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1.
Nucleic Acids Res ; 2021 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-34671803

RESUMO

Eukaryotic cells recognize intracellular pathogens through pattern recognition receptors, including sensors of aberrant nucleic acid structures. Sensors of double-stranded RNA (dsRNA) are known to detect replication intermediates of RNA viruses. It has long been suggested that annealing of mRNA from symmetrical transcription of both top and bottom strands of DNA virus genomes can produce dsRNA during infection. Supporting this hypothesis, nearly all DNA viruses encode inhibitors of dsRNA-recognition pathways. However, direct evidence that DNA viruses produce dsRNA is lacking. Contrary to dogma, we show that the nuclear-replicating DNA virus adenovirus (AdV) does not produce detectable levels of dsRNA during infection. In contrast, abundant dsRNA is detected within the nucleus of cells infected with AdV mutants defective for viral RNA processing. In the presence of nuclear dsRNA, the cytoplasmic dsRNA sensor PKR is relocalized and activated within the nucleus. Accumulation of viral dsRNA occurs in the late phase of infection, when unspliced viral transcripts form intron/exon base pairs between top and bottom strand transcripts. We propose that DNA viruses actively limit dsRNA formation by promoting efficient splicing and mRNA processing, thus avoiding detection and restriction by host innate immune sensors of pathogenic nucleic acids.

2.
Mol Cancer Res ; 2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34593610

RESUMO

Activation of Wnt signaling is among the earliest events in colon cancer development. It is achieved either via activating mutations in the CTNNB1 gene encoding ß-catenin, the key transcription factor in the Wnt pathway, or most commonly by inactivating mutations affecting APC, a major ß-catenin binding partner and negative regulator. However, our analysis of recent Pan Cancer Atlas data revealed that CTNNB1 mutations significantly co-occur with those affecting Wnt receptor complex components (e.g., Frizzled and LRP6), underscoring the importance of additional regulatory events even in the presence of common APC/CTNNB1 mutations. In our effort to identify non-mutational hyperactivating events, we determined that KRAS-transformed murine colonocytes overexpressing direct ß-catenin target MYC show significant upregulation of the Wnt signaling pathway and reduced expression of Dickkopf 3 (DKK3), a reported ligand for Wnt co-receptors. We demonstrate that MYC suppresses DKK3 transcription through one of miR-17-92 cluster miRNAs, miR-92a. We further examined the role of DKK3 by overexpression and knockdown and discovered that DKK3 suppresses Wnt signaling in Apc-null murine colonic organoids and human colon cancer cells despite the presence of downstream activating mutations in the Wnt pathway. Conversely, MYC overexpression in the same cell lines resulted in hyperactive Wnt signaling, acquisition of epithelial-to-mesenchymal transition markers, and enhanced migration/invasion in vitro and metastasis in a syngeneic orthotopic mouse colon cancer model. IMPLICATIONS: Our results suggest that the MYC→miR-92a-|DKK3 axis hyperactivates Wnt signaling, forming a feed-forward oncogenic loop.

3.
EMBO Rep ; 22(9): e52145, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34347354

RESUMO

The APOBEC3 cytidine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluate protein interactors of A3A to identify potential regulators. We find that A3A interacts with the chaperonin-containing TCP-1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT results in A3A-induced DNA damage and cytotoxicity. Evaluation of cancer genomes demonstrates an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity.


Assuntos
Chaperonina com TCP-1 , Citidina Desaminase , Chaperonina com TCP-1/genética , Citidina Desaminase/genética , Mutagênese , Proteínas/genética
4.
Genome Biol ; 22(1): 190, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183059

RESUMO

Resistance to CD19-directed immunotherapies in lymphoblastic leukemia has been attributed, among other factors, to several aberrant CD19 pre-mRNA splicing events, including recently reported excision of a cryptic intron embedded within CD19 exon 2. While "exitrons" are known to exist in hundreds of human transcripts, we discovered, using reporter assays and direct long-read RNA sequencing (dRNA-seq), that the CD19 exitron is an artifact of reverse transcription. Extending our analysis to publicly available datasets, we identified dozens of questionable exitrons, dubbed "falsitrons," that appear only in cDNA-seq, but never in dRNA-seq. Our results highlight the importance of dRNA-seq for transcript isoform validation.

5.
Nat Commun ; 11(1): 6016, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33243990

RESUMO

Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (m6A) to mRNAs. While m6A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m6A mapping using short-read sequencing, here we profile m6A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m6A detection. Although both early and late viral transcripts contain m6A, depletion of m6A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m6A discovery within individual transcripts at nucleotide resolution, and highlight the role of m6A in regulating splicing of a viral pathogen.


