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1.
EMBO J ; 38(14): e101564, 2019 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-31304633

RESUMEN

DOT1L methylates histone H3K79 and is aberrantly regulated in MLL-rearranged leukemia. Inhibitors have been developed to target DOT1L activity in leukemia, but cellular mechanisms that regulate DOT1L are still poorly understood. We have identified the histone deacetylase Rpd3 as a negative regulator of budding yeast Dot1. At its target genes, the transcriptional repressor Rpd3 restricts H3K79 methylation, explaining the absence of H3K79me3 at a subset of genes in the yeast genome. Similar to the crosstalk in yeast, inactivation of the murine Rpd3 homolog HDAC1 in thymocytes led to an increase in H3K79 methylation. Thymic lymphomas that arise upon genetic deletion of Hdac1 retained the increased H3K79 methylation and were sensitive to reduced DOT1L dosage. Furthermore, cell lines derived from Hdac1Δ/Δ thymic lymphomas were sensitive to a DOT1L inhibitor, which induced apoptosis. In summary, we identified an evolutionarily conserved crosstalk between HDAC1 and DOT1L with impact in murine thymic lymphoma development.


Asunto(s)
Histona Desacetilasa 1/genética , Histona Desacetilasa 2/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Linfoma/metabolismo , Neoplasias del Timo/metabolismo , Acetilación , Animales , Línea Celular Tumoral , Eliminación de Gen , Histona Desacetilasas/genética , Humanos , Linfoma/genética , Metilación , Ratones , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Neoplasias del Timo/genética
2.
EMBO Rep ; 22(2): e51184, 2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33410591

RESUMEN

Differentiation of naïve peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B-cell fate remain unclear. Here, we identified a role for the histone H3K79 methyltransferase DOT1L in controlling B-cell differentiation. Mouse B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells in which Dot1L was deleted showed aberrant differentiation and prematurely acquired plasma cell characteristics. Similar results were obtained when DOT1L was chemically inhibited in mature B cells in vitro. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro-proliferative, pro-GC program. In addition, DOT1L indirectly supports the repression of an anti-proliferative plasma cell differentiation program by maintaining the repression of Polycomb Repressor Complex 2 (PRC2) targets. Our findings show that DOT1L is a key modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B-cell naivety and GC B-cell differentiation.


Asunto(s)
Linfocitos B/citología , Diferenciación Celular , N-Metiltransferasa de Histona-Lisina , Histonas , Metiltransferasas , Animales , Epigénesis Genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Histonas/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones
3.
Proc Natl Acad Sci U S A ; 117(34): 20706-20716, 2020 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-32764145

RESUMEN

Cytotoxic T cell differentiation is guided by epigenome adaptations, but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone methyltransferase DOT1L, which marks the nucleosome core on active genes, safeguards normal differentiation of CD8+ T cells. T cell-specific ablation of Dot1L resulted in loss of naïve CD8+ T cells and premature differentiation toward a memory-like state, independent of antigen exposure and in a cell-intrinsic manner. Mechanistically, DOT1L controlled CD8+ T cell differentiation by ensuring normal T cell receptor density and signaling. DOT1L also maintained epigenetic identity, in part by indirectly supporting the repression of developmentally regulated genes. Finally, deletion of Dot1L in T cells resulted in an impaired immune response. Through our study, DOT1L is emerging as a central player in physiology of CD8+ T cells, acting as a barrier to prevent premature differentiation and controlling epigenetic integrity.


