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1.
Cell ; 181(3): 728-744.e21, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32302591

RESUMO

Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor ß (TGF-ß) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.


Assuntos
Técnicas de Introdução de Genes/métodos , Engenharia Genética/métodos , Imunoterapia/métodos , Animais , Células Sanguíneas , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , RNA Guia de Cinetoplastídeos/genética , Análise de Célula Única/métodos , Linfócitos T , Transcriptoma/genética
2.
Cell ; 182(3): 685-712.e19, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32645325

RESUMO

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Pneumonia Viral/metabolismo , Proteômica/métodos , Células A549 , Enzima de Conversão de Angiotensina 2 , Animais , Antivirais/farmacologia , COVID-19 , Células CACO-2 , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Fosforilação , Pneumonia Viral/virologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Receptor Tirosina Quinase Axl
3.
Nature ; 602(7897): 487-495, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34942634

RESUMO

The emergence of SARS-CoV-2 variants of concern suggests viral adaptation to enhance human-to-human transmission1,2. Although much effort has focused on the characterization of changes in the spike protein in variants of concern, mutations outside of spike are likely to contribute to adaptation. Here, using unbiased abundance proteomics, phosphoproteomics, RNA sequencing and viral replication assays, we show that isolates of the Alpha (B.1.1.7) variant3 suppress innate immune responses in airway epithelial cells more effectively than first-wave isolates. We found that the Alpha variant has markedly increased subgenomic RNA and protein levels of the nucleocapsid protein (N), Orf9b and Orf6-all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein that is required for activation of the RNA-sensing adaptor MAVS. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful transmission of the Alpha variant, and may increase in vivo replication and duration of infection4. The importance of mutations outside the spike coding region in the adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the N and Orf9b regulatory regions of the Delta and Omicron variants.


Assuntos
COVID-19/imunologia , COVID-19/virologia , Evolução Molecular , Evasão da Resposta Imune , Imunidade Inata/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , COVID-19/transmissão , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Humanos , Imunidade Inata/genética , Interferons/imunologia , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Proteômica , RNA Viral/genética , RNA-Seq , SARS-CoV-2/classificação , SARS-CoV-2/crescimento & desenvolvimento
4.
Genes Dev ; 34(17-18): 1210-1226, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32820040

RESUMO

Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by initial chemosensitivity followed by emergence of chemoresistant disease. To study roles for MYCN amplification in SCLC progression and chemoresistance, we developed a genetically engineered mouse model of MYCN-overexpressing SCLC. In treatment-naïve mice, MYCN overexpression promoted cell cycle progression, suppressed infiltration of cytotoxic T cells, and accelerated SCLC. MYCN overexpression also suppressed response to cisplatin-etoposide chemotherapy, with similar findings made upon MYCL overexpression. We extended these data to genetically perturb chemosensitive patient-derived xenograft (PDX) models of SCLC. In chemosensitive PDX models, overexpression of either MYCN or MYCL also conferred a switch to chemoresistance. To identify therapeutic strategies for MYCN-overexpressing SCLC, we performed a genome-scale CRISPR-Cas9 sgRNA screen. We identified the deubiquitinase USP7 as a MYCN-associated synthetic vulnerability. Pharmacological inhibition of USP7 resensitized chemoresistant MYCN-overexpressing PDX models to chemotherapy in vivo. Our findings show that MYCN overexpression drives SCLC chemoresistance and provide a therapeutic strategy to restore chemosensitivity.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Xenoenxertos , Humanos , Neoplasias Pulmonares/enzimologia , Camundongos , Proteína Proto-Oncogênica N-Myc/genética , Carcinoma de Pequenas Células do Pulmão/enzimologia , Carcinoma de Pequenas Células do Pulmão/genética
5.
Nat Immunol ; 14(12): 1256-65, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24162774

RESUMO

The humoral immune response after acute infection with HIV-1 is delayed and ineffective. The HIV-1 envelope protein gp120 binds to and signals through integrin α4ß7 on T cells. We found that gp120 also bound to and signaled through α4ß7 on naive B cells, which resulted in an abortive proliferative response. In primary B cells, signaling by gp120 through α4ß7 resulted in increased expression of the immunosuppressive cytokine TGF-ß1 and FcRL4, an inhibitory receptor expressed on B cells. Coculture of B cells with HIV-1-infected autologous CD4(+) T cells also increased the expression of FcRL4 by B cells. Our findings indicated that in addition to mediating chronic activation of the immune system, viral proteins contributed directly to HIV-1-associated B cell dysfunction. Our studies identify a mechanism whereby the virus may subvert the early HIV-1-specific humoral immune response.


