RESUMO
Oocytes are indispensable for mammalian life. Thus, it is important to understand how mature oocytes are generated. As a critical stage of oocytes development, meiosis has been extensively studied, yet how chromatin remodeling contributes to this process is largely unknown. Here, we demonstrate that the ATP-dependent chromatin remodeling factor Snf2h (also known as Smarca5) plays a critical role in regulating meiotic cell cycle progression. Females with oocyte-specific depletion of Snf2h are infertile and oocytes lacking Snf2h fail to undergo meiotic resumption. Mechanistically, depletion of Snf2h results in dysregulation of meiosis-related genes, which causes failure of maturation-promoting factor (MPF) activation. ATAC-seq analysis in oocytes revealed that Snf2h regulates transcription of key meiotic genes, such as Prkar2b, by increasing its promoter chromatin accessibility. Thus, our studies not only demonstrate the importance of Snf2h in oocyte meiotic resumption, but also reveal the mechanism underlying how a chromatin remodeling factor can regulate oocyte meiosis.
Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Fator Promotor de Maturação/genética , Meiose/genética , Oogênese/genética , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Mesotelina , Camundongos , Oócitos/citologia , TranscriptomaRESUMO
The transcription factor PU.1 (Purine-rich DNA binding, SPI1) is a key regulator of hematopoiesis, whose level is influenced by transcription through its enhancers and its post-transcriptional degradation via microRNA-155 (miR-155). The degree of transcriptional regulation of the PU.1 gene is influenced by repression via DNA methylation, as well as other epigenetic factors, such as those related to progenitor maturation status, which is modulated by the transcription factor Myeloblastosis oncogene (MYB). In this work, we show that combinatorial treatment of acute myeloid leukemia (AML) cells with DNA methylation inhibitors (5-Azacytidine), MYB inhibitors (Celastrol), and anti-miR-155 (AM155) ideally leads to overproduction of PU.1. We also show that PU.1 reactivation can be compensated by miR-155 and that only a combined approach leads to sustained PU.1 derepression, even at the protein level. The triple effect on increasing PU.1 levels in myeloblasts stimulates the myeloid transcriptional program while inhibiting cell survival and proliferation, leading to partial leukemic differentiation.
Assuntos
Leucemia Mieloide Aguda , MicroRNAs , Diferenciação Celular/genética , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismoRESUMO
Development of lymphoid progenitors requires a coordinated regulation of gene expression, DNA replication, and gene rearrangement. Chromatin-remodeling activities directed by SWI/SNF2 superfamily complexes play important roles in these processes. In this study, we used a conditional knockout mouse model to investigate the role of Smarca5, a member of the ISWI subfamily of such complexes, in early lymphocyte development. Smarca5 deficiency results in a developmental block at the DN3 stage of αß thymocytes and pro-B stage of early B cells at which the rearrangement of Ag receptor loci occurs. It also disturbs the development of committed (CD73+) γδ thymocytes. The αß thymocyte block is accompanied by massive apoptotic depletion of ß-selected double-negative DN3 cells and premitotic arrest of CD4/CD8 double-positive cells. Although Smarca5-deficient αß T cell precursors that survived apoptosis were able to undergo a successful TCRß rearrangement, they exhibited a highly abnormal mRNA profile, including the persistent expression of CD44 and CD25 markers characteristic of immature cells. We also observed that the p53 pathway became activated in these cells and that a deficiency of p53 partially rescued the defect in thymus cellularity (in contrast to early B cells) of Smarca5-deficient mice. However, the activation of p53 was not primarily responsible for the thymocyte developmental defects observed in the Smarca5 mutants. Our results indicate that Smarca5 plays a key role in the development of thymocytes undergoing ß-selection, γδ thymocytes, and also B cell progenitors by regulating the transcription of early differentiation programs.