Assuntos
Adenosina/análogos & derivados , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Splicing de RNA , RNA Viral/metabolismo , Células A549 , Adenosina/metabolismo , Adenovírus Humanos/patogenicidade , DNA Viral/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , Análise de Sequência de RNA , Replicação Viral
6.
Nat Cell Biol ; 22(10): 1197-1210, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32989251

RESUMO

Alveolar epithelial regeneration is essential for recovery from devastating lung diseases. This process occurs when type II alveolar pneumocytes (AT2 cells) proliferate and transdifferentiate into type I alveolar pneumocytes (AT1 cells). We used genome-wide analysis of chromatin accessibility and gene expression following acute lung injury to elucidate repair mechanisms. AT2 chromatin accessibility changed substantially following injury to reveal STAT3 binding motifs adjacent to genes that regulate essential regenerative pathways. Single-cell transcriptome analysis identified brain-derived neurotrophic factor (Bdnf) as a STAT3 target gene with newly accessible chromatin in a unique population of regenerating AT2 cells. Furthermore, the BDNF receptor tropomyosin receptor kinase B (TrkB) was enriched on mesenchymal alveolar niche cells (MANCs). Loss or blockade of AT2-specific Stat3, Bdnf or mesenchyme-specific TrkB compromised repair and reduced Fgf7 expression by niche cells. A TrkB agonist improved outcomes in vivo following lung injury. These data highlight the biological and therapeutic importance of the STAT3-BDNF-TrkB axis in orchestrating alveolar epithelial regeneration.


Assuntos
Células Epiteliais Alveolares/citologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Lesão Pulmonar/prevenção & controle , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor trkB/metabolismo , Regeneração , Fator de Transcrição STAT3/metabolismo , Células Epiteliais Alveolares/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Feminino , Humanos , Lesão Pulmonar/etiologia , Lesão Pulmonar/patologia , Masculino , Glicoproteínas de Membrana/genética , Proteínas Tirosina Quinases/genética , Receptor trkB/genética , Fator de Transcrição STAT3/genética
7.
Nat Microbiol ; 5(10): 1217-1231, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32661314

RESUMO

Viruses promote infection by hijacking the ubiquitin machinery of the host to counteract or redirect cellular processes. Adenovirus encodes two early proteins, E1B55K and E4orf6, that together co-opt a cellular ubiquitin ligase complex to overcome host defences and promote virus production. Adenovirus mutants lacking E1B55K or E4orf6 display defects in viral RNA processing and protein production, but previously identified substrates of the redirected ligase do not explain these phenotypes. Here, we used a quantitative proteomics approach to identify substrates of E1B55K/E4orf6-mediated ubiquitination that facilitate RNA processing. While all currently known cellular substrates of E1B55K and E4orf6 are degraded by the proteasome, we uncovered RNA-binding proteins as high-confidence substrates that are not decreased in overall abundance. We focused on two RNA-binding proteins, RALY and hnRNP-C, which we confirm are ubiquitinated without degradation. Knockdown of RALY and hnRNP-C increased levels of viral RNA splicing, protein abundance and progeny production during infection with E1B55K-deleted virus. Furthermore, infection with E1B55K-deleted virus resulted in an increased interaction of hnRNP-C with viral RNA and attenuation of viral RNA processing. These data suggest that viral-mediated ubiquitination of RALY and hnRNP-C relieves a restriction on viral RNA processing and reveal an unexpected role for non-degradative ubiquitination in the manipulation of cellular processes during virus infection.


Assuntos
Infecções por Adenoviridae/virologia , Adenoviridae/fisiologia , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Infecções por Adenoviridae/metabolismo , Sequência de Bases , Sítios de Ligação , Biologia Computacional/métodos , Humanos , Motivos de Nucleotídeos , Ligação Proteica , Proteoma , Proteômica/métodos , Processamento Pós-Transcricional do RNA , Splicing de RNA , Ubiquitinação
8.
Nat Commun ; 11(1): 3158, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32572033

RESUMO

Efficient repair of DNA double-strand breaks (DSBs) requires a coordinated DNA Damage Response (DDR), which includes phosphorylation of histone H2Ax, forming γH2Ax. This histone modification spreads beyond the DSB into neighboring chromatin, generating a DDR platform that protects against end disassociation and degradation, minimizing chromosomal rearrangements. However, mechanisms that determine the breadth and intensity of γH2Ax domains remain unclear. Here, we show that chromosomal contacts of a DSB site are the primary determinants for γH2Ax landscapes. DSBs that disrupt a topological border permit extension of γH2Ax domains into both adjacent compartments. In contrast, DSBs near a border produce highly asymmetric DDR platforms, with γH2Ax nearly absent from one broken end. Collectively, our findings lend insights into a basic DNA repair mechanism and how the precise location of a DSB may influence genome integrity.