Assuntos
Linfócitos B/imunologia , Proliferação de Células , Proteína gp120 do Envelope de HIV/imunologia , Receptores Fc/imunologia , Fator de Crescimento Transformador beta1/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células CHO , Células Cultivadas , Técnicas de Cocultura , Cricetinae , Cricetulus , Citometria de Fluxo , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/genética , HIV-1/imunologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Integrinas/genética , Integrinas/imunologia , Integrinas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica/imunologia , Receptores Fc/genética , Receptores Fc/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Transcriptoma/genética , Transcriptoma/imunologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
6.
Nature ; 559(7714): 405-409, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29995861

RESUMO

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.


Assuntos
Reprogramação Celular/genética , Edição de Genes , Genoma Humano/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Autoimunidade/genética , Sistemas CRISPR-Cas/genética , Células Cultivadas , Humanos , Subunidade alfa de Receptor de Interleucina-2/genética , Masculino , Camundongos , Transplante de Neoplasias , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologia
7.
PLoS Pathog ; 17(1): e1009214, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33465157

RESUMO

The precise role of CD4 T cell turnover in maintaining HIV persistence during antiretroviral therapy (ART) has not yet been well characterized. In resting CD4 T cell subpopulations from 24 HIV-infected ART-suppressed and 6 HIV-uninfected individuals, we directly measured cellular turnover by heavy water labeling, HIV reservoir size by integrated HIV-DNA (intDNA) and cell-associated HIV-RNA (caRNA), and HIV reservoir clonality by proviral integration site sequencing. Compared to HIV-negatives, ART-suppressed individuals had similar fractional replacement rates in all subpopulations, but lower absolute proliferation rates of all subpopulations other than effector memory (TEM) cells, and lower plasma IL-7 levels (p = 0.0004). Median CD4 T cell half-lives decreased with cell differentiation from naïve to TEM cells (3 years to 3 months, p<0.001). TEM had the fastest replacement rates, were most highly enriched for intDNA and caRNA, and contained the most clonal proviral expansion. Clonal proviruses detected in less mature subpopulations were more expanded in TEM, suggesting that they were maintained through cell differentiation. Earlier ART initiation was associated with lower levels of intDNA, caRNA and fractional replacement rates. In conclusion, circulating integrated HIV proviruses appear to be maintained both by slow turnover of immature CD4 subpopulations, and by clonal expansion as well as cell differentiation into effector cells with faster replacement rates.


Assuntos
Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/patologia , Diferenciação Celular , Infecções por HIV/virologia , HIV-1/imunologia , Carga Viral , Replicação Viral , Adulto , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Estudos de Casos e Controles , DNA Viral/análise , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/patologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Masculino , Pessoa de Meia-Idade
9.
EMBO J ; 37(18)2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30166453

RESUMO

Multi-subunit cullin-RING ligases (CRLs) are the largest family of ubiquitin E3 ligases in humans. CRL activity is tightly regulated to prevent unintended substrate degradation or autocatalytic degradation of CRL subunits. Using a proteomics strategy, we discovered that CRL4AMBRA1 (CRL substrate receptor denoted in superscript) targets Elongin C (ELOC), the essential adapter protein of CRL5 complexes, for polyubiquitination and degradation. We showed that the ubiquitin ligase function of CRL4AMBRA1 is required to disrupt the assembly and attenuate the ligase activity of human CRL5SOCS3 and HIV-1 CRL5VIF complexes as AMBRA1 depletion leads to hyperactivation of both CRL5 complexes. Moreover, CRL4AMBRA1 modulates interleukin-6/STAT3 signaling and HIV-1 infectivity that are regulated by CRL5SOCS3 and CRL5VIF, respectively. Thus, by discovering a substrate of CRL4AMBRA1, ELOC, the shared adapter of CRL5 ubiquitin ligases, we uncovered a novel CRL cross-regulation pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Elonguina/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Elonguina/genética , Células HEK293 , Infecções por HIV/genética , HIV-1/genética , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Ubiquitina-Proteína Ligases/genética , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética
10.
PLoS Pathog ; 14(8): e1007278, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30153309

RESUMO

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4ß7, a gut-homing receptor. Using both cell-surface expressed α4ß7 and a soluble α4ß7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4ß7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4ß7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4ß7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4ß7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4ß7. It includes the canonical LDV/I α4ß7 binding site, a cryptic epitope that lies 7-9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4ß7 interactions. These mAbs recognize conformations absent from the ß- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4ß7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.