Assuntos
Adenosina Trifosfatases/metabolismo , Linfócitos B/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Células Progenitoras Linfoides/fisiologia , Linfócitos T/fisiologia , Timócitos/fisiologia , Adenosina Trifosfatases/genética , Animais , Diferenciação Celular , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Seleção Clonal Mediada por Antígeno , Rearranjo Gênico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
ISWI chromatin remodeling ATPase SMARCA5 (SNF2H) is a well-known factor for its role in regulation of DNA access via nucleosome sliding and assembly. SMARCA5 transcriptionally inhibits the myeloid master regulator PU.1. Upregulation of SMARCA5 was previously observed in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Since high levels of SMARCA5 are necessary for intensive cell proliferation and cell cycle progression of developing hematopoietic stem and progenitor cells in mice, we reasoned that removal of SMARCA5 enzymatic activity could affect the cycling or undifferentiated state of leukemic progenitor-like clones. Indeed, we observed that CRISPR/cas9-mediated SMARCA5 knockout in AML cell lines (S5KO) inhibited the cell cycle progression. We also observed that the SMARCA5 deletion induced karyorrhexis and nuclear budding as well as increased the ploidy, indicating its role in mitotic division of AML cells. The cytogenetic analysis of S5KO cells revealed the premature chromatid separation. We conclude that deleting SMARCA5 in AML blocks leukemic proliferation and chromatid cohesion.
Assuntos
Adenosina Trifosfatases/deficiência , Proliferação de Células , Cromátides , Proteínas Cromossômicas não Histona/deficiência , Técnicas de Inativação de Genes , Leucemia Mieloide Aguda , Proteínas de Neoplasias , Adenosina Trifosfatases/metabolismo , Linhagem Celular Tumoral , Cromátides/genética , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Feminino , Humanos , Células K562 , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/metabolismoRESUMO
Ocular lens morphogenesis is a model for investigating mechanisms of cellular differentiation, spatial and temporal gene expression control, and chromatin regulation. Brg1 (Smarca4) and Snf2h (Smarca5) are catalytic subunits of distinct ATP-dependent chromatin remodeling complexes implicated in transcriptional regulation. Previous studies have shown that Brg1 regulates both lens fiber cell differentiation and organized degradation of their nuclei (denucleation). Here, we employed a conditional Snf2h(flox) mouse model to probe the cellular and molecular mechanisms of lens formation. Depletion of Snf2h induces premature and expanded differentiation of lens precursor cells forming the lens vesicle, implicating Snf2h as a key regulator of lens vesicle polarity through spatial control of Prox1, Jag1, p27(Kip1) (Cdkn1b) and p57(Kip2) (Cdkn1c) gene expression. The abnormal Snf2h(-/-) fiber cells also retain their nuclei. RNA profiling of Snf2h(-/) (-) and Brg1(-/-) eyes revealed differences in multiple transcripts, including prominent downregulation of those encoding Hsf4 and DNase IIß, which are implicated in the denucleation process. In summary, our data suggest that Snf2h is essential for the establishment of lens vesicle polarity, partitioning of prospective lens epithelial and fiber cell compartments, lens fiber cell differentiation, and lens fiber cell nuclear degradation.
Assuntos
Adenosina Trifosfatases/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Embrião de Mamíferos/metabolismo , Cristalino/citologia , Cristalino/embriologia , Animais , Autofagia , Compartimento Celular , Ciclo Celular , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição de Choque Térmico , Camundongos Knockout , Mitofagia , Modelos Biológicos , Mutação/genética , Proteínas Nucleares/metabolismo , Fator de Transcrição PAX6/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
The imitation switch nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair, and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1-iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells accumulated but their maturation toward erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S15Ph °s ) second with CBP/p300 (K376Ac ), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor-iCre) and observed that animals were anemic postnatally. Furthermore, 4-hydroxytamoxifen-mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis. Stem Cells 2017;35:1614-1623.