Assuntos
Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA , Histonas/metabolismo , Animais , Linhagem Celular Transformada , Cromatina/metabolismo , Camundongos , Fosforilação
9.
J Exp Med ; 217(9)2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32526772

RESUMO

The monoallelic expression of antigen receptor (AgR) genes, called allelic exclusion, is fundamental for highly specific immune responses to pathogens. This cardinal feature of adaptive immunity is achieved by the assembly of a functional AgR gene on one allele, with subsequent feedback inhibition of V(D)J recombination on the other allele. A range of epigenetic mechanisms have been implicated in sequential recombination of AgR alleles; however, we now demonstrate that a genetic mechanism controls this process for Tcrb. Replacement of V(D)J recombinase targets at two different mouse Vß gene segments with a higher quality target elevates Vß rearrangement frequency before feedback inhibition, dramatically increasing the frequency of T cells with TCRß chains derived from both Tcrb alleles. Thus, TCRß allelic exclusion is enforced genetically by the low quality of Vß recombinase targets that stochastically restrict the production of two functional rearrangements before feedback inhibition silences one allele.


Assuntos
Alelos , Sinais Direcionadores de Proteínas , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Recombinação V(D)J/genética , Animais , Sequência de Bases , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Hibridomas , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Linfócitos T/citologia , Timócitos/citologia
10.
Cancer Discov ; 10(4): 552-567, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32001516

RESUMO

Primary resistance to CD19-directed chimeric antigen receptor T-cell therapy (CART19) occurs in 10% to 20% of patients with acute lymphoblastic leukemia (ALL); however, the mechanisms of this resistance remain elusive. Using a genome-wide loss-of-function screen, we identified that impaired death receptor signaling in ALL led to rapidly progressive disease despite CART19 treatment. This was mediated by an inherent resistance to T-cell cytotoxicity that permitted antigen persistence and was subsequently magnified by the induction of CAR T-cell functional impairment. These findings were validated using samples from two CAR T-cell clinical trials in ALL, where we found that reduced expression of death receptor genes was associated with worse overall survival and reduced T-cell fitness. Our findings suggest that inherent dysregulation of death receptor signaling in ALL directly leads to CAR T-cell failure by impairing T-cell cytotoxicity and promoting progressive CAR T-cell dysfunction. SIGNIFICANCE: Resistance to CART19 is a significant barrier to efficacy in the treatment of B-cell malignancies. This work demonstrates that impaired death receptor signaling in tumor cells causes failed CART19 cytotoxicity and drives CART19 dysfunction, identifying a novel mechanism of antigen-independent resistance to CAR therapy.See related commentary by Green and Neelapu, p. 492.


Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Morte Celular/metabolismo , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Transdução de Sinais
13.
Nat Commun ; 10(1): 1909, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015417

RESUMO

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. ß-catenin is widely thought to be a major oncogene in HCC based on the frequency of mutations associated with aberrant Wnt signaling in HCC patients. Challenging this model, our data reveal that ß-catenin nuclear accumulation is restricted to the late stage of the disease. Until then, ß-catenin is primarily located at the plasma membrane in complex with multiple cadherin family members where it drives tumor cell survival by enhancing the signaling of growth factor receptors such as EGFR. Therefore, our study reveals the evolving nature of ß-catenin in HCC to establish it as a compound tumor promoter during the progression of the disease.


Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Proteína Wnt3A/genética , beta Catenina/genética , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Progressão da Doença , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Knockout , Estadiamento de Neoplasias , Fatores Sexuais , Transdução de Sinais , Carga Tumoral , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo
14.
Leukemia ; 33(10): 2429-2441, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30914792