Assuntos
Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/prevenção & controle , Integrinas/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas contra a AIDS/química , Vacinas contra a AIDS/imunologia , Vacinas contra a AIDS/metabolismo , Animais , Anticorpos Monoclonais , Sítios de Ligação/imunologia , Linhagem Celular Tumoral , Epitopos/química , Epitopos/imunologia , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Infecções por HIV/imunologia , HIV-1/imunologia , Macaca , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/imunologia , Vacinas contra a SAIDS/química , Vacinas contra a SAIDS/imunologia , Vacinas contra a SAIDS/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinação/métodos
11.
Mol Cell ; 48(5): 760-70, 2012 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-23142080

RESUMO

MicroRNAs (miRNAs) are essential components of gene regulation, but identification of miRNA targets remains a major challenge. Most target prediction and discovery relies on perfect complementarity of the miRNA seed to the 3' untranslated region (UTR). However, it is unclear to what extent miRNAs target sites without seed matches. Here, we performed a transcriptome-wide identification of the endogenous targets of a single miRNA-miR-155-in a genetically controlled manner. We found that approximately 40% of miR-155-dependent Argonaute binding occurs at sites without perfect seed matches. The majority of these noncanonical sites feature extensive complementarity to the miRNA seed with one mismatch. These noncanonical sites confer regulation of gene expression, albeit less potently than canonical sites. Thus, noncanonical miRNA binding sites are widespread, often contain seed-like motifs, and can regulate gene expression, generating a continuum of targeting and regulation.


Assuntos
MicroRNAs/metabolismo , Transcriptoma , Regiões 3' não Traduzidas , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sítios de Ligação , Linfócitos T CD4-Positivos/metabolismo , Biologia Computacional , Regulação para Baixo , Perfilação da Expressão Gênica/métodos , Genes Reporter , Células HEK293 , Humanos , Ativação Linfocitária , Camundongos , Camundongos Knockout , MicroRNAs/genética , Motivos de Nucleotídeos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Transfecção
12.
Nature ; 500(7461): 207-11, 2013 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-23925245

RESUMO

The HeLa cell line was established in 1951 from cervical cancer cells taken from a patient, Henrietta Lacks. This was the first successful attempt to immortalize human-derived cells in vitro. The robust growth and unrestricted distribution of HeLa cells resulted in its broad adoption--both intentionally and through widespread cross-contamination--and for the past 60 years it has served a role analogous to that of a model organism. The cumulative impact of the HeLa cell line on research is demonstrated by its occurrence in more than 74,000 PubMed abstracts (approximately 0.3%). The genomic architecture of HeLa remains largely unexplored beyond its karyotype, partly because like many cancers, its extensive aneuploidy renders such analyses challenging. We carried out haplotype-resolved whole-genome sequencing of the HeLa CCL-2 strain, examined point- and indel-mutation variations, mapped copy-number variations and loss of heterozygosity regions, and phased variants across full chromosome arms. We also investigated variation and copy-number profiles for HeLa S3 and eight additional strains. We find that HeLa is relatively stable in terms of point variation, with few new mutations accumulating after early passaging. Haplotype resolution facilitated reconstruction of an amplified, highly rearranged region of chromosome 8q24.21 at which integration of the human papilloma virus type 18 (HPV-18) genome occurred and that is likely to be the event that initiated tumorigenesis. We combined these maps with RNA-seq and ENCODE Project data sets to phase the HeLa epigenome. This revealed strong, haplotype-specific activation of the proto-oncogene MYC by the integrated HPV-18 genome approximately 500 kilobases upstream, and enabled global analyses of the relationship between gene dosage and expression. These data provide an extensively phased, high-quality reference genome for past and future experiments relying on HeLa, and demonstrate the value of haplotype resolution for characterizing cancer genomes and epigenomes.


Assuntos
Epigenômica , Genoma Humano/genética , Aneuploidia , Variações do Número de Cópias de DNA , Feminino , Genes myc/genética , Haplótipos , Células HeLa , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/fisiologia , Humanos , Dados de Sequência Molecular , Mutação , Proto-Oncogene Mas , Análise de Sequência de DNA , Ativação Transcricional/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
13.
Genome Res ; 23(5): 843-54, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23382536