Asunto(s)
Linfocitos T CD8-positivos/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Diferenciación Celular/genética , Epigénesis Genética/genética , Epigenómica , Femenino , Histona Metiltransferasas/metabolismo , N-Metiltransferasa de Histona-Lisina/fisiología , Histonas/metabolismo , Masculino , Metiltransferasas/metabolismo , Ratones
4.
Proc Natl Acad Sci U S A ; 117(49): 31343-31352, 2020 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-33229554

RESUMEN

Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged Igh allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive Igh allele is expressed, a phenomenon known as Igh allelic exclusion. In contrast to a productively rearranged Igh allele, the Igh messenger RNA (mRNA) (IgHR) from a nonproductively rearranged Igh allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable IgHR to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the IghTer5H∆TM mouse model from IghTer5H mice having a premature termination codon at position +5 in leader exon of IghTer5H allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of IghTer5H message, indicating that previous conclusions regarding a role of IgHR in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the IghTer5H∆TM knock-in allele, which generated stable IgHR but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not IgHR expression.


Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Alelos , Animales , Biomarcadores/metabolismo , Sitios Genéticos , Ratones Endogámicos C57BL , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados
5.
Blood ; 125(25): 3937-48, 2015 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-25778535

RESUMEN

Interstrand crosslinks (ICLs) are toxic DNA lesions that cause severe genomic damage during replication, especially in Fanconi anemia pathway-deficient cells. This results in progressive bone marrow failure and predisposes to acute myeloid leukemia (AML). The molecular mechanisms responsible for these defects are largely unknown. Using Ercc1-deficient mice, we show that Trp53 is responsible for ICL-induced bone marrow failure and that loss of Trp53 is leukemogenic in this model. In addition, Ercc1-deficient myeloid progenitors gain elevated levels of miR-139-3p and miR-199a-3p with age. These microRNAs exert opposite effects on hematopoiesis. Ectopic expression of miR-139-3p strongly inhibited proliferation of myeloid progenitors, whereas inhibition of miR-139-3p activity restored defective proliferation of Ercc1-deficient progenitors. Conversely, the inhibition of miR-199a-3p functions aggravated the myeloid proliferation defect in the Ercc1-deficient model, whereas its enforced expression enhanced proliferation of progenitors. Importantly, miR-199a-3p caused AML in a pre-leukemic mouse model, supporting its role as an onco-microRNA. Target genes include HuR for miR-139-3p and Prdx6, Runx1, and Suz12 for miR-199a-3p. The latter genes have previously been implicated as tumor suppressors in de novo and secondary AML. These findings show that, in addition to TRP53-controlled mechanisms, miR-139-3p and miR-199a-3p are involved in the defective hematopoietic function of ICL-repair deficient myeloid progenitors.


Asunto(s)
Transformación Celular Neoplásica/genética , Células Madre Hematopoyéticas/patología , Leucemia/genética , MicroARNs/genética , Animales , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Reparación del ADN/genética , Proteínas de Unión al ADN/deficiencia , Modelos Animales de Enfermedad , Endonucleasas/deficiencia , Células Madre Hematopoyéticas/metabolismo , Leucemia/metabolismo , Leucemia/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
6.
Blood ; 119(20): 4723-30, 2012 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-22353998

RESUMEN

MicroRNAs (miRNAs) have the potential to regulate cellular differentiation programs; however, miRNA deficiency in primary hematopoietic stem cells (HSCs) results in HSC depletion in mice, leaving the question of whether miRNAs play a role in early-lineage decisions un-answered. To address this issue, we deleted Dicer1, which encodes an essential RNase III enzyme for miRNA biogenesis, in murine CCAAT/enhancer-binding protein α (C/EBPA)-positive myeloid-committed progenitors in vivo. In contrast to the results in HSCs, we found that miRNA depletion affected neither the number of myeloid progenitors nor the percentage of C/EBPA-positive progenitor cells. Analysis of gene-expression profiles from wild-type and Dicer1-deficient granulocyte-macrophage progenitors (GMPs) revealed that 20 miRNA families were active in GMPs. Of the derepressed miRNA targets in Dicer1-null GMPs, 27% are normally exclusively expressed in HSCs or are specific for multipotent progenitors and erythropoiesis, indicating an altered gene-expression landscape. Dicer1-deficient GMPs were defective in myeloid development in vitro and exhibited an increased replating capacity, indicating the regained self-renewal potential of these cells. In mice, Dicer1 deletion blocked monocytic differentiation, depleted macrophages, and caused myeloid dysplasia with morphologic features of Pelger-Huët anomaly. These results provide evidence for a miRNA-controlled switch for a cellular program of self-renewal and expansion toward myeloid differentiation in GMPs.


