ligação , Diferenciação Celular , Células Cultivadas , Humanos , Megacariócitos/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
LMO2, a critical transcription regulator of hematopoiesis, is involved in human T-cell leukemia. The binding site of proline and acidic amino acid-rich protein (PAR) transcription factors in the promoter of the LMO2 gene plays a central role in hematopoietic-specific expression. E2A-HLF fusion derived from t(17;19) in B-precursor acute lymphoblastic leukemia (ALL) has the transactivation domain of E2A and the basic region/leucine zipper domain of HLF, which is a PAR transcription factor, raising the possibility that E2A-HLF aberrantly induces LMO2 expression. We here demonstrate that cell lines and a primary sample of t(17;19)-ALL expressed LMO2 at significantly higher levels than other B-precursor ALLs did. Transfection of E2A-HLF into a non-t(17;19) B-precursor ALL cell line induced LMO2 gene expression that was dependent on the DNA-binding and transactivation activities of E2A-HLF. The PAR site in the LMO2 gene promoter was critical for E2A-HLF-induced LMO2 expression. Gene silencing of LMO2 in a t(17;19)-ALL cell line by short hairpin RNA induced apoptotic cell death. These observations indicated that E2A-HLF promotes cell survival of t(17;19)-ALL cells by aberrantly up-regulating LMO2 expression. LMO2 could be a target for a new therapeutic modality for extremely chemo-resistant t(17;19)-ALL.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Metaloproteínas/genética , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Células Precursoras de Linfócitos B/fisiologia , Fatores de Transcrição/metabolismo , Translocação Genética , Proteínas Adaptadoras de Transdução de Sinal , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 19 , Sangue Fetal/citologia , Regulação Leucêmica da Expressão Gênica , Humanos , Proteínas com Domínio LIM , Lentivirus/genética , Metaloproteínas/metabolismo , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Células Precursoras de Linfócitos B/citologia , Regiões Promotoras Genéticas/fisiologia , Proteínas Proto-Oncogênicas , RNA Interferente Pequeno , Fatores de Transcrição/genética , Regulação para Cima/genéticaRESUMO
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.
Assuntos
Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Sistema Hematopoético/metabolismo , Proteínas de Homeodomínio , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Transgênicos , Proteína Meis1 , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas , Sequências Reguladoras de Ácido Nucleico , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
Haematopoiesis (or blood formation) in general and haematopoietic stem cells more specifically represent some of the best studied mammalian developmental systems. Sophisticated purification protocols coupled with powerful biological assays permit functional analysis of highly purified cell populations both in vitro and in vivo. However, despite several decades of intensive research, the sheer complexity of the haematopoietic system means that many important questions remain unanswered or even unanswerable with current experimental tools. Scientists have therefore increasingly turned to modelling to tackle complexity at multiple levels ranging from networks of genes to the behaviour of cells and tissues. Early modelling attempts of gene regulatory networks have focused on core regulatory circuits but have more recently been extended to genome-wide datasets such as expression profiling and ChIP-sequencing data. Modelling of haematopoietic cells and tissues has provided insight into the importance of phenotypic heterogeneity for the differentiation of normal progenitor cells as well as a greater understanding of treatment response for particular pathologies such as chronic myeloid leukaemia. Here we will review recent progress in attempts to reconstruct segments of the haematopoietic system. A variety of modelling strategies will be covered from small-scale, protein-DNA or protein-protein interactions to large scale reconstructions. Also discussed will be examples of how stochastic modelling may be applied to multi cell systems such as those seen in normal and malignant haematopoiesis.
Assuntos
Hematopoese/fisiologia , Sistema Hematopoético/fisiologia , Modelos Biológicos , Animais , Hematopoese/genética , Humanos , Mamíferos , Reprodutibilidade dos TestesRESUMO
The Lmo2 gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell acute lymphoblastic leukemia (T-ALL) patients and severe combined immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2 regulation has been published so far. By comparative genomics, we identified 17 highly conserved noncoding elements, 9 of which revealed specific acetylation marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays performed across 250 kb of the Lmo2 locus in 11 cell types covering different stages of hematopoietic differentiation. All candidate regulatory regions were tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed strong endothelial activity, while the distal promoter showed weak forebrain activity. Eight of the 15 distal candidate elements functioned as enhancers, which together recapitulated the full expression pattern of Lmo2, directing expression to endothelium, hematopoietic cells, tail, and forebrain. Interestingly, distinct combinations of specific distal regulatory elements were required to extend endothelial activity of the LMO2 promoter to yolk sac or fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus identifying key upstream regulators and positioning Lmo2 within hematopoietic regulatory networks.