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1.
Blood ; 136(22): 2574-2587, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-32822472

RESUMO

The canonical Wnt signaling pathway is mediated by interaction of ß-catenin with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and subsequent transcription activation of Wnt-target genes. In the hematopoietic system, the function of the pathway has been mainly investigated by rather unspecific genetic manipulations of ß-catenin that yielded contradictory results. Here, we used a mouse expressing a truncated dominant negative form of the human TCF4 transcription factor (dnTCF4) that specifically abrogates ß-catenin-TCF/LEF interaction. Disruption of the ß-catenin-TCF/LEF interaction resulted in the accumulation of immature cells and reduced granulocytic differentiation. Mechanistically, dnTCF4 progenitors exhibited downregulation of the Csf3r gene, reduced granulocyte colony-stimulating factor (G-CSF) receptor levels, attenuation of downstream Stat3 phosphorylation after G-CSF treatment, and impaired G-CSF-mediated differentiation. Chromatin immunoprecipitation assays confirmed direct binding of TCF/LEF factors to the promoter and putative enhancer regions of CSF3R. Inhibition of ß-catenin signaling compromised activation of the emergency granulopoiesis program, which requires maintenance and expansion of myeloid progenitors. Consequently, dnTCF4 mice were more susceptible to Candida albicans infection and more sensitive to 5-fluorouracil-induced granulocytic regeneration. Importantly, genetic and chemical inhibition of ß-catenin-TCF/LEF signaling in human CD34+ cells reduced granulocytic differentiation, whereas its activation enhanced myelopoiesis. Altogether, our data indicate that the ß-catenin-TCF/LEF complex directly regulates G-CSF receptor levels, and consequently controls proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Our results uncover a role for the ß-catenin signaling pathway in fine tuning the granulocytic production, opening venues for clinical intervention that require enhanced or reduced production of neutrophils.


Assuntos
Granulócitos/metabolismo , Mielopoese , Receptores de Fator Estimulador de Colônias/biossíntese , Transdução de Sinais , Fatores de Transcrição TCF/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Regulação para Cima , beta Catenina/metabolismo , Animais , Candida albicans , Candidíase/genética , Candidíase/metabolismo , Camundongos , Camundongos Transgênicos , Receptores de Fator Estimulador de Colônias/genética , Fatores de Transcrição TCF/genética , beta Catenina/genética
2.
Haematologica ; 106(4): 1106-1119, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32527952

RESUMO

The megakaryocyte/erythroid Transient Myeloproliferative Disorder (TMD) in newborns with Down Syndrome (DS) occurs when N-terminal truncating mutations of the hemopoietic transcription factor GATA1, that produce GATA1short protein (GATA1s), are acquired early in development. Prior work has shown that murine GATA1s, by itself, causes a transient yolk sac myeloproliferative disorder. However, it is unclear where in the hemopoietic cellular hierarchy GATA1s exerts its effects to produce this myeloproliferative state. Here, through a detailed examination of hemopoiesis from murine GATA1s ES cells and GATA1s embryos we define defects in erythroid and megakaryocytic differentiation that occur relatively late in hemopoiesis. GATA1s causes an arrest late in erythroid differentiation in vivo, and even more profoundly in ES-cell derived cultures, with a marked reduction of Ter-119 cells and reduced erythroid gene expression. In megakaryopoiesis, GATA1s causes a differentiation delay at a specific stage, with accumulation of immature, kit-expressing CD41hi megakaryocytic cells. In this specific megakaryocytic compartment, there are increased numbers of GATA1s cells in S-phase of cell cycle and reduced number of apoptotic cells compared to GATA1 cells in the same cell compartment. There is also a delay in maturation of these immature GATA1s megakaryocytic lineage cells compared to GATA1 cells at the same stage of differentiation. Finally, even when GATA1s megakaryocytic cells mature, they mature aberrantly with altered megakaryocyte-specific gene expression and activity of the mature megakaryocyte enzyme, acetylcholinesterase. These studies pinpoint the hemopoietic compartment where GATA1s megakaryocyte myeloproliferation occurs, defining where molecular studies should now be focussed to understand the oncogenic action of GATA1s.


