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1.
J Biol Chem ; 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029477

RESUMO

DNA double-stranded breaks (DSBs) are strongly associated with active transcription, and promoter-proximal pausing of RNA polymerase II (Pol II) is a critical step in transcriptional regulation. Mapping the distribution of DSBs along actively expressed genes and identifying the location of DSBs relative to pausing sites can provide mechanistic insights into transcriptional regulation. Using genome-wide DNA break mapping/sequencing techniques at single-nucleotide resolution in human cells, here we found that DSBs are preferentially located around transcription start sites (TSSs) of highly transcribed and paused genes and that Pol II promoter-proximal pausing sites are enriched in DSBs. We observed that DSB frequency at pausing sites increases as the strength of pausing increases, regardless of whether the pausing sites are near or far from annotated TSSs. Inhibition of topoisomerases I and II by camptothecin and etoposide treatment, respectively, increased DSBs at the pausing sites as the concentrations of drugs increased, demonstrating the involvement of topoisomerases in DSB generation at the pausing sites. DNA breaks generated by topoisomerases are short-lived because of the re-ligation activity of these enzymes, which these drugs inhibit; therefore, the observation of increased DSBs with increasing drug doses at pausing sites indicated active recruitment of topoisomerases to these sites. Furthermore, the enrichment and locations of DSBs at pausing sites were shared among different cell types, suggesting that Pol II promoter-proximal pausing is a common regulatory mechanism. Our findings support a model in which topoisomerases participate in Pol II promoter-proximal pausing, and indicated that DSBs at pausing sites contribute to transcriptional activation.

2.
BMC Genomics ; 21(1): 25, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31914926

RESUMO

BACKGROUND: DNA double-stranded breaks (DSBs) are potentially deleterious events in a cell. The end structures (blunt, 3'- and 5'-overhangs) at DSB sites contribute to the fate of their repair and provide critical information concerning the consequences of the damage. Therefore, there has been a recent eruption of DNA break mapping and sequencing methods that aim to map at single-nucleotide resolution where breaks are generated genome-wide. These methods provide high resolution data for the location of DSBs, which can encode the type of end-structure present at these breaks. However, genome-wide analysis of the resulting end structures has not been investigated following these sequencing methods. RESULTS: To address this analysis gap, we develop the use of a coverage-normalized cross correlation analysis (CNCC) to process the high-precision genome-wide break mapping data, and determine genome-wide break end structure distributions at single-nucleotide resolution. We take advantage of the single-nucleotide position and the knowledge of strandness from every mapped break to analyze the relative shifts between positive and negative strand encoded break nucleotides. By applying CNCC we can identify the most abundant end structures captured by a break mapping technique, and further can make comparisons between different samples and treatments. We validate our analysis with restriction enzyme digestions of genomic DNA and establish the sensitivity of the analysis using end structures that only exist as a minor fraction of total breaks. Finally, we demonstrate the versatility of our analysis by applying CNCC to the breaks resulting after treatment with etoposide and study the variety of resulting end structures. CONCLUSION: For the first time, on a genome-wide scale, our analysis revealed the increase in the 5' to 3' end resection following etoposide treatment, and the global progression of the resection. Furthermore, our method distinguished the change in the pattern of DSB end structure with increasing doses of the drug. The ability of this method to determine DNA break end structures without a priori knowledge of break sequences or genomic position should have broad applications in understanding genome instability.

3.
Blood ; 135(1): 41-55, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31697823

RESUMO

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (<9 months from diagnosis), 65% in early relapse (9-36 months), and 32% in late relapse (>36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.