RESUMO

The detection and quantification of genetic heterogeneity in populations of cells is fundamentally important to diverse fields, ranging from microbial evolution to human cancer genetics. However, despite the cost and throughput advances associated with massively parallel sequencing, it remains challenging to reliably detect mutations that are present at a low relative abundance in a given DNA sample. Here we describe smMIP, an assay that combines single molecule tagging with multiplex targeted capture to enable practical and highly sensitive detection of low-frequency or subclonal variation. To demonstrate the potential of the method, we simultaneously resequenced 33 clinically informative cancer genes in eight cell line and 45 clinical cancer samples. Single molecule tagging facilitated extremely accurate consensus calling, with an estimated per-base error rate of 8.4 × 10(-6) in cell lines and 2.6 × 10(-5) in clinical specimens. False-positive mutations in the single molecule consensus base-calls exhibited patterns predominantly consistent with DNA damage, including 8-oxo-guanine and spontaneous deamination of cytosine. Based on mixing experiments with cell line samples, sensitivity for mutations above 1% frequency was 83% with no false positives. At clinically informative sites, we identified seven low-frequency point mutations (0.2%-4.7%), including BRAF p.V600E (melanoma, 0.2% alternate allele frequency), KRAS p.G12V (lung, 0.6%), JAK2 p.V617F (melanoma, colon, two lung, 0.3%-1.4%), and NRAS p.Q61R (colon, 4.7%). We anticipate that smMIP will be broadly adoptable as a practical and effective method for accurately detecting low-frequency mutations in both research and clinical settings.


Assuntos
Sondas de DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Taxa de Mutação , Mutação , Linhagem Celular Tumoral , Células/citologia , Inversão Cromossômica/genética , Humanos , Neoplasias/genética , Reação em Cadeia da Polimerase/métodos
14.
Brain ; 138(Pt 6): 1613-28, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25722288

RESUMO

Malformations of cortical development containing dysplastic neuronal and glial elements, including hemimegalencephaly and focal cortical dysplasia, are common causes of intractable paediatric epilepsy. In this study we performed multiplex targeted sequencing of 10 genes in the PI3K/AKT pathway on brain tissue from 33 children who underwent surgical resection of dysplastic cortex for the treatment of intractable epilepsy. Sequencing results were correlated with clinical, imaging, pathological and immunohistological phenotypes. We identified mosaic activating mutations in PIK3CA and AKT3 in this cohort, including cancer-associated hotspot PIK3CA mutations in dysplastic megalencephaly, hemimegalencephaly, and focal cortical dysplasia type IIa. In addition, a germline PTEN mutation was identified in a male with hemimegalencephaly but no peripheral manifestations of the PTEN hamartoma tumour syndrome. A spectrum of clinical, imaging and pathological abnormalities was found in this cohort. While patients with more severe brain imaging abnormalities and systemic manifestations were more likely to have detected mutations, routine histopathological studies did not predict mutation status. In addition, elevated levels of phosphorylated S6 ribosomal protein were identified in both neurons and astrocytes of all hemimegalencephaly and focal cortical dysplasia type II specimens, regardless of the presence or absence of detected PI3K/AKT pathway mutations. In contrast, expression patterns of the T308 and S473 phosphorylated forms of AKT and in vitro AKT kinase activities discriminated between mutation-positive dysplasia cortex, mutation-negative dysplasia cortex, and non-dysplasia epilepsy cortex. Our findings identify PI3K/AKT pathway mutations as an important cause of epileptogenic brain malformations and establish megalencephaly, hemimegalencephaly, and focal cortical dysplasia as part of a single pathogenic spectrum.


Assuntos
Encéfalo/anormalidades , Hemimegalencefalia/genética , Malformações do Desenvolvimento Cortical/genética , Megalencefalia/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética , Adolescente , Encéfalo/metabolismo , Criança , Pré-Escolar , Classe I de Fosfatidilinositol 3-Quinases , Feminino , Predisposição Genética para Doença/genética , Hemimegalencefalia/metabolismo , Hemimegalencefalia/patologia , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Masculino , Malformações do Desenvolvimento Cortical/metabolismo , Malformações do Desenvolvimento Cortical/patologia , Megalencefalia/metabolismo , Megalencefalia/patologia , Mutação , Neuroimagem , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo
15.
Proc Natl Acad Sci U S A ; 110(14): E1263-72, 2013 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-23509263