Assuntos
Síndrome de Down , Reação Leucemoide , Animais , Diferenciação Celular , Fator de Transcrição GATA1/genética , Humanos , Recém-Nascido , Megacariócitos , Camundongos
3.
IUBMB Life ; 72(1): 89-105, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31769197

RESUMO

GATA1 is considered as the "master" transcription factor in erythropoiesis. It regulates at the transcriptional level all aspects of erythroid maturation and function, as revealed by gene knockout studies in mice and by genome-wide occupancies in erythroid cells. The GATA1 protein contains two zinc finger domains and an N-terminal transactivation domain. GATA1 translation results in the production of the full-length protein and of a shorter variant (GATA1s) lacking the N-terminal transactivation domain, which is functionally deficient in supporting erythropoiesis. GATA1 protein abundance is highly regulated in erythroid cells at different levels, including transcription, mRNA translation, posttranslational modifications, and protein degradation, in a differentiation-stage-specific manner. Maintaining high GATA1 protein levels is essential in the early stages of erythroid maturation, whereas downregulating GATA1 protein levels is a necessary step in terminal erythroid differentiation. The importance of maintaining proper GATA1 protein homeostasis in erythropoiesis is demonstrated by the fact that both GATA1 loss and its overexpression result in lethal anemia. Importantly, alterations in any of those GATA1 regulatory checkpoints have been recognized as an important cause of hematological disorders such as dyserythropoiesis (with or without thrombocytopenia), ß-thalassemia, Diamond-Blackfan anemia, myelodysplasia, or leukemia. In this review, we provide an overview of the multilevel regulation of GATA1 protein homeostasis in erythropoiesis and of its deregulation in hematological disease.


Assuntos
Diferenciação Celular , Células Eritroides/citologia , Eritropoese , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Células Eritroides/metabolismo , Fator de Transcrição GATA1/genética , Humanos , Transdução de Sinais
4.
Biochim Biophys Acta ; 1859(12): 1515-1526, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27693117

RESUMO

DNMT1 is the maintenance DNA methyltransferase shown to be essential for embryonic development and cellular growth and differentiation in many somatic tissues in mammals. Increasing evidence has also suggested a role for DNMT1 in repressing gene expression through interactions with specific transcription factors. Previously, we identified DNMT1 as an interacting partner of the TR2/TR4 nuclear receptor heterodimer in erythroid cells, implicated in the developmental silencing of fetal ß-type globin genes in the adult stage of human erythropoiesis. Here, we extended this work by using a biotinylation tagging approach to characterize DNMT1 protein complexes in mouse erythroleukemic cells. We identified novel DNMT1 interactions with several hematopoietic transcription factors with essential roles in erythroid differentiation, including GATA1, GFI-1b and FOG-1. We provide evidence for DNMT1 forming distinct protein subcomplexes with specific transcription factors and propose the existence of a "core" DNMT1 complex with the transcription factors ZBP-89 and ZNF143, which is also present in non-hematopoietic cells. Furthermore, we identified the short (17a.a.) PCNA Binding Domain (PBD) located near the N-terminus of DNMT1 as being necessary for mediating interactions with the transcription factors described herein. Lastly, we provide evidence for DNMT1 serving as a co-repressor of ZBP-89 and GATA1 acting through upstream regulatory elements of the PU.1 and GATA1 gene loci.


Assuntos
Diferenciação Celular/genética , DNA (Citosina-5-)-Metiltransferases/genética , Complexos Multiproteicos/metabolismo , Fatores de Transcrição/genética , Animais , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Eritroides/química , Células Eritroides/metabolismo , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
5.
Nucleic Acids Res ; 41(9): 4938-48, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23519611

RESUMO

We report the genomic occupancy profiles of the key hematopoietic transcription factor GATA-1 in pro-erythroblasts and mature erythroid cells fractionated from day E12.5 mouse fetal liver cells. Integration of GATA-1 occupancy profiles with available genome-wide transcription factor and epigenetic profiles assayed in fetal liver cells enabled as to evaluate GATA-1 involvement in modulating local chromatin structure of target genes during erythroid differentiation. Our results suggest that GATA-1 associates preferentially with changes of specific epigenetic modifications, such as H4K16, H3K27 acetylation and H3K4 di-methylation. Furthermore, we used random forest (RF) non-linear regression to predict changes in the expression levels of GATA-1 target genes based on the genomic features available for pro-erythroblasts and mature fetal liver-derived erythroid cells. Remarkably, our prediction model explained a high proportion of 62% of variation in gene expression. Hierarchical clustering of the proximity values calculated by the RF model produced a clear separation of upregulated versus downregulated genes and a further separation of downregulated genes in two distinct groups. Thus, our study of GATA-1 genome-wide occupancy profiles in mouse primary erythroid cells and their integration with global epigenetic marks reveals three clusters of GATA-1 gene targets that are associated with specific epigenetic signatures and functional characteristics.


