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1.
Blood ; 113(22): 5456-65, 2009 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-19346495

RESUMO

The basic helix-loop-helix transcription factor Scl/Tal1 controls the development and subsequent differentiation of hematopoietic stem cells (HSCs). However, because few Scl target genes have been validated to date, the underlying mechanisms have remained largely unknown. In this study, we have used ChIP-Seq technology (coupling chromatin immunoprecipitation with deep sequencing) to generate a genome-wide catalog of Scl-binding events in a stem/progenitor cell line, followed by validation using primary fetal liver cells and comprehensive transgenic mouse assays. Transgenic analysis provided in vivo validation of multiple new direct Scl target genes and allowed us to reconstruct an in vivo validated network consisting of 17 factors and their respective regulatory elements. By coupling ChIP-Seq in model cell lines with in vivo transgenic validation and sophisticated bioinformatic analysis, we have identified a widely applicable strategy for the reconstruction of stem cell regulatory networks in which biologic material is otherwise limiting. Moreover, in addition to revealing multiple previously unrecognized links to known HSC regulators, as well as novel links to genes not previously implicated in HSC function, comprehensive transgenic analysis of regulatory elements provided substantial new insights into the transcriptional control of several important hematopoietic regulators, including Cbfa2t3h/Eto2, Cebpe, Nfe2, Zfpm1/Fog1, Erg, Mafk, Gfi1b, and Myb.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Proteínas Proto-Oncogênicas/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação/genética , Células Cultivadas , Embrião de Mamíferos/irrigação sanguínea , Perfilação da Expressão Gênica , Genoma , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Transcrição Gênica
2.
Stem Cells ; 28(10): 1751-9, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20715180

RESUMO

A common feature of early embryo cells from the inner cell mass (ICM) and of ESCs is an absolute dependence on an atypical cell cycle in which the G1 phase is shortened to preserve their self-renewing and pluripotent nature. The transcription factor B-Myb has been attributed a role in proliferation, in particular during the G2/M phases of the cell cycle. Intriguingly, B-Myb levels in ICM/ESCs are greater than 100 times compared with those in normal proliferating cells, suggesting a particularly important function for this transcription factor in pluripotent stem cells. B-Myb is essential for embryo development beyond the preimplantation stage, but its role in ICM/ESCs remains unclear. Using a combination of mouse genetics, single DNA fiber analyses and high-resolution three-dimensional (3D) imaging, we demonstrate that B-Myb has no influence on the expression of pluripotency factors, but instead B-Myb ablation leads to stalling of replication forks and superactivation of replication factories that result in disorganization of the replication program and an increase in double-strand breaks. These effects are partly due to aberrant transcriptional regulation of cell cycle proliferation factors, namely c-Myc and FoxM1, which dictate normal S phase progression. We conclude that B-Myb acts crucially during the S phase in ESCs by facilitating proper progression of replication, thereby protecting the cells from genomic damage. Our findings have particular relevance in the light of the potential therapeutic application of ESCs and the need to maintain their genomic integrity.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fase S/genética , Transativadores/metabolismo , Animais , Western Blotting , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proliferação de Células , Imunoprecipitação da Cromatina , Citometria de Fluxo , Imunofluorescência , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transativadores/genética
3.
Life Sci Alliance ; 2(2)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30877232

RESUMO

Mutations at the N- or C-terminus of C/EBPα are frequent in acute myeloid leukaemia (AML) with normal karyotype. Here, we investigate the role of the transcription factor Myb in AMLs driven by different combinations of CEBPA mutations. Using knockdown of Myb in murine cell lines modelling the spectrum of CEBPA mutations, we show that the effect of reduced Myb depends on the mutational status of the two Cebpa alleles. Importantly, Myb knockdown fails to override the block in myeloid differentiation in cells with biallelic N-terminal C/EBPα mutations, demonstrating for the first time that the dependency on Myb is much lower in AML with this mutational profile. By comparing gene expression following Myb knockdown and chromatin immunoprecipitation sequencing data for the binding of C/EBPα isoforms, we provide evidence for a functional cooperation between C/EBPα and Myb in the maintenance of AML. This co-dependency breaks down when both alleles of CEBPA harbour N-terminal mutations, as a subset of C/EBPα-regulated genes only bind the short p30 C/EBPα isoform and, unlike other C/EBPα-regulated genes, do so without a requirement for Myb.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação/genética , Proteínas Proto-Oncogênicas c-myb/genética , Alelos , Animais , Apoptose/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos , Fenótipo , Isoformas de Proteínas/genética , RNA Interferente Pequeno/genética , Transfecção
4.
Cancer Res ; 78(20): 5767-5779, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30082276