Assuntos
Adenosina Trifosfatases/metabolismo , Diferenciação Celular , Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Adenosina Trifosfatases/deficiência , Anemia/patologia , Animais , Ciclo Celular , Proliferação de Células , Proteínas Cromossômicas não Histona/deficiência , Dano ao DNA/genética , Células Eritroides/citologia , Eritropoese , Deleção de Genes , Genótipo , Hematopoese , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The fusion oncoprotein BCR-ABL1 exhibits aberrant tyrosine kinase activity and it has been proposed that it deregulates signaling networks involving both transcription factors and non-coding microRNAs that result in chronic myeloid leukemia (CML). Previously, microRNA expression profiling showed deregulated expression of miR-150 and miR-155 in CML. In this study, we placed these findings into the broader context of the MYC/miR-150/MYB/miR-155/PU.1 oncogenic network. We propose that up-regulated MYC and miR-155 in CD34+ leukemic stem and progenitor cells, in concert with BCR-ABL1, impair the molecular mechanisms of myeloid differentiation associated with low miR-150 and PU.1 levels. We revealed that MYC directly occupied the -11.7 kb and -0.35 kb regulatory regions in the MIR150 gene. MYC occupancy was markedly increased through BCR-ABL1 activity, causing inhibition of MIR150 gene expression in CML CD34+ and CD34- cells. Furthermore, we found an association between reduced miR-150 levels in CML blast cells and their resistance to tyrosine kinase inhibitors (TKIs). Although TKIs successfully disrupted BCR-ABL1 kinase activity in proliferating CML cells, this treatment did not efficiently target quiescent leukemic stem cells. The study presents new evidence regarding the MYC/miR-150/MYB/miR-155/PU.1 leukemic network established by aberrant BCR-ABL1 activity. The key connecting nodes of this network may serve as potential druggable targets to overcome resistance of CML stem and progenitor cells.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Genes myc/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , MicroRNAs/genética , Adulto , Idoso , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/farmacologiaRESUMO
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/metabolismoRESUMO
Oncogenic microRNAs (oncomiRs) accumulate in serum due to their increased stability and thus serve as biomarkers in breast cancer (BC) pathogenesis. Four oncogenic microRNAs (miR-155, miR-19a, miR-181b, and miR-24) and one tumor suppressor microRNA (let-7a) were shown to differentiate between high- and low-risk early breast cancer (EBC) and reflect the surgical tumor removal and adjuvant therapy. Here we applied the longitudinal multivariate data analyses to stochastically model the serum levels of each of the oncomiRs using the RT-PCR measurements in the EBC patients (N = 133) that were followed up 4 years after diagnosis. This study identifies that two of the studied oncomiRs, miR-155 and miR-24, are highly predictive of EBC relapse. Furthermore, combining the oncomiR level with Ki-67 expression further specifies the relapse probability. Our data move further the notion that oncomiRs in serum enable not only monitoring of EBC but also are a very useful tool for predicting relapse independently of any other currently analyzed characteristics in EBC patients. Our approach can be translated into medical practice to estimate individual relapse risk of EBC patients.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/genética , MicroRNAs/genética , Recidiva Local de Neoplasia/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Estudos de Coortes , Feminino , Humanos , Antígeno Ki-67/metabolismo , Estudos Longitudinais , MicroRNAs/sangue , Pessoa de Meia-Idade , Análise Multivariada , Recidiva Local de Neoplasia/diagnóstico , Fatores de TempoRESUMO
Primary cutaneous T-cell lymphomas (CTCL) affect the skin and tend to transform and spread. CTCL involves primarily the Mycosis fungoides (MF) and more aggressive Sezary syndrome (SS). Oncogenic microRNAs (miRs) are stable epigenetic inhibitors often deregulated in the tumour and detectable as biomarkers in non-cellular fractions of peripheral blood. The tumour-specific expression of miR-155, miR-203, and miR-205 was shown to correctly diagnose CTCL. We herein asked whether these microRNAs can be used as plasma biomarkers for clinical CTCL monitoring. Patients with CTCL (n = 10) and controls with non-malignant conditions (n = 11) repeatedly donated plasma samples every ca. five months. MicroRNAs were detected in the plasma samples by specifically-primed RT-PCR followed by multivariate analyses of the miR expression dynamics. We herein established the plasma miR-classifier for detecting CTCL based on the miR-155 upregulation and miR-203/miR-205 downregulation with 100% specificity and 94% sensitivity. The 3-miR-score in the consecutive samples coincided with the clinical outcome of MF and SS patients such as the therapy response or changes in the clinical stage or tumor size. Quantitation of the selected microRNAs in plasma is a specific and straightforward approach for evaluating CTCL outcome representing, thus, a valuable tool for CTCL diagnostics and therapy response monitoring.
Assuntos
Biomarcadores Tumorais , MicroRNA Circulante , Linfoma Cutâneo de Células T/genética , MicroRNAs/genética , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Humanos , Biópsia Líquida , Linfoma Cutâneo de Células T/sangue , Linfoma Cutâneo de Células T/diagnóstico , Linfoma Cutâneo de Células T/terapia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Pele/patologia , Resultado do TratamentoRESUMO
There is increasing evidence that epigenetic information can be inherited across generations in mammals, despite extensive reprogramming both in the gametes and in the early developing embryo. One corollary to this is that disrupting the establishment of epigenetic state in the gametes of a parent, as a result of heterozygosity for mutations in genes involved in reprogramming, could affect the phenotype of offspring that do not inherit the mutant allele. Here we show that such effects do occur following paternal inheritance in the mouse. We detected changes to transcription and chromosome ploidy in adult animals. Paternal effects of this type have not been reported previously in mammals and suggest that the untransmitted genotype of male parents can influence the phenotype of their offspring.