Asunto(s)
Diferenciación Celular/genética , ARN Helicasas DEAD-box/genética , Células Dendríticas/fisiología , Macrófagos/fisiología , Células Progenitoras Mieloides/fisiología , Neutrófilos/patología , Ribonucleasa III/genética , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/fisiología , Células Cultivadas , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/fisiología , Células Dendríticas/citología , Células Dendríticas/metabolismo , Embrión de Mamíferos , Eliminación de Gen , Recuento de Leucocitos , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/metabolismo , Neutrófilos/citología , Neutrófilos/metabolismo , Neutrófilos/fisiología , Anomalía de Pelger-Huët/genética , Anomalía de Pelger-Huët/patología , Ribonucleasa III/metabolismo , Ribonucleasa III/fisiología
7.
Curr Opin Hematol ; 19(4): 261-7, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22504525

RESUMEN

PURPOSE OF REVIEW: Recent data show that microRNAs play critical roles in the regulation of the developmental process of hematopoietic stem and progenitor cells toward mature myeloid cells. The main focus of the article is the function of some evolutionary conserved microRNAs that are abundantly expressed and tightly regulated during myelopoiesis. RECENT FINDINGS: Global microRNA depletion studies in hematopoietic stem cells have shown the importance of microRNA-controlled pathways for hematopoiesis. Recent insights from genetic mouse models and overexpression or deletion of microRNAs in developmental cell intermediates demonstrate strong evidence for evolutionary conserved microRNA-regulated pathways involved in tight control of cellular processes such as proliferation, differentiation and apoptosis at different stages of blood cell development. It is becoming evident that the myeloid transcription factor PU.1 regulates the expression of critical microRNAs including miR-17∼92 and miR-146a during myelopoiesis. Furthermore, there is evidence for the contribution of aberrant miR-125 activities in hematopoietic disorders including myeloid leukemia. SUMMARY: Despite the large number of articles describing differential microRNA expression during hematopoiesis, microRNA functions and their downstream pathways in myeloid lineage decisions and leukemia are only recently emerging. Here we discuss new findings concerning PU.1-controlled microRNAs and miR-125-regulated networks in normal and malignant myelopoiesis.


Asunto(s)
Neoplasias Hematológicas/genética , Hematopoyesis/genética , MicroARNs/fisiología , Mielopoyesis/genética , Animales , Neoplasias Hematológicas/metabolismo , Hematopoyesis/fisiología , Humanos , Ratones , Mielopoyesis/fisiología
8.
Mol Ther Nucleic Acids ; 23: 1161-1171, 2021 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-33664995

RESUMEN

Emerging data show that microRNA 193a-3p (miR-193a-3p) has a suppressive role in many cancers and is often downregulated in tumors, as compared to surrounding normal tissues. Therefore, mimics of miR-193a-3p could be used as an attractive therapeutic approach in oncology. To better understand and document the molecular mechanism of action of 1B3, a novel synthetic miRNA-193a-3p mimic, RNA sequencing was performed after transfection of 1B3 in six different human tumor cell lines. Genes differentially expressed (DE) in at least three cell lines were mapped by Ingenuity Pathway Analysis (IPA), and interestingly, these results strongly indicated upregulation of the tumor-suppressive phosphatase and tensin homolog (PTEN) pathway, as well as downregulation of many oncogenic growth factor signaling pathways. Importantly, although unsurprisingly, IPA identified miR-193a-3p as a strong upstream regulator of DE genes in an unbiased manner. Furthermore, biological function analysis pointed to an extensive link of 1B3 with cancer, via expected effects on tumor cell survival, proliferation, migration, and cell death. Our data strongly suggest that miR-193a-3p/1B3 is a potent tumor suppressor agent that targets various key oncogenic pathways across cancer types. Therefore, the introduction of 1B3 into tumor cells may represent a promising strategy for cancer treatment.