Assuntos
Epigênese Genética , Eritropoese/genética , Fator de Transcrição GATA1/metabolismo , Fígado/metabolismo , Animais , Células Cultivadas , Células Eritroides/metabolismo , Feto , Genoma , Histonas/metabolismo , Fígado/citologia , Fígado/embriologia , Camundongos
6.
Leukemia ; 36(3): 687-700, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34741119

RESUMO

MIR139 is a tumor suppressor and is commonly silenced in acute myeloid leukemia (AML). However, the tumor-suppressing activities of miR-139 and molecular mechanisms of MIR139-silencing remain largely unknown. Here, we studied the poorly prognostic MLL-AF9 fusion protein-expressing AML. We show that MLL-AF9 expression in hematopoietic precursors caused epigenetic silencing of MIR139, whereas overexpression of MIR139 inhibited in vitro and in vivo AML outgrowth. We identified novel miR-139 targets that mediate the tumor-suppressing activities of miR-139 in MLL-AF9 AML. We revealed that two enhancer regions control MIR139 expression and found that the polycomb repressive complex 2 (PRC2) downstream of MLL-AF9 epigenetically silenced MIR139 in AML. Finally, a genome-wide CRISPR-Cas9 knockout screen revealed RNA Polymerase 2 Subunit M (POLR2M) as a novel MIR139-regulatory factor. Our findings elucidate the molecular control of tumor suppressor MIR139 and reveal a role for POLR2M in the MIR139-silencing mechanism, downstream of MLL-AF9 and PRC2 in AML. In addition, we confirmed these findings in human AML cell lines with different oncogenic aberrations, suggesting that this is a more common oncogenic mechanism in AML. Our results may pave the way for new targeted therapy in AML.


Assuntos
Leucemia Mieloide Aguda/genética , MicroRNAs/genética , RNA Polimerase II/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Epigênese Genética , Regulação Leucêmica da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética
7.
Exp Hematol ; 75: 11-20, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31154069

RESUMO

Translational regulation plays a critical role in erythropoiesis, as it reflects the translational needs of enucleated mature erythroid cells in the absence of transcription and the large translational demands of balanced globin chain synthesis during erythroid maturation. In addition, red blood cells need to respond quickly to changes in their environment and the demands of the organism. Translational regulation occurs at several levels in erythroid cells, including the differential utilization of upstream open reading frames during differentiation and in response to signaling and the employment of RNA-binding proteins in an erythroid cell-specific fashion. Translation initiation is a critical juncture for translational regulation in response to environmental signals such as heme and iron availability, whereas regulatory mechanisms for ribosome recycling are consistent with recent observations highlighting the importance of maintaining adequate ribosome levels in differentiating erythroid cells. Translational deregulation in erythroid cells leads to disease associated with ineffective erythropoiesis, further highlighting the pivotal role translational regulation in erythropoiesis plays in human physiology and homeostasis. Overall, erythropoiesis has served as a unique model that has provided invaluable insight into translational regulation.


Assuntos
Diferenciação Celular/fisiologia , Células Eritroides/metabolismo , Eritropoese/fisiologia , Biossíntese de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Células Eritroides/citologia , Heme/metabolismo , Humanos , Ferro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo
8.
Mol Cell Biol ; 35(12): 2103-18, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25870109

RESUMO

The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs), is a prerequisite for the transcription of protein-coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA, are also necessary for tightly regulated transcription initiation. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. By ablating TAF10 specifically in erythroid cells in vivo, we observed a differentiation block accompanied by deregulated GATA1 target genes, including Gata1 itself, suggesting functional cross talk between GATA1 and TAF10. Additionally, we analyzed by mass spectrometry the composition of TFIID and SAGA complexes in mouse and human cells and found that their global integrity is maintained, with minor changes, during erythroid cell differentiation and development. In agreement with our functional data, we show that TAF10 interacts directly with GATA1 and that TAF10 is enriched on the GATA1 locus in human fetal erythroid cells. Thus, our findings demonstrate a cross talk between canonical TFIID and SAGA complexes and cell-specific transcription activators during development and differentiation.


Assuntos
Células Eritroides/citologia , Eritropoese , Fator de Transcrição GATA1/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Animais , Células Eritroides/metabolismo , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Loci Gênicos , Humanos , Camundongos , Camundongos Knockout , Mapeamento de Interação de Proteínas , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética
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