Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Front Immunol ; 10: 878, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31105700

RESUMO

Common Variable Immunodeficiency (CVID) is characterized by impaired antibody production and poor terminal differentiation of the B cell compartment, yet its pathogenesis is still poorly understood. We first reported the occurrence of epigenetic alterations in CVID by high-throughput methylation analysis in CVID-discordant monozygotic twins. Data from a recent whole DNA methylome analysis throughout different stages of normal B cell differentiation allowed us to design a new experimental approach. We selected CpG sites for analysis based on two criteria: one, CpGs with potential association with the transcriptional status of relevant genes for B cell activation and differentiation; and two, CpGs that undergo significant demethylation from naïve to memory B cells in healthy individuals. DNA methylation was analyzed by bisulfite pyrosequencing of specific CpG sites in sorted naïve and memory B cell subsets from CVID patients and healthy donors. We observed impaired demethylation in two thirds of the selected CpGs in CVID memory B cells, in genes that govern B cell-specific processes or participate in B cell signaling. The degree of demethylation impairment associated with the extent of the memory B cell reduction. The impaired demethylation in such functionally relevant genes as AICDA in switched memory B cells correlated with a lower proliferative rate. Our new results reinforce the hypothesis of altered demethylation during B cell differentiation as a contributing pathogenic mechanism to the impairment of B cell function and maturation in CVID. In particular, deregulated epigenetic control of AICDA could play a role in the defective establishment of a post-germinal center B cell compartment in CVID.

2.
Haematologica ; 104(8): 1572-1579, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30655376

RESUMO

In this study we interrogated the DNA methylome of myelofibrosis patients using high-density DNA methylation arrays. We detected 35,215 differentially methylated CpG, corresponding to 10,253 genes, between myelofibrosis patients and healthy controls. These changes were present both in primary and secondary myelofibrosis, which showed no differences between them. Remarkably, most differentially methylated CpG were located outside gene promoter regions and showed significant association with enhancer regions. This aberrant enhancer hypermethylation was negatively correlated with the expression of 27 genes in the myelofibrosis cohort. Of these, we focused on the ZFP36L1 gene and validated its decreased expression and enhancer DNA hypermethylation in an independent cohort of patients and myeloid cell-lines. In vitro reporter assay and 5'-azacitidine treatment confirmed the functional relevance of hyper-methylation of ZFP36L1 enhancer. Furthermore, in vitro rescue of ZFP36L1 expression had an impact on cell proliferation and induced apoptosis in SET-2 cell line indicating a possible role of ZFP36L1 as a tumor suppressor gene in myelofibrosis. Collectively, we describe the DNA methylation profile of myelofibrosis, identifying extensive changes in enhancer elements and revealing ZFP36L1 as a novel candidate tumor suppressor gene.

3.
Nat Med ; 24(6): 868-880, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29785028

RESUMO

Chronic lymphocytic leukemia (CLL) is a frequent hematological neoplasm in which underlying epigenetic alterations are only partially understood. Here, we analyze the reference epigenome of seven primary CLLs and the regulatory chromatin landscape of 107 primary cases in the context of normal B cell differentiation. We identify that the CLL chromatin landscape is largely influenced by distinct dynamics during normal B cell maturation. Beyond this, we define extensive catalogues of regulatory elements de novo reprogrammed in CLL as a whole and in its major clinico-biological subtypes classified by IGHV somatic hypermutation levels. We uncover that IGHV-unmutated CLLs harbor more active and open chromatin than IGHV-mutated cases. Furthermore, we show that de novo active regions in CLL are enriched for NFAT, FOX and TCF/LEF transcription factor family binding sites. Although most genetic alterations are not associated with consistent epigenetic profiles, CLLs with MYD88 mutations and trisomy 12 show distinct chromatin configurations. Furthermore, we observe that non-coding mutations in IGHV-mutated CLLs are enriched in H3K27ac-associated regulatory elements outside accessible chromatin. Overall, this study provides an integrative portrait of the CLL epigenome, identifies extensive networks of altered regulatory elements and sheds light on the relationship between the genetic and epigenetic architecture of the disease.