Assuntos
Metilação de DNA , Epigênese Genética/genética , Regulação da Expressão Gênica no Desenvolvimento , Padrões de Herança/genética , Camundongos/genética , Fenótipo , Adenosina Trifosfatases , Proteína Agouti Sinalizadora/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Cromossômicas não Histona , Cruzamentos Genéticos , Análise Mutacional de DNA , Citometria de Fluxo , Componentes do Gene , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Mutação Puntual/genética , Alinhamento de Sequência , Espermatogênese/genéticaRESUMO
The oncogenic cluster miR-17-92 encodes seven related microRNAs that regulate cell proliferation, apoptosis and development. Expression of miR-17-92 cluster is decreased upon cell differentiation. Here, we report a novel mechanism of the regulation of miR-17-92 cluster. Using transgenic PU.1(-/-) myeloid progenitors we show that upon macrophage differentiation, the transcription factor PU.1 induces the secondary determinant Egr2 which, in turn, directly represses miR-17-92 expression by recruiting histone demethylase Jarid1b leading to histone H3 lysine K4 demethylation within the CpG island at the miR-17-92 promoter. Conversely, Egr2 itself is targeted by miR-17-92, indicating existence of mutual regulatory relationship between miR-17-92 and Egr2. Furthermore, restoring EGR2 levels in primary acute myeloid leukaemia blasts expressing elevated levels of miR-17-92 and low levels of PU.1 and EGR2 leads to downregulation of miR-17-92 and restored expression of its targets p21CIP1 and BIM. We propose that upon macrophage differentiation PU.1 represses the miR-17-92 cluster promoter by an Egr-2/Jarid1b-mediated H3K4 demethylation mechanism whose deregulation may contribute to leukaemic states.
Assuntos
Diferenciação Celular/genética , Epigênese Genética/fisiologia , Macrófagos/fisiologia , MicroRNAs/genética , Proteínas Proto-Oncogênicas/fisiologia , Transativadores/fisiologia , Animais , Sequência de Bases , Células Cultivadas , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/fisiologia , Inativação Gênica/fisiologia , Técnicas de Transferência de Genes , Células HL-60 , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases com o Domínio Jumonji/fisiologia , Macrófagos/metabolismo , Camundongos , MicroRNAs/metabolismo , Modelos Biológicos , Família Multigênica/genética , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Longo não Codificante , Proteínas Repressoras/metabolismo , Proteínas Repressoras/fisiologia , Homologia de Sequência do Ácido Nucleico , Transativadores/genética , Transativadores/metabolismo , TransfecçãoRESUMO
BACKGROUND: MicroRNAs (miRs) represent a distinct class of posttranscriptional modulators of gene expression with remarkable stability in sera. Several miRs are oncogenic (oncomiRs) and are deregulated in the pathogenesis of breast cancer and function to inhibit tumor suppressors. Routine blood monitoring of these circulating tumor-derived products could be of significant benefit to the diagnosis and relapse detection of early-stage breast cancer (EBC) patients. METHODS: Aim of this project was to determine expression of miR-155, miR-19a, miR-181b, miR-24, relative to let-7a in sera of 63 patients with EBC and 21 healthy controls. Longitudinal multivariate data analysis was performed to stochastically model the serum levels of each of the oncomiRs during disease phases: from diagnosis, after surgery, and following chemo/radiotherapy. Moreover, this analysis was utilized to evaluate oncomiR levels in EBC patients subgrouped using current clinical prognostic factors including HER2, Ki-67, and grade III. RESULTS: EBC patients significantly over-express the oncomiRs at the time of diagnosis. Following surgical resection the serum levels of miR-155, miR-181b, and miR-24 significantly decreased (p = 1.89e-05, 5.41e-06, and 0.00638, respectively) whereas the miR-19a decreased significantly after the therapy (p = 0.00869). Furthermore, in case of high-risk patients serum levels of miR-155, miR-19a, miR-181b, and miR-24 are significantly more abundant in comparison to low-risk group (p = 0.026, 0.02567, 0.0250, and 0.00990) and show a decreasing trend upon therapy. CONCLUSIONS: OncomiRs are significantly more abundant in the sera of EBC patients compared to controls at diagnosis. Differences in oncomiR levels reflecting EBC risk were also observed. Testing the oncomiRs may be useful for diagnostic purpose and possibly also for relapse detection in follow-up studies of EBC.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , MicroRNAs/sangue , MicroRNAs/genética , Adulto , Idoso , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Neoplasias da Mama/terapia , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , PrognósticoRESUMO
