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1.
Nat Commun ; 9(1): 271, 2018 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-29348659

RESUMO

Bloom syndrome is a cancer predisposition disorder caused by mutations in the BLM helicase gene. Cells from persons with Bloom syndrome exhibit striking genomic instability characterized by excessive sister chromatid exchange events (SCEs). We applied single-cell DNA template strand sequencing (Strand-seq) to map the genomic locations of SCEs. Our results show that in the absence of BLM, SCEs in human and murine cells do not occur randomly throughout the genome but are strikingly enriched at coding regions, specifically at sites of guanine quadruplex (G4) motifs in transcribed genes. We propose that BLM protects against genome instability by suppressing recombination at sites of G4 structures, particularly in transcribed regions of the genome.


Assuntos
Síndrome de Bloom/genética , Quadruplex G , Neoplasias/etiologia , RecQ Helicases/metabolismo , Troca de Cromátide Irmã , Animais , Síndrome de Bloom/complicações , Linhagem Celular , Instabilidade Genômica , Humanos , Perda de Heterozigosidade , Camundongos
2.
Elife ; 62017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29231811

RESUMO

Homologous recombination involving sister chromatids is the most accurate, and thus most frequently used, form of recombination-mediated DNA repair. Despite its importance, sister chromatid recombination is not easily studied because it does not result in a change in DNA sequence, making recombination between sister chromatids difficult to detect. We have previously developed a novel DNA template strand sequencing technique, called Strand-seq, that can be used to map sister chromatid exchange (SCE) events genome-wide in single cells. An increase in the rate of SCE is an indicator of elevated recombination activity and of genome instability, which is a hallmark of cancer. In this study, we have adapted Strand-seq to detect SCE in the yeast Saccharomyces cerevisiae. We provide the first quantifiable evidence that most spontaneous SCE events in wild-type cells are not due to the repair of DNA double-strand breaks.


Assuntos
Genoma Fúngico , Biologia Molecular/métodos , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , Troca de Cromátide Irmã
4.
Genome Biol ; 17(1): 115, 2016 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-27246460

RESUMO

BACKGROUND: Chromosome instability leads to aneuploidy, a state in which cells have abnormal numbers of chromosomes, and is found in two out of three cancers. In a chromosomal instable p53 deficient mouse model with accelerated lymphomagenesis, we previously observed whole chromosome copy number changes affecting all lymphoma cells. This suggests that chromosome instability is somehow suppressed in the aneuploid lymphomas or that selection for frequently lost/gained chromosomes out-competes the CIN-imposed mis-segregation. RESULTS: To distinguish between these explanations and to examine karyotype dynamics in chromosome instable lymphoma, we use a newly developed single-cell whole genome sequencing (scWGS) platform that provides a complete and unbiased overview of copy number variations (CNV) in individual cells. To analyse these scWGS data, we develop AneuFinder, which allows annotation of copy number changes in a fully automated fashion and quantification of CNV heterogeneity between cells. Single-cell sequencing and AneuFinder analysis reveals high levels of copy number heterogeneity in chromosome instability-driven murine T-cell lymphoma samples, indicating ongoing chromosome instability. Application of this technology to human B cell leukaemias reveals different levels of karyotype heterogeneity in these cancers. CONCLUSION: Our data show that even though aneuploid tumours select for particular and recurring chromosome combinations, single-cell analysis using AneuFinder reveals copy number heterogeneity. This suggests ongoing chromosome instability that other platforms fail to detect. As chromosome instability might drive tumour evolution, karyotype analysis using single-cell sequencing technology could become an essential tool for cancer treatment stratification.


Assuntos
Heterogeneidade Genética , Cariótipo , Neoplasias/genética , Análise de Célula Única , Aneuploidia , Animais , Instabilidade Cromossômica , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Biologia Computacional , Variações do Número de Cópias de DNA , Humanos , Camundongos , Camundongos Knockout , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Análise de Célula Única/métodos , Software
5.
Genome Biol ; 17(1): 116, 2016 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-27246599

RESUMO

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease of the brain and the most common form of dementia in the elderly. Aneuploidy, a state in which cells have an abnormal number of chromosomes, has been proposed to play a role in neurodegeneration in AD patients. Several studies using fluorescence in situ hybridization have shown that the brains of AD patients contain an increased number of aneuploid cells. However, because the reported rate of aneuploidy in neurons ranges widely, a more sensitive method is needed to establish a possible role of aneuploidy in AD pathology. RESULTS: In the current study, we used a novel single-cell whole genome sequencing (scWGS) approach to assess aneuploidy in isolated neurons from the frontal cortex of normal control individuals (n = 6) and patients with AD (n = 10). The sensitivity and specificity of our method was shown by the presence of three copies of chromosome 21 in all analyzed neuronal nuclei of a Down's syndrome sample (n = 36). Very low levels of aneuploidy were found in the brains from control individuals (n = 589) and AD patients (n = 893). In contrast to other studies, we observe no selective gain of chromosomes 17 or 21 in neurons of AD patients. CONCLUSION: scWGS showed no evidence for common aneuploidy in normal and AD neurons. Therefore, our results do not support an important role for aneuploidy in neuronal cells in the pathogenesis of AD. This will need to be confirmed by future studies in larger cohorts.