Assuntos
Cromatina/metabolismo , Epigenômica , Leucemia Linfocítica Crônica de Células B/genética , Linfócitos B/metabolismo , Sequência de Bases , Estudos de Coortes , Humanos
4.
Clin Cancer Res ; 24(6): 1355-1363, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29351917

RESUMO

Purpose: The classification of medulloblastoma into WNT, SHH, group 3, and group 4 subgroups has become of critical importance for patient risk stratification and subgroup-tailored clinical trials. Here, we aimed to develop a simplified, clinically applicable classification approach that can be implemented in the majority of centers treating patients with medulloblastoma.Experimental Design: We analyzed 1,577 samples comprising previously published DNA methylation microarray data (913 medulloblastomas, 457 non-medulloblastoma tumors, 85 normal tissues), and 122 frozen and formalin-fixed paraffin-embedded medulloblastoma samples. Biomarkers were identified applying stringent selection filters and Linear Discriminant Analysis (LDA) method, and validated using DNA methylation microarray data, bisulfite pyrosequencing, and direct-bisulfite sequencing.Results: Using a LDA-based approach, we developed and validated a prediction method (EpiWNT-SHH classifier) based on six epigenetic biomarkers that allowed for rapid classification of medulloblastoma into the clinically relevant subgroups WNT, SHH, and non-WNT/non-SHH with excellent concordance (>99%) with current gold-standard methods, DNA methylation microarray, and gene signature profiling analysis. The EpiWNT-SHH classifier showed high prediction capacity using both frozen and formalin-fixed material, as well as diverse DNA methylation detection methods. Similarly, we developed a classifier specific for group 3 and group 4 tumors, based on five biomarkers (EpiG3-G4) with good discriminatory capacity, allowing for correct assignment of more than 92% of tumors. EpiWNT-SHH and EpiG3-G4 methylation profiles remained stable across tumor primary, metastasis, and relapse samples.Conclusions: The EpiWNT-SHH and EpiG3-G4 classifiers represent a new simplified approach for accurate, rapid, and cost-effective molecular classification of single medulloblastoma DNA samples, using clinically applicable DNA methylation detection methods. Clin Cancer Res; 24(6); 1355-63. ©2018 AACR.


Assuntos
Biomarcadores Tumorais , Neoplasias Cerebelares/diagnóstico , Neoplasias Cerebelares/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Meduloblastoma/diagnóstico , Meduloblastoma/genética , Biópsia , Ilhas de CpG , Metilação de DNA , Epigênese Genética , Epigenômica/métodos , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Reprodutibilidade dos Testes
5.
Cancer Cell ; 30(5): 806-821, 2016 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-27846393

RESUMO

We analyzed the in silico purified DNA methylation signatures of 82 mantle cell lymphomas (MCL) in comparison with cell subpopulations spanning the entire B cell lineage. We identified two MCL subgroups, respectively carrying epigenetic imprints of germinal-center-inexperienced and germinal-center-experienced B cells, and we found that DNA methylation profiles during lymphomagenesis are largely influenced by the methylation dynamics in normal B cells. An integrative epigenomic approach revealed 10,504 differentially methylated regions in regulatory elements marked by H3K27ac in MCL primary cases, including a distant enhancer showing de novo looping to the MCL oncogene SOX11. Finally, we observed that the magnitude of DNA methylation changes per case is highly variable and serves as an independent prognostic factor for MCL outcome.


Assuntos
Metilação de DNA , Elementos Facilitadores Genéticos , Epigenômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Linfoma de Célula do Manto/genética , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula , Simulação por Computador , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores de Transcrição SOXC/genética
6.
Cell Rep ; 13(5): 1059-71, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26565917

RESUMO

Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-?1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxy)methylation, and cell fate determination.