The formation of hematopoietic cells relies on the chromatin remodeling activities of ISWI ATPase SMARCA5 (SNF2H) and its complexes. The Smarca5 null and conditional alleles have been used to study its functions in embryonic and organ development in mice. These mouse model phenotypes vary from embryonic lethality of constitutive knockout to less severe phenotypes observed in tissue-specific Smarca5 deletions, e.g., in the hematopoietic system. Here we show that, in a gene dosage-dependent manner, the hypomorphic allele of SMARCA5 (S5tg) can rescue not only the developmental arrest in hematopoiesis in the hCD2iCre model but also the lethal phenotypes associated with constitutive Smarca5 deletion or Vav1iCre-driven conditional knockout in hematopoietic progenitor cells. Interestingly, the latter model also provided evidence for the role of SMARCA5 expression level in hematopoietic stem cells, as the Vav1iCre S5tg animals accumulate stem and progenitor cells. Furthermore, their hematopoietic stem cells exhibited impaired lymphoid lineage entry and differentiation. This observation contrasts with the myeloid lineage which is developing without significant disturbances. Our findings indicate that animals with low expression of SMARCA5 exhibit normal embryonic development with altered lymphoid entry within the hematopoietic stem cell compartment.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Camundongos , Animais , Células-Tronco Hematopoéticas/metabolismo , Hematopoese/genética , Diferenciação Celular/genética , Adenosina Trifosfatases/metabolismoRESUMO
Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In this study, we present evidence that MYB (v-myb myeloblastosis viral oncogene homolog) is overexpressed in a subset of B-CLL patients. MYB physically associates with the promoter of miR-155 host gene (MIR155HG, also known as BIC, B-cell integration cluster) and stimulates its transcription. This coincides with the hypermethylated histone H3K4 residue and spread hyperacetylation of H3K9 at MIR155HG promoter. Our data provide evidence of oncogenic activities of MYB in B-CLL that include its stimulatory role in MIR155HG transcription.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , MicroRNAs/genética , Proteínas Oncogênicas v-myb/fisiologia , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Células HeLa , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Análise em Microsséries , Proteínas Oncogênicas v-myb/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transcrição Gênica/fisiologia , Transfecção , Células Tumorais CultivadasRESUMO
Chromatin remodeling complexes are required for many distinct nuclear processes such as transcription, DNA replication, and DNA repair. However, the contribution of these complexes to the development of complex tissues within an organism is poorly characterized. Imitation switch (ISWI) proteins are among the most evolutionarily conserved ATP-dependent chromatin remodeling factors and are represented by yeast Isw1/Isw2, and their vertebrate counterparts Snf2h (Smarca5) and Snf2l (Smarca1). In this study, we focused on the role of the Snf2h gene during the development of the mammalian retina. We show that Snf2h is expressed in both retinal progenitors and post-mitotic retinal cells. Using Snf2h conditional knockout mice (Snf2h cKO), we found that when Snf2h is deleted, the laminar structure of the adult retina is not retained, the overall thickness of the retina is significantly reduced compared with controls, and the outer nuclear layer (ONL) is completely missing. The depletion of Snf2h did not influence the ability of retinal progenitors to generate all the differentiated retinal cell types. Instead, the Snf2h function is critical for the proliferation of retinal progenitor cells. Cells lacking Snf2h have a defective S-phase, leading to the entire cell division process impairments. Although all retinal cell types appear to be specified in the absence of the Snf2h function, cell-cycle defects and concomitantly increased apoptosis in Snf2h cKO result in abnormal retina lamination, complete destruction of the photoreceptor layer, and consequently, a physiologically non-functional retina.
