RESUMEN
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.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción GATA/metabolismo , Leucemia/metabolismo , Metaloproteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Telomerasa/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Factores de Transcripción GATA/genética , Regulación del Desarrollo de la Expresión Génica , Genoma/genética , Proteínas con Dominio LIM , Leucemia/genética , Metaloproteínas/genética , Ratones , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Telomerasa/genética , Análisis de Matrices Tisulares , Transactivadores/genéticaRESUMEN
Hematopoietic stem cell (HSC) development is regulated by several signaling pathways and a number of key transcription factors, which include Scl/Tal1, Runx1, and members of the Smad family. However, it remains unclear how these various determinants interact. Using a genome-wide computational screen based on the well characterized Scl +19 HSC enhancer, we have identified a related Smad6 enhancer that also targets expression to blood and endothelial cells in transgenic mice. Smad6, Bmp4, and Runx1 transcripts are concentrated along the ventral aspect of the E10.5 dorsal aorta in the aorta-gonad-mesonephros region from which HSCs originate. Moreover, Smad6, an inhibitor of Bmp4 signaling, binds and inhibits Runx1 activity, whereas Smad1, a positive mediator of Bmp4 signaling, transactivates the Runx1 promoter. Taken together, our results integrate three key determinants of HSC development; the Scl transcriptional network, Runx1 activity, and the Bmp4/Smad signaling pathway.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas Morfogenéticas Óseas/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/genética , Biología Computacional , Secuencia Conservada , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Células Endoteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Familia de Multigenes , Células 3T3 NIH , Unión Proteica , Elementos Reguladores de la Transcripción/genética , Alineación de Secuencia , Proteína smad6/química , Proteína smad6/genética , Proteína smad6/metabolismoRESUMEN
Transcription factors are key regulators of hematopoietic stem cells (HSCs), yet the molecular mechanisms that control their expression are largely unknown. Previously, we demonstrated that expression of Scl/Tal1, a transcription factor required for the specification of HSCs, is controlled by Ets and GATA factors. Here we characterize the molecular mechanisms controlling expression of Lyl1, a paralog of Scl also required for HSC function. Two closely spaced promoters directed expression to hematopoietic progenitor, megakaryocytic, and endothelial cells in transgenic mice. Conserved binding sites required for promoter activity were bound in vivo by GATA-2 and the Ets factors Fli1, Elf1, Erg, and PU.1. However, despite coregulation of Scl and Lyl1 by the same Ets and GATA factors, Scl expression was initiated prior to Lyl1 in embryonic stem (ES) cell differentiation assays. Moreover, ectopic expression of Scl but not Lyl1 rescued hematopoietic differentiation in Scl-/- ES cells, thus providing a molecular explanation for the vastly different phenotypes of Scl-/- and Lyl1-/- mouse embryos. Furthermore, coregulation of Scl and Lyl1 later during development may explain the mild phenotype of Scl-/- adult HSCs.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factor de Transcripción GATA2/metabolismo , Hematopoyesis , Proteínas de Neoplasias/metabolismo , Proteína Proto-Oncogénica c-ets-1/metabolismo , Proteínas Proto-Oncogénicas/deficiencia , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , Secuencia Conservada , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Células Endoteliales/metabolismo , Expresión Génica , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fenotipo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Alineación de Secuencia , Proteína 1 de la Leucemia Linfocítica T Aguda , Factores de TiempoRESUMEN
L3mbtl encodes a member of the Polycomb group of proteins, which function as transcriptional repressors in large protein complexes. The Drosophila D-l(3)mbt protein is considered a tumor suppressor since its inactivation results in brain tumors. The human L3MBTL gene lies in a region of chromosome 20 frequently deleted in patients with myeloid malignancies and has been proposed as a candidate 20q tumor suppressor gene. Recently we have shown that L3MBTL undergoes monoallelic methylation in hematopoietic tissues and is transcribed from the paternally derived allele. The mouse L3mbtl gene is located on chromosome 2, a region of syntenic homology with human chromosome 20, and in a region containing a number of genes subject to epigenetic regulation. Here we analyze the genomic structure and alternative splicing of L3mbtl and assess its imprinting status in mouse. L3mbtl displays a complex pattern of alternative splicing involving both 5' noncoding and coding exons and is transcribed from two promoters. Unlike its human counterpart, L3mbtl escapes imprinting and there is no differential methylation of its CpG island.
