RESUMEN
In higher eukaryotes, many genes are regulated by enhancers that are 104-106 base pairs (bp) away from the promoter. Enhancers contain transcription-factor-binding sites (which are typically around 7-22 bp), and physical contact between the promoters and enhancers is thought to be required to modulate gene expression. Although chromatin architecture has been mapped extensively at resolutions of 1 kilobase and above; it has not been possible to define physical contacts at the scale of the proteins that determine gene expression. Here we define these interactions in detail using a chromosome conformation capture method (Micro-Capture-C) that enables the physical contacts between different classes of regulatory elements to be determined at base-pair resolution. We find that highly punctate contacts occur between enhancers, promoters and CCCTC-binding factor (CTCF) sites and we show that transcription factors have an important role in the maintenance of the contacts between enhancers and promoters. Our data show that interactions between CTCF sites are increased when active promoters and enhancers are located within the intervening chromatin. This supports a model in which chromatin loop extrusion1 is dependent on cohesin loading at active promoters and enhancers, which explains the formation of tissue-specific chromatin domains without changes in CTCF binding.
Asunto(s)
Emparejamiento Base/genética , Genoma/genética , Animales , Sitios de Unión , Factor de Unión a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Elementos de Facilitación Genéticos/genética , Células Eritroides/citología , Células Eritroides/metabolismo , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , Globinas alfa/genética , CohesinasAsunto(s)
Elementos de Facilitación Genéticos , Hemoglobina H/genética , Globinas alfa/genética , Talasemia alfa/genética , Adulto , Alelos , Cromatina/genética , Cromatina/ultraestructura , Cromosomas Humanos Par 16/genética , Cromosomas Humanos Par 16/ultraestructura , Eritroblastos/patología , Eritropoyesis , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Genotipo , Hemoglobina E/genética , Hemoglobina H/análisis , Humanos , Masculino , Linaje , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Eliminación de Secuencia , Suriname/etnología , Globinas alfa/biosíntesis , Talasemia alfa/sangre , Globinas beta/genéticaRESUMEN
Diffuse large B-cell lymphoma with secondary involvement of the central nervous system (SCNS-DLBCL) is a rare condition carrying a poor prognosis. No optimal therapeutic regimen has been identified. We retrospectively analysed 23 patients with SCNS-DLBCL treated with R-IDARAM (rituximab 375 mg/m(2) IV day 1; methotrexate 12·5 mg by intrathecal injection day 1; idarubicin 10 mg/m(2) /day IV days 1 and 2; dexamethasone 100 mg/day IV infusion over 12 h days 1-3; cytosine arabinoside 1000 mg/m(2) /day IV over 1 h days 1 and 2; and methotrexate 2000 mg/m(2) IV over 2 h day 3. Ten out of 23 (44%) patients had CNS involvement at initial presentation ('new disease'), 10/23 (44%) had relapsed disease and 3/23 (13%) had primary refractory disease. 14/23 (61%) of patients responded - 6 (26%) complete response, 8 (35%) partial response. Grade 3-4 haematological toxicity was seen in all cycles, with no grade 3-4 or long-term neurological toxicity. Median follow-up for surviving patients was 49 months. At 2 years, estimated progression-free survival (PFS) was 39% and overall survival (OS) was 52%. Encouraging outcomes were reported in patients with new disease, with 5-year estimated PFS of 50% and OS 75%. R-IDARAM is a well-tolerated regimen with encouraging efficacy in patients with SCNS-DLBCL, although patients with relapsed or refractory disease continue to fare poorly.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Adulto , Anciano , Citarabina/administración & dosificación , Dexametasona/administración & dosificación , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Humanos , Idarrubicina/administración & dosificación , Infusiones Intravenosas , Inyecciones Espinales , Masculino , Metotrexato/administración & dosificación , Persona de Mediana Edad , Estudios Retrospectivos , Rituximab/administración & dosificación , Resultado del TratamientoRESUMEN
Haemoglobin E (HbE) ß-thalassaemia causes approximately 50% of all severe thalassaemia worldwide; equating to around 30,000 births per year. HbE ß-thalassaemia is due to a point mutation in codon 26 of the human HBB gene on one allele (GAG; glutamatic acid â AAG; lysine, E26K), and any mutation causing severe ß-thalassaemia on the other. When inherited together in compound heterozygosity these mutations can cause a severe thalassaemic phenotype. However, if only one allele is mutated individuals are carriers for the respective mutation and have an asymptomatic phenotype (ß-thalassaemia trait). Here we describe a base editing strategy which corrects the HbE mutation either to wildtype (WT) or a normal variant haemoglobin (E26G) known as Hb Aubenas and thereby recreates the asymptomatic trait phenotype. We have achieved editing efficiencies in excess of 90% in primary human CD34 + cells. We demonstrate editing of long-term repopulating haematopoietic stem cells (LT-HSCs) using serial xenotransplantation in NSG mice. We have profiled the off-target effects using a combination of circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) and deep targeted capture and have developed machine-learning based methods to predict functional effects of candidate off-target mutations.