9.
Front Pharmacol ; 12: 596535, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33679394

RESUMEN

The antihelmintic drug ABZ and its metabolites belong to the chemical family of benzimidazoles (BZM) that act as potent tubulin polymerization inhibitors, suggesting a potential re-direction of BZMs for cancer therapy. Applying UV-Vis spectrometry we here demonstrate ABZ as a DNA intercalator. This insight led us to determine the primary mode of ABZ action in mammalian cells. As revealed by RNA sequencing, ABZ did neither grossly affect replication as analyzed by survival and replication stress signaling, nor the transcriptome. Actually, unbiased transcriptome analysis revealed a marked cell cycle signature in ABZ exposed cells. Indeed, short-term exposure to ABZ arrested mammalian cells in G2/M cell cycle stages associated with frequent gains and losses of chromatin. Cellular analyses revealed ABZ as a potent mammalian spindle poison for normal and malignant cells, explaining the serious chromosome segregation defects. Since chromosomal aberrations promote both cancer development and cell death, we determined if besides its general cytotoxicity, ABZ could predispose to tumor development. As measured by loss of heterozygosity (LOH) in vitro and in vivo ABZ was found as a potent inducer of LOH and accelerator of chromosomal missegregation.

10.
Oncotarget ; 12(5): 422-439, 2021 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-33747358

RESUMEN

Compelling evidence demonstrates that miR-193a-3p is a tumor suppressor microRNA in many cancer types, and its reduced expression is linked to cancer initiation and progression, metastasis, and therapy resistance. However, its mechanism of action is not consistently described between studies, and often contradicts the pleiotropic role of a microRNA in manipulating several different mRNA targets. We therefore comprehensively investigated miRNA-193a-3p's mode of action in a panel of human cancer cell lines, with a variety of genetic backgrounds, using 1B3, a synthetic microRNA mimic. Interestingly, the exact mechanism through which 1B3 reduced cell proliferation varied between cell lines. 1B3 efficiently reduced target gene expression, leading to reduced cell proliferation/survival, cell cycle arrest, induction of apoptosis, increased cell senescence, DNA damage, and inhibition of migration. SiRNA silencing of 1B3 target mRNAs further highlighted the advantage of the pleiotropic mechanism of 1B3 action, as repression of individual targets did not achieve the same robust effect on cell proliferation in all cell lines. Importantly, a novel lipid nanoparticle-based formulation of 1B3, INT-1B3, demonstrated marked anti-tumor activity as a single agent following systemic administration in tumor-bearing mice. Together, these data strongly support the development of 1B3 as a novel therapeutic agent for treatment of human cancer.