RESUMO

Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify the molecular determinants within E3 ligases that modulate activity, we scored each member of a library of nearly 100,000 protein variants of the murine ubiquitination factor E4B (Ube4b) U-box domain for auto-ubiquitination activity in the presence of the E2 UbcH5c. This assay identified mutations that enhance activity both in vitro and in cellular p53 degradation assays. The activity-enhancing mutations fall into two distinct mechanistic classes: One increases the U-box:E2-binding affinity, and the other allosterically stimulates the formation of catalytically active conformations of the E2∼Ub conjugate. The same mutations enhance E3 activity in the presence of another E2, Ube2w, implying a common allosteric mechanism, and therefore the general applicability of our observations to other E3s. A comparison of the E3 activity with the two different E2s identified an additional variant that exhibits E3:E2 specificity. Our results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Modelos Moleculares , Mutagênese/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina/metabolismo , Regulação Alostérica/genética , Sequência de Aminoácidos , Animais , Western Blotting , Técnicas de Visualização da Superfície Celular , Imunoprecipitação , Camundongos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Oligonucleotídeos/genética , Alinhamento de Sequência , Ubiquitina-Proteína Ligases/metabolismo
16.
Proc Natl Acad Sci U S A ; 109(46): 18749-54, 2012 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-23112150

RESUMO

The relatively short read lengths associated with the most cost-effective DNA sequencing technologies have limited their use in de novo genome assembly, structural variation detection, and haplotype-resolved genome sequencing. Consequently, there is a strong need for methods that capture various scales of contiguity information at a throughput commensurate with the current scale of massively parallel sequencing. We propose in situ library construction and optical sequencing on the flow cells of currently available massively parallel sequencing platforms as an efficient means of capturing both contiguity information and primary sequence with a single technology. In this proof-of-concept study, we demonstrate basic feasibility by generating >30,000 Escherichia coli paired-end reads separated by 1, 2, or 3 kb using in situ library construction on standard Illumina flow cells. We also show that it is possible to stretch single molecules ranging from 3 to 8 kb on the surface of a flow cell before in situ library construction, thereby enabling the production of clusters whose physical relationship to one another on the flow cell is related to genomic distance.


Assuntos
DNA Bacteriano/genética , Escherichia coli/genética , Genoma Bacteriano , Biblioteca Genômica , Análise de Sequência de DNA/métodos
17.
Proc Natl Acad Sci U S A ; 109(36): 14508-13, 2012 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-22853953

RESUMO

Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ~1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when "deep sequencing" genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenoma/genética , Mutação/genética , Neoplasias/genética , Projetos de Pesquisa/estatística & dados numéricos , Dano ao DNA/genética , Humanos , Oligonucleotídeos/genética
18.
Sci Adv ; 10(15): eadk2082, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38598634

RESUMO

We report an approach for cancer phenotyping based on targeted sequencing of cell-free DNA (cfDNA) for small cell lung cancer (SCLC). In SCLC, differential activation of transcription factors (TFs), such as ASCL1, NEUROD1, POU2F3, and REST defines molecular subtypes. We designed a targeted capture panel that identifies chromatin organization signatures at 1535 TF binding sites and 13,240 gene transcription start sites and detects exonic mutations in 842 genes. Sequencing of cfDNA from SCLC patient-derived xenograft models captured TF activity and gene expression and revealed individual highly informative loci. Prediction models of ASCL1 and NEUROD1 activity using informative loci achieved areas under the receiver operating characteristic curve (AUCs) from 0.84 to 0.88 in patients with SCLC. As non-SCLC (NSCLC) often transforms to SCLC following targeted therapy, we applied our framework to distinguish NSCLC from SCLC and achieved an AUC of 0.99. Our approach shows promising utility for SCLC subtyping and transformation monitoring, with potential applicability to diverse tumor types.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Ácidos Nucleicos Livres , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma de Pequenas Células do Pulmão/metabolismo , Neoplasias Pulmonares/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Sequências Reguladoras de Ácido Nucleico , Regulação Neoplásica da Expressão Gênica
19.
mBio ; 15(4): e0222223, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38411080

RESUMO

During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies. IMPORTANCE: HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell's ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or "latent" cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Infecções por HIV , HIV , Fator 2 Associado a Receptor de TNF , Ubiquitina-Proteína Ligases , Latência Viral , Humanos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linfócitos T CD4-Positivos , Sistemas CRISPR-Cas , Fator 2 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Replicação Viral , HIV/fisiologia
20.
Nat Methods ; 7(2): 119-22, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20081835

RESUMO

We demonstrate subassembly, an in vitro library construction method that extends the utility of short-read sequencing platforms to applications requiring long, accurate reads. A long DNA fragment library is converted to a population of nested sublibraries, and a tag sequence directs grouping of short reads derived from the same long fragment, enabling localized assembly of long fragment sequences. Subassembly may facilitate accurate de novo genome assembly and metagenome sequencing.


Assuntos
Mapeamento Cromossômico/métodos , Análise de Sequência de DNA/métodos , Sequência de Bases , Etiquetas de Sequências Expressas , Dados de Sequência Molecular
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