Asunto(s)
Hemoglobina E , Talasemia , Talasemia beta , Humanos , Animales , Ratones , Talasemia beta/genética , Hemoglobina E/genética , Talasemia/genética , Mutación , Mutación PuntualRESUMEN
Generation of mature cells from progenitors requires tight coupling of differentiation and metabolism. During erythropoiesis, erythroblasts are required to massively upregulate globin synthesis then clear extraneous material and enucleate to produce erythrocytes1-3. Nprl3 has remained in synteny with the α-globin genes for >500 million years4, and harbours the majority of the α-globin enhancers5. Nprl3 is a highly conserved inhibitor of mTORC1, which controls cellular metabolism. However, whether Nprl3 itself serves an erythroid role is unknown. Here, we show that Nprl3 is a key regulator of erythroid metabolism. Using Nprl3-deficient fetal liver and adult competitive bone marrow - fetal liver chimeras, we show that NprI3 is required for sufficient erythropoiesis. Loss of Nprl3 elevates mTORC1 signalling, suppresses autophagy and disrupts erythroblast glycolysis and redox control. Human CD34+ progenitors lacking NPRL3 produce fewer enucleated cells and demonstrate dysregulated mTORC1 signalling in response to nutrient availability and erythropoietin. Finally, we show that the α-globin enhancers upregulate NprI3 expression, and that this activity is necessary for optimal erythropoiesis. Therefore, the anciently conserved linkage of NprI3, α-globin and their associated enhancers has enabled coupling of metabolic and developmental control in erythroid cells. This may enable erythropoiesis to adapt to fluctuating nutritional and environmental conditions.
Asunto(s)
Ceruloplasmina/deficiencia , Deferoxamina/administración & dosificación , Cardiopatías/tratamiento farmacológico , Trastornos del Metabolismo del Hierro/tratamiento farmacológico , Sobrecarga de Hierro/tratamiento farmacológico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Piridonas/administración & dosificación , Sideróforos/administración & dosificación , Adulto , Deferiprona , Quimioterapia Combinada , Cardiopatías/sangre , Cardiopatías/complicaciones , Humanos , Trastornos del Metabolismo del Hierro/sangre , Trastornos del Metabolismo del Hierro/complicaciones , Sobrecarga de Hierro/sangre , Sobrecarga de Hierro/complicaciones , Masculino , Enfermedades Neurodegenerativas/sangre , Enfermedades Neurodegenerativas/complicacionesRESUMEN
ß-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and ß-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with ß-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem/progenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia. When edited CD34+ cells are differentiated into erythroid cells, we observe the expected reduction in α-globin expression and a correction of the pathologic globin chain imbalance in cells from patients with ß-thalassemia. Xenograft assays show that a proportion of the edited CD34+ cells are long-term repopulating hematopoietic stem cells, demonstrating the potential of this approach for translation into a therapy for ß-thalassemia.ß-thalassemia is characterised by the presence of an excess of α-globin chains, which contribute to erythrocyte pathology. Here the authors use CRISP/Cas9 to reduce α-globin expression in hematopoietic precursors, and show effectiveness in xenograft assays in mice.