11.
PLoS One ; 14(1): e0210526, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30629682

RESUMEN

C9orf82 protein, or conserved anti-apoptotic protein 1 or caspase activity and apoptosis inhibitor 1 (CAAP1) has been implicated as a negative regulator of the intrinsic apoptosis pathway by modulating caspase expression and activity. In contrast, an independent genome wide screen for factors capable of driving drug resistance to the topoisomerase II (Topo II) poisons doxorubicin and etoposide, implicated a role for the nuclear protein C9orf82 in delaying DSBs repair downstream of Topo II, hereby sensitizing cells to DSB induced apoptosis. To determine its function in a genetically defined setting in vivo and ex vivo, we here employed CRISPR/Cas9 technology in zygotes to generate a C9orf82 knockout mouse model. C9orf82ko/ko mice were born at a Mendelian ratio and did not display any overt macroscopic or histological abnormalities. DSBs repair dependent processes like lymphocyte development and class switch recombination (CSR) appeared normal, arguing against a link between the C9orf82 encoded protein and V(D)J recombination or CSR. Most relevant, primary pre-B cell cultures and Tp53 transformed mouse embryo fibroblasts (MEFs) derived from C9orf82ko/ko E14.5 and wild type embryos displayed comparable sensitivity to a number of DNA lesions, including DSBs breaks induced by the topoisomerase II inhibitors, etoposide and doxorubicin. Likewise, the kinetics of γH2AX formation and resolution in response to etoposide of C9orf82 protein proficient, deficient and overexpressing MEFs were indistinguishable. These data argue against a direct role of C9orf82 protein in delaying repair of Topo II generated DSBs and regulating apoptosis. The genetically defined systems generated in this study will be of value to determine the actual function of C9orf82 protein.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/fisiología , Roturas del ADN de Doble Cadena , ADN-Topoisomerasas de Tipo II/metabolismo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Linfocitos B/citología , Linfocitos B/metabolismo , Sistemas CRISPR-Cas , Caspasa 3/metabolismo , Proliferación Celular , Células Cultivadas , Daño del ADN , Reparación del ADN , Cambio de Clase de Inmunoglobulina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T/citología , Linfocitos T/metabolismo
12.
Cell Stem Cell ; 19(3): 383-96, 2016 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-27424784

RESUMEN

Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the self-renewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols.


Asunto(s)
Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , MicroARNs/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Antígenos CD34/metabolismo , Proliferación Celular , Autorrenovación de las Células/genética , Redes Reguladoras de Genes , Trasplante de Células Madre Hematopoyéticas , Humanos , Marcaje Isotópico , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , Modelos Biológicos , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Células Madre Multipotentes/trasplante , Reproducibilidad de los Resultados , Factores de Tiempo
13.
Cell Cycle ; 11(15): 2799-807, 2012 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-22801545

RESUMEN

MicroRNAs (miRNAs) belong to an abundant class of highly conserved small (22nt) non-coding RNAs. MiRNA profiling studies indicate that their expression is highly cell type-dependent. DICER1 is an essential RNase III endoribonuclease for miRNA processing. Hematopoietic cell type- and developmental stage-specific Dicer1 deletion models show that miRNAs are essential regulators of cellular survival, differentiation and function. For instance, miRNA deficiency in hematopoietic stem cells and progenitors of different origins results in decreased cell survival, dramatic developmental aberrations or dysfunctions in mice. We recently found that homozygous Dicer1 deletion in myeloid-committed progenitors results in an aberrant expression of stem cell genes and induces a regained self-renewal capacity. Moreover, Dicer1 deletion causes a block in macrophage development and myeloid dysplasia, a cellular condition that may be considered as a preleukemic state. However, Dicer1-null cells do not develop leukemia in mice, indicating that depletion of miRNAs is not enough for tumorigenesis. Surprisingly, we found that heterozygous Dicer1 deletion in myeloid-committed progenitors, but not Dicer1 knockout, collaborates with p53 deletion in leukemic progression and results in various types of leukemia. Our data indicate that Dicer1 is a haploinsufficient tumorsuppressor in hematopoietic neoplasms, which is consistent with the observed downregulation of miRNA expression in human leukemia samples. Here, we review the various hematopoietic specific Dicer1 deletion mouse models and the phenotypes observed within the different hematopoietic lineages and cell developmental stages. Finally, we discuss the role for DICER1 in mouse and human malignant hematopoiesis.


Asunto(s)
ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Leucemia/genética , MicroARNs/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Animales , Diferenciación Celular/genética , ARN Helicasas DEAD-box/deficiencia , Regulación hacia Abajo , Genes Supresores de Tumor , Células Madre Hematopoyéticas/citología , Humanos , Macrófagos , Ratones , Ratones Endogámicos C57BL , Ribonucleasa III/deficiencia , Eliminación de Secuencia , Proteína p53 Supresora de Tumor/genética
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