Asunto(s)
Empalme Alternativo/genética , Genes Supresores de Tumor , Impresión Genómica/genética , Ratones/genética , Proteínas de Neoplasias/genética , Animales , Secuencia de Bases , Proteínas Cromosómicas no Histona , Islas de CpG , Metilación de ADN , Exones , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras , Especificidad de la Especie , Proteínas Supresoras de TumorRESUMEN
Appropriate transcriptional regulation is critical for the biological functions of many key regulatory genes, including the stem cell leukemia (SCL) gene. As part of a systematic dissection of SCL transcriptional regulation, we have previously identified a 5,245-bp SCL +18/19 enhancer that targeted embryonic endothelium together with embryonic and adult hematopoietic progenitors and stem cells (HSCs). This enhancer is proving to be a powerful tool for manipulating hematopoietic progenitors and stem cells, but the design and interpretation of such transgenic studies require a detailed understanding of enhancer activity in vivo. In this study, we demonstrate that the +18/19 enhancer is active in mast cells, megakaryocytes, and adult endothelium. A 644-bp +19 core enhancer exhibited similar temporal and spatial activity to the 5,245-bp +18/19 fragment both during development and in adult mice. Unlike the +18/19 enhancer, the +19 core enhancer was only active in adult mice when linked to the eukaryotic reporter gene human placental alkaline phosphatase. Activity of a single core enhancer in HSCs, endothelium, mast cells, and megakaryocytes suggests possible overlaps in their respective transcriptional programs. Moreover, activity in a proportion of thymocytes and other SCL-negative cell types suggests the existence of a silencer elsewhere in the SCL locus.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Elementos de Facilitación Genéticos/genética , Células Madre Hematopoyéticas/citología , Proteínas Proto-Oncogénicas/genética , Animales , Células de la Médula Ósea/citología , Linaje de la Célula , Células Endoteliales/citología , Células Endoteliales/enzimología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/genética , Masculino , Mastocitos/citología , Mastocitos/enzimología , Megacariocitos/citología , Megacariocitos/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Proteína 1 de la Leucemia Linfocítica T Aguda , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/sangre , beta-Galactosidasa/genéticaRESUMEN
The development of blood has long served as a model for mammalian cell type specification and differentiation, and yet the underlying transcriptional networks remain ill defined. Characterization of such networks will require genome-wide identification of cis-regulatory sequences and an understanding of how regulatory information is encoded in the primary DNA sequence. Despite progress in lower organisms, genome-wide computational identification of mammalian cis-regulatory sequences has been hindered by increased genomic complexity and cumbersome transgenic assays. Starting with a well-characterized blood stem cell enhancer from the SCL gene, we have developed computational tools for the identification of functionally related gene regulatory sequences. Two candidate enhancers discovered in this way were located in intron 1 of the Fli-1 and PRH/Hex genes, both transcription factors previously implicated in controlling blood and endothelial development. Subsequent transgenic and biochemical analysis demonstrated that the two computationally identified enhancers are functionally related to the SCL stem cell enhancer. The approach developed here may therefore be useful for identifying additional enhancers involved in the control of early blood and endothelial development, and may be adapted to decipher transcriptional regulatory codes controlling a broad range of mammalian developmental programmes.