4.
Nucleic Acids Res ; 47(4): 1615-1627, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30576466

RESUMO

Antimalarial resistance is a major obstacle in the eradication of the human malaria parasite, Plasmodium falciparum. Genome amplifications, a type of DNA copy number variation (CNV), facilitate overexpression of drug targets and contribute to parasite survival. Long monomeric A/T tracks are found at the breakpoints of many Plasmodium resistance-conferring CNVs. We hypothesize that other proximal sequence features, such as DNA hairpins, act with A/T tracks to trigger CNV formation. By adapting a sequence analysis pipeline to investigate previously reported CNVs, we identified breakpoints in 35 parasite clones with near single base-pair resolution. Using parental genome sequence, we predicted the formation of stable hairpins within close proximity to all future breakpoint locations. Especially stable hairpins were predicted to form near five shared breakpoints, establishing that the initiating event could have occurred at these sites. Further in-depth analyses defined characteristics of these 'trigger sites' across the genome and detected signatures of error-prone repair pathways at the breakpoints. We propose that these two genomic signals form the initial lesion (hairpins) and facilitate microhomology-mediated repair (A/T tracks) that lead to CNV formation across this highly repetitive genome. Targeting these repair pathways in P. falciparum may be used to block adaptation to antimalarial drugs.


Assuntos
DNA/genética , Genômica , Plasmodium falciparum/genética , Análise de Sequência de DNA/métodos , DNA/química , Variações do Número de Cópias de DNA , Genoma de Protozoário/genética , Humanos , Malária Falciparum/parasitologia , Conformação de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico/genética
5.
Nat Commun ; 9(1): 4275, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30323222

RESUMO

Predicting the response and identifying additional targets that will improve the efficacy of chemotherapy is a major goal in cancer research. Through large-scale in vivo and in vitro CRISPR knockout screens in pancreatic ductal adenocarcinoma cells, we identified genes whose genetic deletion or pharmacologic inhibition synergistically increase the cytotoxicity of MEK signaling inhibitors. Furthermore, we show that CRISPR viability scores combined with basal gene expression levels could model global cellular responses to the drug treatment. We develop drug response evaluation by in vivo CRISPR screening (DREBIC) method and validated its efficacy using large-scale experimental data from independent experiments. Comparative analyses demonstrate that DREBIC predicts drug response in cancer cells from a wide range of tissues with high accuracy and identifies therapeutic vulnerabilities of cancer-causing mutations to MEK inhibitors in various cancer types.


Assuntos
Antineoplásicos/farmacologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Técnicas de Química Combinatória , Sistemas de Liberação de Medicamentos , Técnicas de Inativação de Genes , Testes Genéticos , Modelos Biológicos , Neoplasias Pancreáticas/genética , Animais , Pontos de Checagem do Ciclo Celular , Morte Celular , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Camundongos Nus , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Reprodutibilidade dos Testes
6.
BMC Cancer ; 18(1): 960, 2018 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-30305041

RESUMO

BACKGROUND: The cellular effects of androgen are transduced through the androgen receptor, which controls the expression of genes that regulate biosynthetic processes, cell growth, and metabolism. Androgen signaling also impacts DNA damage signaling through mechanisms involving gene expression and transcription-associated DNA damaging events. Defining the contributions of androgen signaling to DNA repair is important for understanding androgen receptor function, and it also has translational implications. METHODS: We generated RNA-seq data from multiple prostate cancer lines and used bioinformatic analyses to characterize androgen-regulated gene expression. We compared the results from cell lines with gene expression data from prostate cancer xenografts, and patient samples, to query how androgen signaling and prostate cancer progression influences the expression of DNA repair genes. We performed whole genome sequencing to help characterize the status of the DNA repair machinery in widely used prostate cancer lines. Finally, we tested a DNA repair enzyme inhibitor for effects on androgen-dependent transcription. RESULTS: Our data indicates that androgen signaling regulates a subset of DNA repair genes that are largely specific to the respective model system and disease state. We identified deleterious mutations in the DNA repair genes RAD50 and CHEK2. We found that inhibition of the DNA repair enzyme MRE11 with the small molecule mirin inhibits androgen-dependent transcription and growth of prostate cancer cells. CONCLUSIONS: Our data supports the view that crosstalk between androgen signaling and DNA repair occurs at multiple levels, and that DNA repair enzymes in addition to PARPs, could be actionable targets in prostate cancer.