RESUMO

Therapeutic targeting of initiating oncogenes is the mainstay of precision medicine. Considerable efforts have been expended toward silencing MYC, which drives many human cancers including Burkitt lymphomas (BL). Yet, the effects of MYC silencing on standard-of-care therapies are poorly understood. Here we found that inhibition of MYC transcription renders B-lymphoblastoid cells refractory to chemotherapeutic agents. This suggested that in the context of chemotherapy, stabilization of Myc protein could be more beneficial than its inactivation. We tested this hypothesis by pharmacologically inhibiting glycogen synthase kinase 3ß (GSK-3ß), which normally targets Myc for proteasomal degradation. We discovered that chemorefractory BL cell lines responded better to doxorubicin and other anti-cancer drugs when Myc was transiently stabilized. In vivo, GSK3 inhibitors (GSK3i) enhanced doxorubicin-induced apoptosis in BL patient-derived xenografts (BL-PDX), as well as in murine MYC-driven lymphoma allografts. This enhancement was accompanied by and required deregulation of several key genes acting in the extrinsic, death-receptor-mediated apoptotic pathway. Consistent with this mechanism of action, GSK3i also facilitated lymphoma cell killing by a death ligand TRAIL and by a death receptor agonist mapatumumab. Thus, GSK3i synergizes with both standard chemotherapeutics and direct engagers of death receptors and could improve outcomes in patients with refractory lymphomas.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linfoma de Células B/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Linfoma de Células B/metabolismo , Masculino , Camundongos , Transdução de Sinais
16.
Nucleic Acids Res ; 46(21): 11357-11369, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30357359

RESUMO

Aberrant splicing is a hallmark of leukemias with mutations in splicing factor (SF)-encoding genes. Here we investigated its prevalence in pediatric B-cell acute lymphoblastic leukemias (B-ALL), where SFs are not mutated. By comparing these samples to normal pro-B cells, we found thousands of aberrant local splice variations (LSVs) per sample, with 279 LSVs in 241 genes present in every comparison. These genes were enriched in RNA processing pathways and encoded ∼100 SFs, e.g. hnRNPA1. HNRNPA1 3'UTR was most pervasively mis-spliced, yielding the transcript subject to nonsense-mediated decay. To mimic this event, we knocked it down in B-lymphoblastoid cells and identified 213 hnRNPA1-regulated exon usage events comprising the hnRNPA1 splicing signature in pediatric leukemia. Some of its elements were LSVs in DICER1 and NT5C2, known cancer drivers. We searched for LSVs in other leukemia and lymphoma drivers and discovered 81 LSVs in 41 additional genes. Seventy-seven LSVs out of 81 were confirmed using two large independent B-ALL RNA-seq datasets, and the twenty most common B-ALL drivers, including NT5C2, showed higher prevalence of aberrant splicing than of somatic mutations. Thus, post-transcriptional deregulation of SF can drive widespread changes in B-ALL splicing and likely contributes to disease pathogenesis.


Assuntos
Processamento Alternativo , Linfócitos B/metabolismo , Regulação Leucêmica da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea A1/genética , Degradação do RNAm Mediada por Códon sem Sentido , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Regiões 3' não Traduzidas , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Adulto , Linfócitos B/patologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Criança , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Éxons , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Íntrons , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Cultura Primária de Células , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Fatores de Processamento de Serina-Arginina/antagonistas & inibidores , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Transativadores/genética , Transativadores/metabolismo
17.
Cell Rep ; 23(3): 878-887, 2018 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-29669291

RESUMO

Sensory experiences dynamically modify whether animals respond to a given stimulus, but it is unclear how innate behavioral thresholds are established. Here, we identify molecular and circuit-level mechanisms underlying the innate threshold of the zebrafish startle response. From a forward genetic screen, we isolated five mutant lines with reduced innate startle thresholds. Using whole-genome sequencing, we identify the causative mutation for one line to be in the fragile X mental retardation protein (FMRP)-interacting protein cyfip2. We show that cyfip2 acts independently of FMRP and that reactivation of cyfip2 restores the baseline threshold after phenotype onset. Finally, we show that cyfip2 regulates the innate startle threshold by reducing neural activity in a small group of excitatory hindbrain interneurons. Thus, we identify a selective set of genes critical to establishing an innate behavioral threshold and uncover a circuit-level role for cyfip2 in this process.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Interneurônios/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Estimulação Acústica , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Axônios/metabolismo , Comportamento Animal , Cálcio/metabolismo , Citoesqueleto/metabolismo , Potenciais Pós-Sinápticos Excitadores , Proteína do X Frágil de Retardo Mental/metabolismo , Hipersensibilidade/metabolismo , Hipersensibilidade/patologia , Larva/metabolismo , Mutagênese , Reflexo de Sobressalto/fisiologia , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
18.
BMC Genomics ; 18(1): 602, 2017 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-28797240