Asunto(s)
Diferenciación Celular/genética , Endotelio/fisiología , Elementos de Facilitación Genéticos , Células Madre Hematopoyéticas/fisiología , Antígenos CD34/genética , Antígenos CD34/metabolismo , Secuencia de Bases , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endotelio/citología , Células Madre Hematopoyéticas/citología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Datos de Secuencia Molecular , Proteína Proto-Oncogénica c-fli-1 , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Programas Informáticos , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Analysis of cis-regulatory elements is central to understanding the genomic program for development. The scl/tal-1 transcription factor is essential for lineage commitment to blood cell formation and previous studies identified an scl enhancer (the +18/19 element) which was sufficient to target the vast majority of hematopoietic stem cells, together with hematopoietic progenitors and endothelium. Moreover, expression of scl under control of the +18/19 enhancer rescued blood progenitor formation in scl(-/-) embryos. However, here we demonstrate by using a knockout approach that, within the endogenous scl locus, the +18/19 enhancer is not necessary for the initiation of scl transcription or for the formation of hematopoietic cells. These results led to the identification of a bifunctional 5' enhancer (-3.8 element), which targets expression to hematopoietic progenitors and endothelium, contains conserved critical Ets sites, and is bound by Ets family transcription factors, including Fli-1 and Elf-1. These data demonstrate that two geographically distinct but functionally related enhancers regulate scl transcription in hematopoietic progenitors and endothelial cells and suggest that enhancers with dual hematopoietic-endothelial activity may represent a general strategy for regulating blood and endothelial development.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Linaje de la Célula , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/fisiología , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas Nucleares , Proteína Proto-Oncogénica c-fli-1 , Proteínas Proto-Oncogénicas/genética , Alineación de Secuencia , Proteína 1 de la Leucemia Linfocítica T Aguda , Transactivadores/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Stem cells are a central feature of metazoan biology. Haematopoietic stem cells (HSCs) represent the best-characterized example of this phenomenon, but the molecular mechanisms responsible for their formation remain obscure. The stem cell leukaemia (SCL) gene encodes a basic helix-loop-helix (bHLH) transcription factor with an essential role in specifying HSCs. Here we have addressed the transcriptional hierarchy responsible for HSC formation by characterizing an SCL 3' enhancer that targets expression to HSCs and endothelium and their bipotential precursors, the haemangioblast. We have identified three critical motifs, which are essential for enhancer function and bind GATA-2, Fli-1 and Elf-1 in vivo. Our results suggest that these transcription factors are key components of an enhanceosome responsible for activating SCL transcription and establishing the transcriptional programme required for HSC formation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos/genética , Células Madre Hematopoyéticas/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Embrión de Mamíferos/fisiología , Embrión no Mamífero , Factor de Transcripción GATA2 , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Secuencias Hélice-Asa-Hélice/genética , Humanos , Sustancias Macromoleculares , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Complejos Multiproteicos , Unión Proteica , Proteína Proto-Oncogénica c-fli-1 , Proteínas Proto-Oncogénicas/metabolismo , Alineación de Secuencia , Proteína 1 de la Leucemia Linfocítica T Aguda , Transactivadores/metabolismo , Xenopus laevis/fisiologíaRESUMEN
The stem cell leukemia (SCL) gene encodes a basic helix-loop-helix transcription factor with a critical role in the development of both blood and endothelium. Loss-of-function studies have shown that SCL is essential for the formation of hematopoietic stem cells, for subsequent erythroid development and for yolk sac angiogenesis. SCL exhibits a highly conserved pattern of expression from mammals to teleost fish. Several murine SCL enhancers have been identified, each of which directs reporter gene expression in vivo to a subdomain of the normal SCL expression pattern. However, regulatory elements necessary for SCL expression in erythroid cells remain to be identified and the size of the chromosomal domain needed to support appropriate SCL transcription is unknown. Here we demonstrate that a 130-kilobase (kb) yeast artificial chromosome (YAC) containing the human SCL locus completely rescued the embryonic lethal phenotype of scl(-/-) mice. Rescued YAC(+) scl(-/-) mice were born in appropriate Mendelian ratios, were healthy and fertile, and exhibited no detectable abnormality of yolk sac, fetal liver, or adult hematopoiesis. The human SCL protein can therefore substitute for its murine homologue. In addition, our results demonstrate that the human SCL YAC contains the chromosomal domain necessary to direct expression to the erythroid lineage and to all other tissues in which SCL performs a nonredundant essential function.