RESUMO

Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases characterized by blood cytopenias that occur as a result of somatic mutations in hematopoietic stem cells (HSC). MDS leads to ineffective hematopoiesis, and as many as 30% of patients progress to acute myeloid leukemia (AML). The mechanisms by which mutations accumulate in HSC during aging remain poorly understood. Here we identify a novel role for MYBL2 in DNA double-strand break (DSB) repair in HSC. In patients with MDS, low MYBL2 levels associated with and preceded transcriptional deregulation of DNA repair genes. Stem/progenitor cells from these patients display dysfunctional DSB repair kinetics after exposure to ionizing radiation (IR). Haploinsufficiency of Mybl2 in mice also led to a defect in the repair of DSBs induced by IR in HSC and was characterized by unsustained phosphorylation of the ATM substrate KAP1 and telomere fragility. Our study identifies MYBL2 as a crucial regulator of DSB repair and identifies MYBL2 expression levels as a potential biomarker to predict cellular response to genotoxic treatments in MDS and to identify patients with defects in DNA repair. Such patients with worse prognosis may require a different therapeutic regimen to prevent progression to AML.Significance: These findings suggest MYBL2 levels may be used as a biological biomarker to determine the DNA repair capacity of hematopoietic stem cells from patients with MDS and as a clinical biomarker to inform decisions regarding patient selection for treatments that target DNA repair.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/20/5767/F1.large.jpg Cancer Res; 78(20); 5767-79. ©2018 AACR.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Células-Tronco Hematopoéticas/metabolismo , Transativadores/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Ensaio Cometa , Reparo do DNA , Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Síndromes Mielodisplásicas/metabolismo , Fosforilação , Radiação Ionizante
5.
Exp Hematol ; 34(5): 654-63, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16647571

RESUMO

OBJECTIVE: The differentiation of megakaryocytes is characterized by polyploidization and cytoplasmic maturation leading to platelet production. Studying these processes is hindered by the paucity of bone marrow megakaryocytes and their precursors. We describe a method for the expansion and purification of committed megakaryocyte progenitors and demonstrate their usefulness by studying changes in the expression of Ets and GATA family transcription factors throughout megakaryocytopoiesis. METHODS: A two-step serum-free method was developed. Cells isolated using this method were analyzed for surface marker expression by flow cytometry, and for their ability to differentiate using single-cell culture. Purified progenitors were induced to differentiate and analyzed with respect to their ploidy by flow cytometry and expression of specific genes by RT-PCR. RESULTS: A population of Lin- c-kit+ CD45+ CD41+ CD31+ CD34low CD9low FcgammaRII/IIIlow Sca-1med/low committed megakaryocyte progenitors was purified. These cells could be differentiated efficiently, achieving ploidy of up to 128N. Analysis of RNA demonstrated the expected increases in expression of key megakaryocyte-associated genes. RT-PCR analysis also revealed that a range of Ets and GATA factors are expressed, their individual levels and patterns of expression varying widely. Surprisingly, we find that GATA-6 is specifically expressed in late differentiated megakaryocytes and has the potential to regulate megakaryocyte-expressed genes in cooperation with Ets factors. CONCLUSION: Purified primary megakaryocytic progenitors are able to differentiate as a cohort into fully mature megakaryocytes. The number of cells obtainable, and the synchrony of the differentiation process, facilitates analysis of the dynamics of molecular processes involved in megakaryocytopoiesis. The expression pattern of Ets and GATA family transcription factors reveals the complexity of the involvement of these key megakaryocytic regulators. The finding of GATA-6 expression and demonstration of its functional activity suggests a novel mechanism for the regulation of certain genes late in megakaryocytopoiesis.