Assuntos
Ciclo Celular , Metilação de DNA , Linfopoese , Plasmócitos/citologia , Células Cultivadas , Humanos , Plasmócitos/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Receptores de IgE/genética , Receptores de IgE/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
7.
Nat Genet ; 47(11): 1316-1325, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26437030

RESUMO

Although Burkitt lymphomas and follicular lymphomas both have features of germinal center B cells, they are biologically and clinically quite distinct. Here we performed whole-genome bisulfite, genome and transcriptome sequencing in 13 IG-MYC translocation-positive Burkitt lymphoma, nine BCL2 translocation-positive follicular lymphoma and four normal germinal center B cell samples. Comparison of Burkitt and follicular lymphoma samples showed differential methylation of intragenic regions that strongly correlated with expression of associated genes, for example, genes active in germinal center dark-zone and light-zone B cells. Integrative pathway analyses of regions differentially methylated in Burkitt and follicular lymphomas implicated DNA methylation as cooperating with somatic mutation of sphingosine phosphate signaling, as well as the TCF3-ID3 and SWI/SNF complexes, in a large fraction of Burkitt lymphomas. Taken together, our results demonstrate a tight connection between somatic mutation, DNA methylation and transcriptional control in key B cell pathways deregulated differentially in Burkitt lymphoma and other germinal center B cell lymphomas.


Assuntos
Linfoma de Burkitt/genética , Metilação de DNA , Linfoma Folicular/genética , Mutação , Transcriptoma/genética , Adolescente , Adulto , Idoso , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Criança , Pré-Escolar , Feminino , Genoma Humano/genética , Centro Germinativo/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/genética , Translocação Genética , Adulto Jovem
8.
Nat Genet ; 47(7): 746-56, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26053498

RESUMO

We analyzed the DNA methylome of ten subpopulations spanning the entire B cell differentiation program by whole-genome bisulfite sequencing and high-density microarrays. We observed that non-CpG methylation disappeared upon B cell commitment, whereas CpG methylation changed extensively during B cell maturation, showing an accumulative pattern and affecting around 30% of all measured CpG sites. Early differentiation stages mainly displayed enhancer demethylation, which was associated with upregulation of key B cell transcription factors and affected multiple genes involved in B cell biology. Late differentiation stages, in contrast, showed extensive demethylation of heterochromatin and methylation gain at Polycomb-repressed areas, and genes with apparent functional impact in B cells were not affected. This signature, which has previously been linked to aging and cancer, was particularly widespread in mature cells with an extended lifespan. Comparing B cell neoplasms with their normal counterparts, we determined that they frequently acquire methylation changes in regions already undergoing dynamic methylation during normal B cell differentiation.


Assuntos
Linfócitos B/fisiologia , Metilação de DNA , Epigênese Genética/imunologia , Sequência de Bases , Diferenciação Celular , Células Cultivadas , Ilhas de CpG , Regulação Leucêmica da Expressão Gênica , Genoma Humano , Humanos , Leucemia de Células B/genética , Análise de Sequência de DNA
9.
Genome Res ; 25(4): 478-87, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25644835

RESUMO

While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM.


Assuntos
Metilação de DNA/genética , Elementos Facilitadores Genéticos/genética , Mieloma Múltiplo/genética , Células-Tronco Neoplásicas/citologia , Plasmócitos/citologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , Ilhas de CpG/genética , DNA de Neoplasias/genética , Regulação para Baixo/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
10.
Genome Res ; 24(2): 212-26, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24265505