RESUMO

BACKGROUND: Though Illumina has largely dominated the RNA-Seq field, the simultaneous availability of Ion Torrent has left scientists wondering which platform is most effective for differential gene expression (DGE) analysis. Previous investigations of this question have typically used reference samples derived from cell lines and brain tissue, and do not involve biological variability. While these comparisons might inform studies of tissue-specific expression, marked by large-scale transcriptional differences, this is not the common use case. RESULTS: Here we employ a standard treatment/control experimental design, which enables us to evaluate these platforms in the context of the expression differences common in differential gene expression experiments. Specifically, we assessed the hepatic inflammatory response of mice by assaying liver RNA from control and IL-1ß treated animals with both the Illumina HiSeq and the Ion Torrent Proton sequencing platforms. We found the greatest difference between the platforms at the level of read alignment, a moderate level of concordance at the level of DGE analysis, and nearly identical results at the level of differentially affected pathways. Interestingly, we also observed a strong interaction between sequencing platform and choice of aligner. By aligning both real and simulated Illumina and Ion Torrent data with the twelve most commonly-cited aligners in the literature, we observed that different aligner and platform combinations were better suited to probing different genomic features; for example, disentangling the source of expression in gene-pseudogene pairs. CONCLUSIONS: Taken together, our results indicate that while Illumina and Ion Torrent have similar capacities to detect changes in biology from a treatment/control experiment, these platforms may be tailored to interrogate different transcriptional phenomena through careful selection of alignment software.


Assuntos
Perfilação da Expressão Gênica , Análise de Sequência de RNA/métodos , Algoritmos , Sequenciamento de Nucleotídeos em Larga Escala
19.
Cancer Res ; 77(17): 4579-4588, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28655787

RESUMO

Mutational signatures in cancer genomes have implicated the APOBEC3 cytosine deaminases in oncogenesis, possibly offering a therapeutic vulnerability. Elevated APOBEC3B expression has been detected in solid tumors, but expression of APOBEC3A (A3A) in cancer has not been described to date. Here, we report that A3A is highly expressed in subsets of pediatric and adult acute myelogenous leukemia (AML). We modeled A3A expression in the THP1 AML cell line by introducing an inducible A3A gene. A3A expression caused ATR-dependent phosphorylation of Chk1 and cell-cycle arrest, consistent with replication checkpoint activation. Further, replication checkpoint blockade via small-molecule inhibition of ATR kinase in cells expressing A3A led to apoptosis and cell death. Although DNA damage checkpoints are broadly activated in response to A3A activity, synthetic lethality was specific to ATR signaling via Chk1 and did not occur with ATM inhibition. Our findings identify elevation of A3A expression in AML cells, enabling apoptotic sensitivity to inhibitors of the DNA replication checkpoint and suggesting it as a candidate biomarker for ATR inhibitor therapy. Cancer Res; 77(17); 4579-88. ©2017 AACR.


Assuntos
Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Citidina Desaminase/metabolismo , Replicação do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mieloide Aguda/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas/metabolismo , Adulto , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular , Quinase 1 do Ponto de Checagem/metabolismo , Criança , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas
20.
J Exp Med ; 214(7): 1901-1912, 2017 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-28550162

RESUMO

Prolonged exit from quiescence by hematopoietic stem cells (HSCs) progressively impairs their homeostasis in the bone marrow through an unidentified mechanism. We show that Rb proteins, which are major enforcers of quiescence, maintain HSC homeostasis by positively regulating thrombopoietin (Tpo)-mediated Jak2 signaling. Rb family protein inactivation triggers the progressive E2f-mediated transactivation of Socs3, a potent inhibitor of Jak2 signaling, in cycling HSCs. Aberrant activation of Socs3 impairs Tpo signaling and leads to impaired HSC homeostasis. Therefore, Rb proteins act as a central hub of quiescence and homeostasis by coordinating the regulation of both cell cycle and Jak2 signaling in HSCs.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Homeostase/genética , Proteína do Retinoblastoma/genética , Proteína p107 Retinoblastoma-Like/genética , Proteína p130 Retinoblastoma-Like/genética , Proteína 3 Supressora da Sinalização de Citocinas/genética , Animais , Ciclo Celular/genética , Divisão Celular/genética , Proliferação de Células/genética , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Perfilação da Expressão Gênica/métodos , Immunoblotting , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos , Interferência de RNA , Proteína do Retinoblastoma/metabolismo , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína p130 Retinoblastoma-Like/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Trombopoetina/farmacologia , Ativação Transcricional
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