Assuntos
Doença de Alzheimer/genética , Aneuploidia , Genoma Humano/genética , Neurônios/metabolismo , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/patologia
6.
PLoS One ; 8(9): e76647, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24073292

RESUMO

Processing of miRNAs occurs simultaneous with the transcription and splicing of their primary transcripts. For the small subset of exonic miRNAs it is unclear if the unspliced and/or spliced transcripts are used for miRNA biogenesis. We assessed endogenous levels and cellular location of primary transcripts of three exonic miRNAs. The ratio between unspliced and spliced transcripts varied markedly, i.e. >1 for BIC, <1 for pri-miR-146a and variable for pri-miR-22. Endogenous unspliced transcripts were located almost exclusively in the nucleus and thus available for miRNA processing for all three miRNAs. Endogenous spliced pri-miRNA transcripts were present both in the nucleus and in the cytoplasm and thus only partly available for miRNA processing. Overexpression of constructs containing the 5' upstream exonic or intronic sequence flanking pre-miR-155 resulted in strongly enhanced miR-155 levels, indicating that the flanking sequence does not affect processing efficiency. Exogenously overexpressed full-length spliced BIC transcripts were present both in the nucleus and in the cytoplasm, were bound by the Microprocessor complex and resulted in enhanced miR-155 levels. We conclude that both unspliced and spliced transcripts of exonic miRNAs can be used for pre-miRNA cleavage. Splicing and cytoplasmic transport of spliced transcripts may present a mechanism to regulate levels of exonic microRNAs.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Éxons/genética , Linfoma de Células B/genética , MicroRNAs/genética , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , Núcleo Celular/genética , Citoplasma/genética , Humanos , Imunoprecipitação , Íntrons/genética , Splicing de RNA/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares , Transcrição Gênica , Células Tumorais Cultivadas
7.
Methods ; 58(2): 113-7, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22836127

RESUMO

MicroRNA (miRNA) sponges are RNA molecules with repeated miRNA antisense sequences that can sequester miRNAs from their endogenous targets and thus serve as a decoy. Stably expressed miRNA sponges are especially valuable for long-term loss-of-function studies and can be used in vitro and in vivo. We describe here a straightforward method to generate retroviral miRNA sponge constructs using a single directional ligation reaction. This approach allows generation of sponges containing more than 20 miRNA binding sites. We provide a basis for the design of the sponge constructs with respect to the sequence of the miRNA binding site and the sequences flanking the miRNA binding sites. In-silico validation approaches are presented to test the predicted efficiencies of the sponges in comparison to known target genes. In addition, we describe in vitro validation experiments to confirm the effectiveness of the miRNA sponges. Finally, we describe how the here described procedure can be adapted to easily generate sponges that target multiple miRNAs simultaneously. In summary, our approach allows rapid generation of single or combination miRNA sponges that can be used for long-term miRNA loss-of-function studies.


Assuntos
MicroRNAs , RNA Antissenso , Regiões 3' não Traduzidas/genética , Sequência de Bases , Sítios de Ligação , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/isolamento & purificação , Dados de Sequência Molecular , RNA Antissenso/química , RNA Antissenso/genética
8.
PLoS One ; 7(1): e29275, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22238599

RESUMO

MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies.


Assuntos
Ligação Competitiva , Clonagem Molecular/métodos , MicroRNAs/antagonistas & inibidores , MicroRNAs/síntese química , Interferência de RNA , RNA Antissenso/síntese química , RNA Antissenso/metabolismo , Animais , Sequência de Bases , Ligação Competitiva/genética , Ligação Competitiva/fisiologia , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Interferência de RNA/fisiologia , RNA Antissenso/genética , Fatores de Tempo , Células Tumorais Cultivadas
9.
J Pathol ; 225(4): 609-17, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21953646

RESUMO

Hodgkin's lymphoma (HL) is a B cell-derived lymphoma characterized by a minority of malignant Hodgkin Reed-Sternberg (HRS) cells that have lost their normal B cell phenotype. Alterations in the cell cycle and apoptosis pathways might contribute to their resistance to apoptosis and sustained cell cycle progression. A key player in both cell cycle arrest and apoptosis is CDKN1A, encoding p21$^{{\rm{waf/cip1}}}$ (p21). P21 is regulated by p53 and can function as a cell cycle inhibitor when in the nucleus or as an apoptosis inhibitor when localized in the cytoplasm. We observed expression of p53, p21 and p-p21 in a variable number of HRS cells in 24 of 40 cases. Expression of miR-17 and miR-106a was detected in HRS cells of 10 HL cases. MiR-17/106b seed family members, CDKN1A RNA and p21 protein levels were variable in HL cell lines. We showed effective targeting of the CDKN1A 3' UTR by miR-17/106b in HL cell lines in a luciferase reporter assay and up-regulation of p21 protein levels upon anti-miR-17 treatment of KM-H2 cells. Functional studies indicated a p21-mediated G(1) arrest after miR-17/106b down-regulation in KM-H2, whereas no G(1) arrest was observed for U-HO1 and L428. This difference could not be explained by differences in the 3' UTR, the cellular location of p21 or expression variation during cell cycle progression. A strong correlation was observed for the miR-17/106b:CDKN1A ratio and the responsiveness to miR-17 inhibition, ie a low ratio in KM-H2 and an extremely high ratio in the two unresponsive HL cell lines. In conclusion, we show that miR-17/106b regulates p21 protein levels in HL and that the effect of miR-17/106b-mediated inhibition depends on the miRNA : target gene ratio. Thus, in HL high miR-17/106b expression contributes to a dysfunctional p53 pathway and thereby also to the malignant phenotype.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica , Doença de Hodgkin/genética , MicroRNAs/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citometria de Fluxo , Marcação de Genes , Doença de Hodgkin/metabolismo , Doença de Hodgkin/patologia , Humanos , Hibridização In Situ/métodos , MicroRNAs/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
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