RESUMO

Chronic lymphocytic leukemia (CLL) has heterogeneous clinical and biological behavior. Whole-genome and -exome sequencing has contributed to the characterization of the mutational spectrum of the disease, but the underlying transcriptional profile is still poorly understood. We have performed deep RNA sequencing in different subpopulations of normal B-lymphocytes and CLL cells from a cohort of 98 patients, and characterized the CLL transcriptional landscape with unprecedented resolution. We detected thousands of transcriptional elements differentially expressed between the CLL and normal B cells, including protein-coding genes, noncoding RNAs, and pseudogenes. Transposable elements are globally derepressed in CLL cells. In addition, two thousand genes-most of which are not differentially expressed-exhibit CLL-specific splicing patterns. Genes involved in metabolic pathways showed higher expression in CLL, while genes related to spliceosome, proteasome, and ribosome were among the most down-regulated in CLL. Clustering of the CLL samples according to RNA-seq derived gene expression levels unveiled two robust molecular subgroups, C1 and C2. C1/C2 subgroups and the mutational status of the immunoglobulin heavy variable (IGHV) region were the only independent variables in predicting time to treatment in a multivariate analysis with main clinico-biological features. This subdivision was validated in an independent cohort of patients monitored through DNA microarrays. Further analysis shows that B-cell receptor (BCR) activation in the microenvironment of the lymph node may be at the origin of the C1/C2 differences.


Assuntos
Linfócitos B , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Leucemia Linfocítica Crônica de Células B/genética , Idoso , Sequência de Bases , Feminino , Perfilação da Expressão Gênica , Humanos , Região Variável de Imunoglobulina , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Ribossomos/genética , Spliceossomos/genética
11.
PLoS One ; 8(8): e74885, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24015320

RESUMO

ERH is a small, highly evolutionarily conserved nuclear protein of unknown function. Its three-dimensional structure is absolutely unique and it can form a homodimer through a ß sheet surface. ERH has been shown to interact, among others, with PDIP46/SKAR and Ciz1. When coexpressed with the latter protein, ERH accumulates in replication foci in the nucleus of HeLa cells. Here, we report that when ERH is coexpressed with PDIP46/SKAR in HeLa cells, it is recruited to nuclear speckles, and identify amino acid residues critical for targeting ERH to both these subnuclear structures. ERH H3A Q9A shows a diminished recruitment to nuclear speckles but it is recruited to replication foci. ERH E37A T51A is very poorly recruited to replication foci while still accumulating in nuclear speckles. Consequently, ERH H3A Q9A E37A T51A is recruited neither to nuclear speckles nor to replication foci. The lack of interactions of these three ERH forms with PDIP46/SKAR and/or Ciz1 was further confirmed in vitro by GST pull-down assay. The residues whose substitutions interfere with the accumulation in nuclear speckles are situated on the ß sheet surface of ERH, indicating that only the monomer of ERH can interact with PDIP46/SKAR. Substitutions affecting the recruitment to replication foci map to the other side of ERH, near a long loop between the α1 and α2 helices, thus both the monomer and the dimer of ERH could interact with Ciz1. The construction of the ERH mutants not recruited to nuclear speckles or replication foci will facilitate further studies on ERH actions in these subnuclear structures.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Estruturas do Núcleo Celular/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Substituição de Aminoácidos , Proteínas de Ciclo Celular/genética , Estruturas do Núcleo Celular/genética , Células HeLa , Humanos , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Secundária de Proteína , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/genética
12.
Biochim Biophys Acta ; 1829(11): 1161-74, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23938249

RESUMO

Ever since the discovery of DNA methylation at cytosine residues, the role of this so called fifth base has been extensively studied and debated. Until recently, the majority of DNA methylation studies focused on the analysis of CpG islands associated to promoter regions. However, with the upcoming possibilities to study DNA methylation in a genome-wide context, this epigenetic mark can now be studied in an unbiased manner. As a result, recent studies have shown that not only promoters but also intragenic and intergenic regions are widely modulated during physiological processes and disease. In particular, it is becoming increasingly clear that DNA methylation in the gene body is not just a passive witness of gene transcription but it seems to be actively involved in multiple gene regulation processes. In this review we discuss the potential role of intragenic DNA methylation in alternative promoter usage, regulation of short and long non-coding RNAs, alternative RNA processing, as well as enhancer activity. Furthermore, we summarize how the intragenic DNA methylome is modified both during normal cell differentiation and neoplastic transformation.


