RESUMEN
Gene therapy for beta-thalassemia requires stable transfer of a beta-globin gene into hematopoietic stem cells (HSCs) and high and regulated hemoglobin expression in the erythroblastic progeny. We developed an erythroid-specific lentiviral vector driving the expression of the human beta-globin gene from a minimal promoter/enhancer element containing two hypersensitive sites from the beta-globin locus control region. Transplantation of transduced HSCs into thalassemic mice leads to stable and long-term correction of anemia with all red blood cells expressing the transgene. A frequency of 30-50% of transduced HSCs, harboring an average vector copy number per cell of 1, was sufficient to fully correct the thalassemic phenotype. In the mouse model of Cooley's anemia transplantation of transduced cells rescues lethality, leading to either a normal or a thalassemia intermedia phenotype. We show that genetically corrected erythroblasts undergo in vivo selection with preferential survival of progenitors harboring proviral integrations in genome sites more favorable to high levels of vector-derived expression. These data provide a rationale for a gene therapy approach to beta-thalassemia based on partially myeloablative transplantation protocols.
Asunto(s)
Eritroblastos/metabolismo , Regulación de la Expresión Génica , Terapia Genética/métodos , Globinas/metabolismo , Células Madre Hematopoyéticas/citología , Talasemia beta/genética , Talasemia beta/terapia , Animales , Trasplante de Células , Clonación Molecular , Vectores Genéticos , Humanos , Ratones , Ratones Endogámicos C57BL , Fenotipo , Células Madre/citologíaRESUMEN
The lambda5 and VpreB genes encode the components of the surrogate light-chain which forms part of the pre-B cell receptor and plays a key role in B cell development. In the mouse, the lambda5 and VpreB1 genes are closely linked and are co-regulated by a multi-component locus control region. To identify the sequences that regulate lambda5 and VpreB1 expression during B cell development, we have comprehensively mapped the DNaseI hypersensitive sites (HS) in the lambda5-VpreB1 functional domain. The active domain contains 12 HS that are distributed at high density across the 18.3 kb region that forms the lambda5 and VpreB1 functional unit. Analysis of a reporter gene driven by the VpreB1 promoter in transgenic mice identified a novel enhancer associated with two HS located upstream of lambda5. The lambda5-VpreB1 locus was also found to be closely linked to the ubiquitously expressed Topoisomerase-3beta (Topo3beta) gene. The VpreB1 and Topo3beta genes have entirely different expression patterns despite the fact that the two promoters are separated by a distance of only 1.5 kb.
Asunto(s)
Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Glicoproteínas de Membrana/genética , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Secuencia de Bases , Diferenciación Celular , Línea Celular , Mapeo Cromosómico , ADN Complementario/genética , Desoxirribonucleasa I , Elementos de Facilitación Genéticos , Genes Reguladores , Inmunoglobulina de Cadenas Ligeras Subrogadas , Ratones , Ratones Transgénicos , Plásmidos/genética , Receptores de Células Precursoras de Linfocitos B , Regiones Promotoras Genéticas , Receptores de Antígenos de Linfocitos BRESUMEN
Variant histone H3.3 is incorporated into nucleosomes by a mechanism that does not require DNA replication and has also been implicated as a potential mediator of epigenetic memory of active transcriptional states. In this study, we have used chromatin immunoprecipitation analysis to show that H3.3 is found mainly at the promoters of transcriptionally active genes. We also show that H3.3 combines with H3 acetylation and K4 methylation to form a stable mark that persists during mitosis. Our results suggest that H3.3 is deposited principally through the action of chromatin-remodelling complexes associated with transcriptional initiation, with deposition mediated by RNA polymerase II elongation having only a minor role.
Asunto(s)
División Celular/fisiología , Histonas/metabolismo , Regiones Promotoras Genéticas/genética , Transcripción Genética/fisiología , Animales , Línea Celular , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiología , Inmunoprecipitación de Cromatina , Técnica del Anticuerpo Fluorescente Indirecta , Histonas/genética , Hibridación Fluorescente in Situ , Ratones , Nucleosomas/metabolismo , Transcripción Genética/genéticaRESUMEN
The differentiation potential of stem cells is determined by the ability of these cells to establish and maintain developmentally regulated gene expression programs that are specific to different lineages. Although transcriptionally potentiated epigenetic states of genes have been described for haematopoietic progenitors, the developmental stage at which the formation of lineage-specific gene expression domains is initiated remains unclear. In this study, we show that an intergenic cis-acting element in the mouse lambda5-VpreB1 locus is marked by histone H3 acetylation and histone H3 lysine 4 methylation at a discrete site in embryonic stem (ES) cells. The epigenetic modifications spread from this site toward the VpreB1 and lambda5 genes at later stages of B-cell development, and a large, active chromatin domain is established in pre-B cells when the genes are fully expressed. In early B-cell progenitors, the binding of haematopoietic factor PU.1 coincides with the expansion of the marked region, and the region becomes a center for the recruitment of general transcription factors and RNA polymerase II. In pre-B cells, E2A also binds to the locus, and general transcription factors are distributed across the active domain, including the gene promoters and the intergenic region. These results suggest that localized epigenetic marking is important for establishing the transcriptional competence of the lambda5 and VpreB1 genes as early as the pluripotent ES cell stage.