Assuntos
Androgênios/metabolismo , Reparo do DNA/genética , DNA de Neoplasias/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Células PC-3 , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
7.
Genome Biol ; 19(1): 89, 2018 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-30001206

RESUMO

BACKGROUND: Alternative DNA secondary structures can arise from single-stranded DNA when duplex DNA is unwound during DNA processes such as transcription, resulting in the regulation or perturbation of these processes. We identify sites of high propensity to form stable DNA secondary structure across the human genome using Mfold and ViennaRNA programs with parameters for analyzing DNA. RESULTS: The promoter-proximal regions of genes with paused transcription are significantly and energetically more favorable to form DNA secondary structure than non-paused genes or genes without RNA polymerase II (Pol II) binding. Using Pol II ChIP-seq, GRO-seq, NET-seq, and mNET-seq data, we arrive at a robust set of criteria for Pol II pausing, independent of annotation, and find that a highly stable secondary structure is likely to form about 10-50 nucleotides upstream of a Pol II pausing site. Structure probing data confirm the existence of DNA secondary structures enriched at the promoter-proximal regions of paused genes in human cells. Using an in vitro transcription assay, we demonstrate that Pol II pausing at HSPA1B, a human heat shock gene, is affected by manipulating DNA secondary structure upstream of the pausing site. CONCLUSIONS: Our results indicate alternative DNA secondary structure formation as a mechanism for how GC-rich sequences regulate RNA Pol II promoter-proximal pausing genome-wide.


Assuntos
DNA/genética , Genoma Humano/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , Humanos , Nucleotídeos/genética , Transcrição Genética/genética
8.
Nat Methods ; 14(7): 710-712, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28581493

RESUMO

CRISPR-Cas9-induced DNA damage may have deleterious effects at high-copy-number genomic regions. Here, we use CRISPR base editors to knock out genes by changing single nucleotides to create stop codons. We show that the CRISPR-STOP method is an efficient and less deleterious alternative to wild-type Cas9 for gene-knockout studies. Early stop codons can be introduced in ∼17,000 human genes. CRISPR-STOP-mediated targeted screening demonstrates comparable efficiency to WT Cas9, which indicates the suitability of our approach for genome-wide functional screenings.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Códon de Terminação/genética , Inativação Gênica , Códon sem Sentido , Regulação da Expressão Gênica , Marcação de Genes/métodos , Vetores Genéticos , Células HEK293 , Humanos , Plasmídeos
9.
Nat Commun ; 8: 14725, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28290446

RESUMO

Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation. Recently, the RNA-guidable feature of CRISPR-Cas9 has been utilized for imaging of chromatin within live cells. However, these methods are mostly applicable to highly repetitive regions, whereas imaging regions with low or no repeats remains as a challenge. To address this challenge, we design single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification. These engineered sgRNAs enable multicolour labelling of low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four unique sgRNAs. We achieve tracking of native chromatin loci throughout the cell cycle and determine differential positioning of transcriptionally active and inactive regions in the nucleus. These results demonstrate the feasibility of our approach to monitor the position and dynamics of both repetitive and non-repetitive genomic regions in live cells.


Assuntos
Ciclo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , RNA Guia/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Estudos de Viabilidade , Loci Gênicos , Células HEK293 , Células HeLa , Humanos , Microscopia Intravital , Microscopia Confocal , Epitélio Pigmentado da Retina/citologia
10.
Mol Immunol ; 71: 143-151, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26896718

RESUMO

Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1Å. Cetirizine is bound in two sites--a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizine binding sites. We show that the residues forming the binding pockets in ESA are highly conserved in human serum albumin (HSA), and suggest that binding of cetirizine to HSA will be similar. In support of that hypothesis, we show that the dissociation constants for cetirizine binding to CBS2 in ESA and HSA are identical using tryptophan fluorescence quenching. Presence of lysine and arginine residues that have been previously reported to undergo nonenzymatic glycosylation in CBS1 and CBS2 suggests that cetirizine transport in patients with diabetes could be altered. A review of all available SA structures from the PDB shows that in addition to the novel drug binding site we present here (CBS1), there are two pockets on SA capable of binding drugs that do not overlap with fatty acid binding sites and have not been discussed in published reviews.