Assuntos
Diferenciação Celular/genética , Metilação de DNA , Regulação da Expressão Gênica , Neoplasias/genética , Transcrição Genética , Elementos de DNA Transponíveis , Elementos Facilitadores Genéticos , Humanos , Neoplasias/patologia , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA
13.
PLoS One ; 7(11): e48401, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23144874

RESUMO

Neuroblastoma (NB) pathogenesis has been reported to be closely associated with numerous genetic alterations. However, underlying DNA methylation patterns have not been extensively studied in this developmental malignancy. Here, we generated microarray-based DNA methylation profiles of primary neuroblastic tumors. Stringent supervised differential methylation analyses allowed us to identify epigenetic changes characteristic for NB tumors as well as for clinical and biological subtypes of NB. We observed that gene-specific loss of DNA methylation is more prevalent than promoter hypermethylation. Remarkably, such hypomethylation affected cancer-related biological functions and genes relevant to NB pathogenesis such as CCND1, SPRR3, BTC, EGF and FGF6. In particular, differential methylation in CCND1 affected mostly an evolutionary conserved functionally relevant 3' untranslated region, suggesting that hypomethylation outside promoter regions may play a role in NB pathogenesis. Hypermethylation targeted genes involved in cell development and proliferation such as RASSF1A, POU2F2 or HOXD3, among others. The results derived from this study provide new candidate epigenetic biomarkers associated with NB as well as insights into the molecular pathogenesis of this tumor, which involves a marked gene-specific hypomethylation.


Assuntos
Metilação de DNA/genética , Genes Neoplásicos/genética , Neuroblastoma/genética , Criança , Pré-Escolar , Ciclina D1/genética , Ciclina D1/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Temperatura Ambiente
14.
Nat Genet ; 44(11): 1236-42, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23064414

RESUMO

We have extensively characterized the DNA methylomes of 139 patients with chronic lymphocytic leukemia (CLL) with mutated or unmutated IGHV and of several mature B-cell subpopulations through the use of whole-genome bisulfite sequencing and high-density microarrays. The two molecular subtypes of CLL have differing DNA methylomes that seem to represent epigenetic imprints from distinct normal B-cell subpopulations. DNA hypomethylation in the gene body, targeting mostly enhancer sites, was the most frequent difference between naive and memory B cells and between the two molecular subtypes of CLL and normal B cells. Although DNA methylation and gene expression were poorly correlated, we identified gene-body CpG dinucleotides whose methylation was positively or negatively associated with expression. We have also recognized a DNA methylation signature that distinguishes new clinico-biological subtypes of CLL. We propose an epigenomic scenario in which differential methylation in the gene body may have functional and clinical implications in leukemogenesis.


Assuntos
Linfócitos B/metabolismo , Metilação de DNA/genética , Epigênese Genética/genética , Leucemia Linfocítica Crônica de Células B/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo , Ilhas de CpG/genética , Feminino , Regulação da Expressão Gênica , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade
15.
Bioorg Med Chem ; 20(5): 1699-710, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22316555

RESUMO

We describe synthesis and properties of eight dinucleotide mRNA 5' cap analogs containing imidodiphosphate moiety within 5',5'-tri- or tetraphosphate bridge (NH-analogs). The compounds were obtained by coupling an appropriate nucleoside 5'-imidodiphosphate with nucleotide P-imidazolide mediated by divalent metal chloride in anhydrous DMF. To evaluate the novel compounds as tools for studying cap-dependent processes, we determined their binding affinities for eukaryotic translation initiation factor 4E, susceptibilities to decapping pyrophosphatase DcpS and, for non-hydrolysable analogs, binding affinities to this enzyme. The results indicate that the O to NH substitution in selected positions of oligophosphate bridge ensures resistance to enzymatic decapping and suggest that interactions of NH-analogs with cap binding proteins fairly mimic interactions of unmodified parent compounds. Finally, we identified NH-analogs as potent inhibitors of cap-dependent translation in cell free system, and evaluated their utility as reagents for obtaining 5' capped mRNAs in vitro to be rather moderate.