Assuntos
Adenosina Trifosfatases , Montagem e Desmontagem da Cromatina , Cromatina , Proteínas Cromossômicas não Histona , Animais , Camundongos , Núcleo Celular/metabolismo , Proliferação de Células , Cromatina/metabolismo , Camundongos Knockout , Retina , Proteínas Cromossômicas não Histona/metabolismo , Adenosina Trifosfatases/metabolismoRESUMO
Chromatin remodeling complexes are required for many distinct nuclear processes such as transcription, DNA replication and DNA repair. However, how these complexes contribute to the development of complex tissues within an organism is poorly characterized. Imitation switch (ISWI) proteins are among the most evolutionarily conserved ATP-dependent chromatin remodeling factors and are represented by yeast Isw1/Isw2, and their vertebrate counterparts Snf2h (Smarca5) and Snf2l (Smarca1). In this study, we focused on the role of the Snf2h gene during development of the mammalian retina. We show that Snf2h is expressed in both retinal progenitors and post-mitotic retinal cells. Using Snf2h conditional knockout mice ( Snf2h cKO), we found that when Snf2h is deleted the laminar structure of the adult retina is not retained, the overall thickness of the retina is significantly reduced compared with controls, and the outer nuclear layer (ONL) is completely missing. Depletion of Snf2h did not influence the ability of retinal progenitors to generate all of the differentiated retinal cell types. Instead, Snf2h function is critical for proliferation of retinal progenitor cells. Cells lacking Snf2h have a defective S-phase, leading to the entire cell division process impairments. Although, all retinal cell types appear to be specified in the absence of Snf2h function, cell cycle defects and concomitantly increased apoptosis in Snf2h cKO result in abnormal retina lamination, complete destruction of the photoreceptor layer and, consequently, in a physiologically non-functional retina.
RESUMO
Smarca5, an ATPase of the ISWI class of chromatin remodelers, is a key regulator of chromatin structure, cell cycle and DNA repair. Smarca5 is deregulated in leukemia and breast, lung and gastric cancers. However, its role in oncogenesis is not well understood. Chromatin remodelers often play dosage-dependent roles in cancer. We therefore investigated the epigenomic and phenotypic impact of controlled stepwise attenuation of Smarca5 function in the context of primary cell transformation, a process relevant to tumor formation. Upon conditional single- or double-allele Smarca5 deletion, the cells underwent both accelerated growth arrest and senescence entry and displayed gradually increased sensitivity to genotoxic insults. These phenotypic characteristics were explained by specific remodeling of the chromatin structure and the transcriptome in primary cells prior to the immortalization onset. These molecular programs implicated Smarca5 requirement in DNA damage repair, telomere maintenance, cell cycle progression and in restricting apoptosis and cellular senescence. Consistent with the molecular programs, we demonstrate for the first time that Smarca5-deficient primary cells exhibit dramatically decreased capacity to bypass senescence and immortalize, an indispensable step during cell transformation and cancer development. Thus, Smarca5 plays a crucial role in key homeostatic processes and sustains cancer-promoting molecular programs and cellular phenotypes.
Assuntos
Cromatina , Neoplasias , Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Dano ao DNA , Reparo do DNARESUMO
TRIAL REGISTRATION: GA trial is registered under EudraCT#: 2013-001639-38.
Assuntos
Azacitidina , Síndromes Mielodisplásicas , Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Fator Estimulador de Colônias de Granulócitos , Humanos , Síndromes Mielodisplásicas/tratamento farmacológicoRESUMO
Lymphoma with secondary central nervous system (CNS) involvement represents one of the most aggressive malignancies, with poor prognosis and high mortality. New diagnostic tools for its early detection, response evaluation, and CNS relapse prediction are needed. We analyzed circulating microRNAs in the cerebrospinal fluid (CSF) and plasma of 162 patients with aggressive B-cell non-Hodgkin's lymphomas (B-NHL) and compared their levels in CNS-involving lymphomas versus in systemic lymphomas, at diagnosis and during treatment and CNS relapse. We identified a set of five oncogenic microRNAs (miR-19a, miR-20a, miR-21, miR-92a, and miR-155) in CSF that detect, with high sensitivity, secondary CNS lymphoma involvement in aggressive B-NHL, including DLBCL, MCL, and Burkitt lymphoma. Their combination into an oncomiR index enables the separation of CNS lymphomas from systemic lymphomas or nonmalignant controls with high sensitivity and specificity, and high Receiver Operating Characteristics (DLBCL AUC = 0.96, MCL = 0.93, BL = 1.0). Longitudinal analysis showed that oncomiR levels reflect treatment efficacy and clinical outcomes, allowing their monitoring and prediction. In contrast to conventional methods, CSF oncomiRs enable detection of early and residual CNS involvement, as well as parenchymal involvement. These circulating oncomiRs increase 1-4 months before CNS relapse, allowing its early detection and improving the prediction of CNS relapse risk in DLBCL. Similar effects were detectable, to a lesser extent, in plasma.