Assuntos
Cetirizina/química , Albumina Sérica/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/fisiologia , Cetirizina/metabolismo , Cristalografia por Raios X , Antagonistas dos Receptores Histamínicos H1 não Sedativos/química , Antagonistas dos Receptores Histamínicos H1 não Sedativos/metabolismo , Cavalos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Albumina Sérica/metabolismo
11.
J Neural Transm (Vienna) ; 122(6): 863-5, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25204278

RESUMO

Accumulating data confirm the usefulness of transcranial sonography (TCS) in the diagnosis of Parkinson's disease. The relevance of basal ganglia abnormalities depicted by TCS in atypical parkinsonian syndromes still needs further assessment. In the present study, 20 patients with progressive supranuclear palsy (PSP) and 13 patients with corticobasal syndrome (CBS) were studied with the use of transcranial sonography. Echogenicity of the substantia nigra (SN) and lenticular nucleus (LN) were assessed. 0/20 patients with PSP and 8/12 (66.6 %) patients with CBS were characterized with SN hyperechogenicity. LN hyperechogenicity was observed in 9/20 patients diagnosed with PSP and 0/11 of CBS patients. The combination of SN isoechogenicity and LN hyperechogenicity reached 100 % sensitivity and positive predictive value for the diagnosis of PSP. The results of this study point out that CBS has to be taken into consideration when SN hyperechogenicity is depicted in a patient with parkinsonian syndrome. Normal echogenicity of the SN coexisting with LN hyperechogenicity practically excludes CBS.


Assuntos
Corpo Estriado/diagnóstico por imagem , Paralisia Supranuclear Progressiva/diagnóstico por imagem , Tauopatias/diagnóstico por imagem , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Substância Negra/diagnóstico por imagem , Ultrassonografia
12.
J Phys Condens Matter ; 24(24): 244106, 2012 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-22595616

RESUMO

The possible role of iron in neurodegeneration was studied by various techniques: electron microscopy, enzyme-linked immunosorbent assay, Mössbauer spectroscopy, atomic absorption, ultrasonography and magnetic resonance imaging. The measurements were made on human tissues extracted from liver and from brain structures involved in diseases of the human brain: substantia nigra (Parkinson's, PD), hippocampal cortex (Alzheimer's, AD) and globus pallidus (progressive supranuclear palsy, PSP). The sizes of the iron cores of ferritin, the main iron storage compound in tissues, were found to be smaller in brain than in liver. Brain ferritin has a higher proportion of H to L chains compared to liver. A significant decrease of the concentration of L chains in PD compared to control was found. No increase in the concentration of iron in PD versus control was detected; however, there was an increase of labile iron, which constitutes only 2‰ of brain iron. In AD an increase in the concentration of ferritin was noticed, without a significant increase in iron concentration. In PSP an increase of total iron was observed. Our findings suggest that the mechanisms leading to the death of nerve cells in these three diseases may be different, although all may be related to iron mediated oxidative stress.


Assuntos
Ferro/metabolismo , Microscopia Eletrônica , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/metabolismo , Neuroimagem , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/ultraestrutura , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Humanos , Imagem por Ressonância Magnética , Doenças Neurodegenerativas/diagnóstico por imagem , Doenças Neurodegenerativas/patologia , Estresse Oxidativo , Espectroscopia de Mossbauer , Ultrassonografia
13.
J Neural Transm (Vienna) ; 119(3): 363-7, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21881837

RESUMO

The aim of the present study was to assess the origin of the substantia nigra hyperechogenicity in Parkinson disease patients. The cause of hyperechogenicity was tested on an animal model. Fresh porcine brains were injected consecutively with ferritin, apoferritin and water. Then, glioma samples were inserted into animal model. The echogenicity of the region of interest was assessed before and after experimental procedures. We observed the same echogenicity of porcine brain before and after injections of iron-loaded ferritin, apoferritin and water. Increased echogenicity of glioma samples compared to surrounding porcine brain tissue could be clearly seen. We postulate that the relative gliosis might be, at least partially, responsible for the increased echogenicity of the substantia nigra in Parkinson disease patients. Keeping in mind all limitations and inaccuracies of animal model used, it seems that hyperechogenicity of substantia nigra is caused rather by structural changes within the brain tissue than by increased iron concentration.


Assuntos
Doença de Parkinson/diagnóstico por imagem , Substância Negra/diagnóstico por imagem , Animais , Ferritinas/análise , Gliose/diagnóstico por imagem , Ferro/análise , Substância Negra/química , Suínos , Ultrassonografia Doppler Transcraniana
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