Assuntos
Difosfonatos/química , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/síntese química , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Humanos , Hidrólise , Ligação Proteica , Biossíntese de Proteínas , Análogos de Capuz de RNA/metabolismo
16.
RNA ; 17(5): 978-88, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21447710

RESUMO

Decapping is an essential step in multiple pathways of mRNA degradation. Previously, we synthesized mRNAs containing caps that were resistant to decapping, both to dissect the various pathways for mRNA degradation and to stabilize mRNA for more sustained protein expression. mRNAs containing an α-ß CH(2) group are resistant to in vitro cleavage by the decapping enzyme hDcp2 but poorly translated. mRNAs containing an S substitution at the ß-phosphate are well translated but only partially resistant to hDcp2. We now describe seven new cap analogs substituted at the ß-phosphate with BH(3) or Se, or substituted at either the α-ß or ß-γ O with NH. The analogs differ in affinity for eIF4E and efficiency of in vitro incorporation into mRNA by T7 RNA polymerase. Luciferase mRNAs capped with these analogs differ in resistance to hDcp2 hydrolysis in vitro, translational efficiency in rabbit reticulocyte lysate and in HeLa cells, and stability in HeLa cells. Whereas mRNAs capped with m(2)(7,2'-O)Gpp(S)pG were previously found to have the most favorable properties of translational efficiency and stability in mammalian cells, mRNAs capped with m(7)Gpp(BH3)pm(7)G are translated with the same efficiency but are more stable. Interestingly, some mRNAs exhibit a lag of up to 60 min before undergoing first-order decay (t(1/2) ≅ 25 min). Only mRNAs that are efficiently capped, resistant to decapping in vitro, and actively translated have long lag phases.


Assuntos
Ácidos Bóricos/metabolismo , Compostos de Nitrogênio/metabolismo , Polifosfatos/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/análise , Selênio/metabolismo , Animais , RNA Polimerases Dirigidas por DNA/metabolismo , Endorribonucleases/metabolismo , Células HeLa , Humanos , Camundongos , Estrutura Molecular , Polifosfatos/química , Capuzes de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Coelhos , Reticulócitos/química , Estereoisomerismo , Especificidade por Substrato , Proteínas Virais/metabolismo
17.
Adv Genet ; 70: 27-56, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20920744

RESUMO

DNA methylation is one of the most intensely studied epigenetic modifications in mammals. In normal cells, it assures the proper regulation of gene expression and stable gene silencing. DNA methylation is associated with histone modifications and the interplay of these epigenetic modifications is crucial to regulate the functioning of the genome by changing chromatin architecture. The covalent addition of a methyl group occurs generally in cytosine within CpG dinucleotides which are concentrated in large clusters called CpG islands. DNA methyltransferases are responsible for establishing and maintenance of methylation pattern. It is commonly known that inactivation of certain tumor-suppressor genes occurs as a consequence of hypermethylation within the promoter regions and a numerous studies have demonstrated a broad range of genes silenced by DNA methylation in different cancer types. On the other hand, global hypomethylation, inducing genomic instability, also contributes to cell transformation. Apart from DNA methylation alterations in promoter regions and repetitive DNA sequences, this phenomenon is associated also with regulation of expression of noncoding RNAs such as microRNAs that may play role in tumor suppression. DNA methylation seems to be promising in putative translational use in patients and hypermethylated promoters may serve as biomarkers. Moreover, unlike genetic alterations, DNA methylation is reversible what makes it extremely interesting for therapy approaches. The importance of DNA methylation alterations in tumorigenesis encourages us to decode the human epigenome. Different DNA methylome mapping techniques are indispensable to realize this project in the future.