Assuntos
Diferenciação Celular , Fator de Transcrição GATA6/fisiologia , Células-Tronco Hematopoéticas/citologia , Megacariócitos/citologia , Proteínas Proto-Oncogênicas c-ets/fisiologia , Animais , Sequência de Bases , Primers do DNA , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica
6.
PLoS One ; 10(9): e0138257, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26382271

RESUMO

The MYB transcription factor plays critical roles in normal and malignant haematopoiesis. We previously showed that MYB was a direct activator of FLT3 expression within the context of acute myeloid leukaemia. During normal haematopoiesis, increasing levels of FLT3 expression determine a strict hierarchy within the haematopoietic stem and early progenitor compartment, which associates with lymphoid and myeloid commitment potential. We use the conditional deletion of the Myb gene to investigate the influence of MYB in Flt3 transcriptional regulation within the haematopoietic stem cell (HSC) hierarchy. In accordance with previous report, in vivo deletion of Myb resulted in rapid biased differentiation of HSC with concomitant loss of proliferation capacity. We find that loss of MYB activity also coincided with decreased FLT3 expression. At the chromatin level, the Flt3 promoter is primed in immature HSC, but occupancy of further intronic elements determines expression. Binding to these locations, MYB and C/EBPα need functional cooperation to activate transcription of the locus. This cooperation is cell context dependent and indicates that MYB and C/EBPα activities are inter-dependent in controlling Flt3 expression to influence lineage commitment of multipotential progenitors.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Oncogênicas v-myb/fisiologia , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Células Cultivadas , Regulação da Expressão Gênica , Hematopoese/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Oncogênicas v-myb/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo
7.
PLoS One ; 7(8): e43300, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22952660

RESUMO

Product of the Itga2b gene, CD41 contributes to hematopoietic stem cell (HSC) and megakaryocyte/platelet functions. CD41 expression marks the onset of definitive hematopoiesis in the embryo where it participates in regulating the numbers of multipotential progenitors. Key to platelet aggregation, CD41 expression also characterises their precursor, the megakaryocyte, and is specifically up regulated during megakaryopoiesis. Though phenotypically unique, megakaryocytes and HSC share numerous features, including key transcription factors, which could indicate common sub-regulatory networks. In these respects, Itga2b can serve as a paradigm to study features of both developmental-stage and HSC- versus megakaryocyte-specific regulations. By comparing different cellular contexts, we highlight a mechanism by which internal promoters participate in Itga2b regulation. A developmental process connects epigenetic regulation and promoter switching leading to CD41 expression in HSC. Interestingly, a similar process can be observed at the Mpl locus, which codes for another receptor that defines both HSC and megakaryocyte identities. Our study shows that Itga2b expression is controlled by lineage-specific networks and associates with H4K8ac in megakaryocyte or H3K27me3 in the multipotential hematopoietic cell line HPC7. Correlating with the decrease in H3K27me3 at the Itga2b Iocus, we find that following commitment to megakaryocyte differentiation, the H3K27 demethylase Jmjd3 up-regulation influences both Itga2b and Mpl expression.


Assuntos
Hematopoese/fisiologia , Integrina alfa2/metabolismo , Receptores de Trombopoetina/biossíntese , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Imunoprecipitação da Cromatina , Análise por Conglomerados , DNA/metabolismo , Epigênese Genética , Regulação da Expressão Gênica , Hematopoese/genética , Humanos , Megacariócitos/citologia , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/metabolismo
8.
Mol Cell Proteomics ; 6(3): 548-64, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17186946

RESUMO

The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68, and 22 surface membrane, intracellular membrane, and membrane proteins of unknown subcellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomics studies, we analyzed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing importance of mutant mouse models in establishing protein function in platelets. This approach identified all of the major classes of platelet transmembrane receptors, including multitransmembrane proteins. Strikingly 17 of the 25 most megakaryocyte-specific genes (relative to 30 other serial analysis of gene expression libraries) were transmembrane proteins, illustrating the unique nature of the megakaryocyte/platelet surface. The list of novel plasma membrane proteins identified using proteomics includes the immunoglobulin superfamily member G6b, which undergoes extensive alternate splicing. Specific antibodies were used to demonstrate expression of the G6b-B isoform, which contains an immunoreceptor tyrosine-based inhibition motif. G6b-B undergoes tyrosine phosphorylation and association with the SH2 domain-containing phosphatase, SHP-1, in stimulated platelets suggesting that it may play a novel role in limiting platelet activation.


Assuntos
Plaquetas/química , Megacariócitos/química , Proteínas de Membrana/análise , Animais , Membrana Celular/química , Células Cultivadas , Cromatografia de Afinidade , Expressão Gênica , Genômica , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ativação Plaquetária , Proteômica , RNA/metabolismo , Receptores Imunológicos/análise , Receptores Imunológicos/metabolismo , Espectrometria de Massas em Tandem
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