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1.
Genome Res ; 29(4): 564-575, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30796038

RESUMEN

The epigenetic regulator TET2 is frequently mutated in hematological diseases. Mutations have been shown to arise in hematopoietic stem cells early in disease development and lead to altered DNA methylation landscapes and an increased risk of hematopoietic malignancy. Here, we show by genome-wide mapping of TET2 binding sites in different cell types that TET2 localizes to regions of open chromatin and cell-type-specific enhancers. We find that deletion of Tet2 in native hematopoiesis as well as fully transformed acute myeloid leukemia (AML) results in changes in transcription factor (TF) activity within these regions, and we provide evidence that loss of TET2 leads to attenuation of chromatin binding of members of the basic helix-loop-helix (bHLH) TF family. Together, these findings demonstrate that TET2 activity shapes the local chromatin environment at enhancers to facilitate TF binding and provides an example of how epigenetic dysregulation can affect gene expression patterns and drive disease development.


Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Células Madre Hematopoyéticas/metabolismo , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogénicas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular , Células Cultivadas , Dioxigenasas , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Leucemia Mieloide Aguda/metabolismo , Ratones , Unión Proteica
2.
Nat Med ; 23(8): 954-963, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28671689

RESUMEN

The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.


Asunto(s)
Conductos Biliares Extrahepáticos/fisiología , Células Epiteliales/citología , Vesícula Biliar/fisiología , Organoides/fisiología , Regeneración/fisiología , Ingeniería de Tejidos/métodos , Animales , Conductos Biliares Extrahepáticos/citología , Conductos Biliares Extrahepáticos/lesiones , Sistema Biliar/citología , Sistema Biliar/lesiones , Sistema Biliar/fisiología , Trasplante de Células , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Vesícula Biliar/lesiones , Humanos , Técnicas In Vitro , Queratina-19/metabolismo , Queratina-7/metabolismo , Ratones , Organoides/citología , Organoides/efectos de los fármacos , Organoides/metabolismo , Secretina/farmacología , Somatostatina/farmacología , Andamios del Tejido , gamma-Glutamiltransferasa/metabolismo
3.
Genes Dev ; 29(9): 910-22, 2015 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-25886910

RESUMEN

DNA methylation is tightly regulated throughout mammalian development, and altered DNA methylation patterns are a general hallmark of cancer. The methylcytosine dioxygenase TET2 is frequently mutated in hematological disorders, including acute myeloid leukemia (AML), and has been suggested to protect CG dinucleotide (CpG) islands and promoters from aberrant DNA methylation. In this study, we present a novel Tet2-dependent leukemia mouse model that closely recapitulates gene expression profiles and hallmarks of human AML1-ETO-induced AML. Using this model, we show that the primary effect of Tet2 loss in preleukemic hematopoietic cells is progressive and widespread DNA hypermethylation affecting up to 25% of active enhancer elements. In contrast, CpG island and promoter methylation does not change in a Tet2-dependent manner but increases relative to population doublings. We confirmed this specific enhancer hypermethylation phenotype in human AML patients with TET2 mutations. Analysis of immediate gene expression changes reveals rapid deregulation of a large number of genes implicated in tumorigenesis, including many down-regulated tumor suppressor genes. Hence, we propose that TET2 prevents leukemic transformation by protecting enhancers from aberrant DNA methylation and that it is the combined silencing of several tumor suppressor genes in TET2 mutated hematopoietic cells that contributes to increased stem cell proliferation and leukemogenesis.


Asunto(s)
Carcinogénesis/genética , Metilación de ADN/genética , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos/genética , Regulación Neoplásica de la Expresión Génica , Células Madre Hematopoyéticas/patología , Proteínas Proto-Oncogénicas/genética , Animales , Proliferación Celular/genética , Dioxigenasas , Células Madre Hematopoyéticas/citología , Humanos , Ratones , Mutación/genética , Translocación Genética/genética
4.
Haematologica ; 98(12): 1912-20, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23831920

RESUMEN

The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers and recent investigations have shown TET2 mutations in diffuse large B-cell lymphoma. However, the detection rates and the types of TET2 mutations vary, and the relation to global methylation patterns has not been investigated. Here, we show TET2 mutations in 12 of 100 diffuse large B-cell lymphomas with 7% carrying loss-of-function and 5% carrying missense mutations. Genome-wide methylation profiling using 450K Illumina arrays identified 315 differentially methylated genes between TET2 mutated and TET2 wild-type cases. TET2 mutations are primarily associated with hypermethylation within CpG islands (70%; P<0.0001), and at CpG-rich promoters (60%; P<0.0001) of genes involved in hematopoietic differentiation and cellular development. Hypermethylated loci in TET2 mutated samples overlap with the bivalent (H3K27me3/H3K4me3) silencing mark in human embryonic stem cells (P=1.5×10(-30)). Surprisingly, gene expression profiling showed that only 11% of the hypermethylated genes were down-regulated, among which there were several genes previously suggested to be tumor suppressors. A meta-analysis suggested that the 35 hypermethylated and down-regulated genes are associated with the activated B-cell-like type of diffuse large B-cell lymphoma in other studies. In conclusion, our data suggest that TET2 mutations may cause aberrant methylation mainly of genes involved in hematopoietic development, which are silenced but poised for activation in human embryonic stem cells.


Asunto(s)
Metilación de ADN/genética , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica/métodos , Estudio de Asociación del Genoma Completo/métodos , Linfoma de Células B Grandes Difuso/genética , Mutación/genética , Proteínas Proto-Oncogénicas/genética , Anciano , Dioxigenasas , Femenino , Humanos , Linfoma de Células B Grandes Difuso/diagnóstico , Masculino , Persona de Mediana Edad
5.
J Immunol ; 179(12): 8235-42, 2007 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-18056367

RESUMEN

In this study, we characterize the molecular signal pathways that lead to MHC class I chain-related protein A (MICA) expression after histone deacetylase (HDAC)-inhibitor (HDAC-i) treatment of Jurkat T cells. Chelating calcium with BAPTA-AM or EGTA potently inhibited HDAC- and CMV-mediated MICA/B expression. It was further observed that endoplasmic reticulum calcium stores were depleted after HDAC treatment. NF-kappaB activity can be induced by HDAC treatment. However, nuclear translocation of NF-kappaB p65 was not observed after HDAC treatment of Jurkat T cells and even though we could effectively inhibit p65 expression by siRNA, it did not modify MICA/B expression. To identify important elements in MICA regulation, we made a promoter construct consisting of approximately 3 kb of the proximal MICA promoter in front of GFP. Deletion analysis showed that a germinal center-box containing a putative Sp1 site from position -113 to -93 relative to the mRNA start site was important for HDAC and CMV-induced promoter activity. Sp1 was subsequently shown to be important, as targeted mutation of the Sp1 binding sequence or siRNA mediated down modulation of Sp1-inhibited MICA promoter activity and surface-expression.


Asunto(s)
Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/genética , Inhibidores de Histona Desacetilasas , Linfocitos T/inmunología , Transporte Activo de Núcleo Celular , Calcio/antagonistas & inhibidores , Calcio/metabolismo , Quelantes/farmacología , Citomegalovirus/genética , Depsipéptidos/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/farmacología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Células Jurkat , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Factor de Transcripción Sp1/metabolismo , Linfocitos T/enzimología , Factor de Transcripción ReIA/metabolismo
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