Assuntos
Metilação de DNA/genética , DNA de Neoplasias/metabolismo , Epigênese Genética , Neoplasias/genética , Neoplasias/terapia , Animais , Antineoplásicos/uso terapêutico , Azacitidina/análogos & derivados , Azacitidina/uso terapêutico , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , DNA de Neoplasias/genética , Decitabina , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Supressores de Tumor , Humanos
18.
Epigenetics ; 5(7): 656-63, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20716963

RESUMO

MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate post-transcriptional gene expression. They influence a wide range of physiological functions, including neuronal processes, and are regulated by various mechanisms, such as DNA methylation. This epigenetic mark is recognized by transcriptional regulators such as the methyl CpG binding protein Mecp2. Rett syndrome is a complex neurological disorder that has been associated with mutations in the gene coding for Mecp2. Thus, we examined the possible miRNA misregulation caused by Mecp2 absence in a mouse model of Rett syndrome. Using miRNA expression microarrays, we observed that the brain of Rett syndrome mice undergoes a disruption of the expression profiles of miRNAs. Among the significantly altered miRNAs (26%, 65 of 245), overall downregulation of these transcripts was the most common feature (71%), whilst the remaining 30% were upregulated. Further validation by quantitative RT-PCR demonstrated that the most commonly disrupted miRNAs were miR-146a, miR-146b, miR-130, miR-122a, miR-342 and miR-409 (downregulated), and miR-29b, miR329, miR-199b, miR-382, miR-296, miR-221 and miR-92 (upregulated). Most importantly, transfection of miR-146a in a neuroblastoma cell line caused the downregulation of IL-1 receptor-associated kinase 1 (Irak1) levels, suggesting that the identified defect of miR-146a in Rett syndrome mice brains might be responsible for the observed upregulation of Irak1 in this model of the human disease. Overall, we provide another level of molecular deregulation occurring in Rett syndrome that might be useful for understanding the disease and for designing targeted therapies.


Assuntos
Proteína 2 de Ligação a Metil-CpG/deficiência , Proteína 2 de Ligação a Metil-CpG/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Animais , Sequência de Bases , Encéfalo/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , Metilação de DNA , Primers do DNA/genética , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Processamento Pós-Transcricional do RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
FEBS J ; 275(2): 332-40, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18081865

RESUMO

Enhancer of rudimentary homolog (Drosophila) (ERH) is a small, highly conserved, nuclear protein with a unique three-dimensional structure, whose gene has been identified in animals, plants and protists, but not in fungi. Involvement of ERH in fundamental processes such as regulation of pyrimidine metabolism, cell cycle progression, transcription and cell growth control has been suggested. Here, employing a yeast two-hybrid system, a glutathione S-transferase pull-down assay and tandem MS, we demonstrate that Ciz1 is a bona fide interactor of human ERH. Ciz1 is a nuclear zinc finger protein interacting with p21(Cip1/Waf1), a universal inhibitor of cyclin-dependent kinases, and is a DNA replication factor. The region of Ciz1 necessary for the interaction with ERH spans residues 531-644, encompassing its first zinc finger motif. This region overlaps the p21(Cip1/Waf1)-binding site, suggesting that the interaction with ERH could block the binding of p21(Cip1/Waf1) by Ciz1 in the cell. When ERH and Ciz1 are coexpressed in HeLa cells, Ciz1 recruits ERH to DNA replication foci.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Ciclo Celular/química , Primers do DNA , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/química , Ligação Proteica , Espectrometria de Massas em Tandem , Fatores de Transcrição/química , Técnicas do Sistema de Duplo-Híbrido
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA