RESUMO
Coordinated induction, but also repression, of genes are key to normal differentiation. Although the role of lineage-specific transcription regulators has been studied extensively, their functional integration with chromatin remodelers, one of the key enzymatic machineries that control chromatin accessibility, remains ill-defined. Here we investigate the role of Mi-2ß, a SNF-2-like nucleosome remodeler and key component of the nucleosome remodeling and histone deacetylase (NuRD) complex in early B cells. Inactivation of Mi-2ß arrested differentiation at the large pre-B-cell stage and caused derepression of cell adhesion and cell migration signaling factors by increasing chromatin access at poised enhancers and chromosome architectural elements. Mi-2ß also supported IL-7R signaling, survival, and proliferation by repressing negative effectors of this pathway. Importantly, overexpression of Bcl2, a mitochondrial prosurvival gene and target of IL-7R signaling, partly rescued the differentiation block caused by Mi-2ß loss. Mi-2ß stably associated with chromatin sites that harbor binding motifs for IKAROS and EBF1 and physically associated with these transcription factors both on and off chromatin. Notably, Mi-2ß shared loss-of-function cellular and molecular phenotypes with IKAROS and EBF1, albeit in a distinct fashion. Thus, the nucleosome remodeler Mi-2ß promotes pre-B-cell differentiation by providing repression capabilities to distinct lineage-specific transcription factor-based regulatory networks.
Assuntos
Linfócitos B/citologia , Diferenciação Celular/genética , Cromatina/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Linhagem da Célula , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Camundongos , Fatores de TranscriçãoRESUMO
Double-strand DNA breaks (DSB) induce chromosomal translocations and gene amplification in cell culture, but mechanisms by which DSB cause genomic instability in vivo are poorly understood. We show that RAG-1/2-induced DSB cause IgH/c-Myc translocations in leukemic pro-B cells from p53/Prkdc-deficient mice. Strikingly, these translocations were complex, clonally heterogeneous and amplified. We observed reiterated IgH/c-Myc fusions on dicentric chromosomes, suggesting that amplification occurred by repeated cycles of bridge, breakage and fusion. Leukemogenesis was not mitigated in RAG-2/p53/Prkdc-deficient mice, but leukemic pro-B cells lacked IgH/c-Myc translocations. Thus, global genomic instability conferred by p53/Prkdc disruption efficiently transforms pro-B cells lacking RAG-1/2-induced DSB. Unexpectedly, RAG-2/p53/Prkdc-deficient mice also developed leptomeningeal leukemia, providing a novel spontaneous model for this frequent complication of human lymphoblastic malignancies.
Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Homeodomínio/genética , Leucemia Linfoide/genética , Translocação Genética , Animais , Northern Blotting , Southern Blotting , Doenças do Sistema Nervoso Central/etiologia , Doenças do Sistema Nervoso Central/patologia , Proteínas de Ligação a DNA/deficiência , Citometria de Fluxo , Amplificação de Genes/genética , Genes myc/genética , Transplante de Células-Tronco Hematopoéticas , Cadeias Pesadas de Imunoglobulinas/genética , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Leucemia Linfoide/complicações , Leucemia Linfoide/fisiopatologia , Neoplasias Meníngeas/etiologia , Neoplasias Meníngeas/genética , Camundongos , Modelos Animais , Proteína Supressora de Tumor p53/deficiênciaRESUMO
The ability of somatic stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin environments that keep stem cell-specific genes active and key differentiation factors repressed but poised for activation. The epigenetic factors that provide this type of regulation remain ill-defined. Here we provide the first evidence that the SNF2-like ATPase Mi-2beta of the Nucleosome Remodeling Deacetylase (NuRD) complex is required for maintenance of and multilineage differentiation in the early hematopoietic hierarchy. Shortly after conditional inactivation of Mi-2beta, there is an increase in cycling and a decrease in quiescence in an HSC (hematopoietic stem cell)-enriched bone marrow population. These cycling mutant cells readily differentiate into the erythroid lineage but not into the myeloid and lymphoid lineages. Together, these effects result in an initial expansion of mutant HSC and erythroid progenitors that are later depleted as more differentiated proerythroblasts accumulate at hematopoietic sites exhibiting features of erythroid leukemia. Examination of gene expression in the mutant HSC reveals changes in the expression of genes associated with self-renewal and lineage priming and a pivotal role of Mi-2beta in their regulation. Thus, Mi-2beta provides the hematopoietic system with immune cell capabilities as well as with an extensive regenerative capacity.
Assuntos
Adenosina Trifosfatases/metabolismo , Diferenciação Celular/fisiologia , Cromatina/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Adenosina Trifosfatases/genética , Animais , Antígenos CD/análise , Antígenos CD34/análise , Apoptose , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Ciclo Celular , Diferenciação Celular/genética , Linhagem da Célula , Proliferação de Células , Células Cultivadas , DNA Helicases , Eritrócitos/citologia , Eritrócitos/metabolismo , Feminino , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Linfócitos/citologia , Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Células Mieloides/citologia , Células Mieloides/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Receptores da Transferrina/análiseRESUMO
Lineage commitment is induced by changes in gene expression dictated by the intimate interaction between transcription factors and chromatin regulators. Here, we revealed the antagonistic interplay between Ikaros and its associate the chromatin remodeler Mi-2beta during T cell development, as exemplified by the regulation of Cd4 expression. Loss of Ikaros or Mi-2beta led to activation or repression, respectively, of the Cd4 locus at inappropriate stages of development. Their combined mutation reverted to normal CD4 expression. In double-negative thymocytes, Ikaros binding to the Cd4 silencer contributed to its repressive activity. In double-positive thymocytes, concomitant binding of Mi-2beta with Ikaros to the Cd4 silencer caused silencer inactivation, thereby allowing for CD4 expression. Mi-2beta facilitated recruitment of histone acetyl transferases to the silencer. This recruitment possibly antagonized Ikaros and associated repressive activities. Thus, concomitant interactions between functionally opposing chromatin-regulating machineries are an important mode of gene regulation during lineage determination.
Assuntos
Adenosina Trifosfatases/metabolismo , Antígenos CD4/genética , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Fator de Transcrição Ikaros/metabolismo , Linfócitos T/citologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/imunologia , Animais , Antígenos CD4/biossíntese , Linhagem da Célula , DNA Helicases , Citometria de Fluxo , Expressão Gênica , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/imunologia , Imunoprecipitação , Camundongos , Mutação , Elementos Silenciadores Transcricionais , Linfócitos T/imunologiaRESUMO
Changes in chromatin structure underlie the activation or silencing of genes during development. The chromatin remodeler Mi-2beta is highly expressed in thymocytes and is presumed to be a transcriptional repressor because of its presence in the nucleosome remodeling deacetylase (NuRD) complex. Using conditional inactivation, we show that Mi-2beta is required at several steps during T cell development: for differentiation of beta selected immature thymocytes, for developmental expression of CD4, and for cell divisions in mature T cells. We further show that Mi-2beta plays a direct role in promoting CD4 gene expression. Mi-2beta associates with the CD4 enhancer as well as the E box binding protein HEB and the histone acetyltransferase (HAT) p300, enabling their recruitment to the CD4 enhancer and causing histone H3-hyperacetylation to this regulatory region. These findings provide important insights into the regulation of CD4 expression during T cell development and define a role for Mi-2beta in gene activation.