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Nat Genet ; 51(4): 581-583, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30926964

Hematopoese , Humanos
G3 (Bethesda) ; 8(9): 2923-2940, 2018 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-30021829


Alternative splicing leverages genomic content by allowing the synthesis of multiple transcripts and, by implication, protein isoforms, from a single gene. However, estimating the abundance of transcripts produced in a given tissue from short sequencing reads is difficult and can result in both the construction of transcripts that do not exist, and the failure to identify true transcripts. An alternative approach is to catalog the events that make up isoforms (splice junctions and exons). We present here the Event Analysis (EA) approach, where we project transcripts onto the genome and identify overlapping/unique regions and junctions. In addition, all possible logical junctions are assembled into a catalog. Transcripts are filtered before quantitation based on simple measures: the proportion of the events detected, and the coverage. We find that mapping to a junction catalog is more efficient at detecting novel junctions than mapping in a splice aware manner. We identify 99.8% of true transcripts while iReckon identifies 82% of the true transcripts and creates more transcripts not included in the simulation than were initially used in the simulation. Using PacBio Iso-seq data from a mouse neural progenitor cell model, EA detects 60% of the novel junctions that are combinations of existing exons while only 43% are detected by STAR. EA further detects ∼5,000 annotated junctions missed by STAR. Filtering transcripts based on the proportion of the transcript detected and the number of reads on average supporting that transcript captures 95% of the PacBio transcriptome. Filtering the reference transcriptome before quantitation, results in is a more stable estimate of isoform abundance, with improved correlation between replicates. This was particularly evident when EA is applied to an RNA-seq study of type 1 diabetes (T1D), where the coefficient of variation among subjects (n = 81) in the transcript abundance estimates was substantially reduced compared to the estimation using the full reference. EA focuses on individual transcriptional events. These events can be quantitate and analyzed directly or used to identify the probable set of expressed transcripts. Simple rules based on detected events and coverage used in filtering result in a dramatic improvement in isoform estimation without the use of ancillary data (e.g., ChIP, long reads) that may not be available for many studies.

Diabetes Mellitus Tipo 1 , Modelos Genéticos , Células-Tronco Neurais/metabolismo , RNA Mensageiro , Transcriptoma , Animais , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Camundongos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
Nat Commun ; 9(1): 575, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29422508


One drawback of chemotherapy is poor drug delivery to tumor cells, due in part to hyperpermeability of the tumor vasculature. Extracellular superoxide dismutase (SOD3) is an antioxidant enzyme usually repressed in the tumor milieu. Here we show that specific SOD3 re-expression in tumor-associated endothelial cells (ECs) increases doxorubicin (Doxo) delivery into and chemotherapeutic effect on tumors. Enhanced SOD3 activity fostered perivascular nitric oxide accumulation and reduced vessel leakage by inducing vascular endothelial cadherin (VEC) transcription. SOD3 reduced HIF prolyl hydroxylase domain protein activity, which increased hypoxia-inducible factor-2α (HIF-2α) stability and enhanced its binding to a specific VEC promoter region. EC-specific HIF-2α ablation prevented both the SOD3-mediated increase in VEC transcription and the enhanced Doxo effect. SOD3, VEC, and HIF-2α levels correlated positively in primary colorectal cancers, which suggests a similar interconnection of these proteins in human malignancy.

Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Doxorrubicina/administração & dosagem , Células Endoteliais/metabolismo , Neoplasias/tratamento farmacológico , Superóxido Dismutase/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antineoplásicos/administração & dosagem , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Caderinas/genética , Caderinas/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Tratamento Farmacológico , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Neoplasias/metabolismo , Estabilidade Proteica , Superóxido Dismutase/genética
Genome Res ; 2018 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-29440222


High-throughput sequencing of full-length transcripts using long reads has paved the way for the discovery of thousands of novel transcripts, even in well-annotated mammalian species. The advances in sequencing technology have created a need for studies and tools that can characterize these novel variants. Here, we present SQANTI, an automated pipeline for the classification of long-read transcripts that can assess the quality of data and the preprocessing pipeline using 47 unique descriptors. We apply SQANTI to a neuronal mouse transcriptome using Pacific Biosciences (PacBio) long reads and illustrate how the tool is effective in characterizing and describing the composition of the full-length transcriptome. We perform extensive evaluation of ToFU PacBio transcripts by PCR to reveal that an important number of the novel transcripts are technical artifacts of the sequencing approach and that SQANTI quality descriptors can be used to engineer a filtering strategy to remove them. Most novel transcripts in this curated transcriptome are novel combinations of existing splice sites, resulting more frequently in novel ORFs than novel UTRs, and are enriched in both general metabolic and neural-specific functions. We show that these new transcripts have a major impact in the correct quantification of transcript levels by state-of-the-art short-read-based quantification algorithms. By comparing our iso-transcriptome with public proteomics databases, we find that alternative isoforms are elusive to proteogenomics detection. SQANTI allows the user to maximize the analytical outcome of long-read technologies by providing the tools to deliver quality-evaluated and curated full-length transcriptomes.

Sci Rep ; 6: 20223, 2016 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-26838552


Evidence links aryl hydrocarbon receptor (AHR) activation to rheumatoid arthritis (RA) pathogenesis, although results are inconsistent. AHR agonists inhibit pro-inflammatory cytokine expression in macrophages, pivotal cells in RA aetiopathogenesis, which hints at specific circuits that regulate the AHR pathway in RA macrophages. We compared microRNA (miR) expression in CD14(+) cells from patients with active RA or with osteoarthritis (OA). Seven miR were downregulated and one (miR-223) upregulated in RA compared to OA cells. miR-223 upregulation correlated with reduced Notch3 and Notch effector expression in RA patients. Overexpression of the Notch-induced repressor HEY-1 and co-culture of healthy donor monocytes with Notch ligand-expressing cells showed direct Notch-mediated downregulation of miR-223. Bioinformatics predicted the AHR regulator ARNT (AHR nuclear translocator) as a miR-223 target. Pre-miR-223 overexpression silenced ARNT 3'UTR-driven reporter expression, reduced ARNT (but not AHR) protein levels and prevented AHR/ARNT-mediated inhibition of pro-inflammatory cytokine expression. miR-223 counteracted AHR/ARNT-induced Notch3 upregulation in monocytes. Levels of ARNT and of CYP1B1, an AHR/ARNT signalling effector, were reduced in RA compared to OA synovial tissue, which correlated with miR-223 levels. Our results associate Notch signalling to miR-223 downregulation in RA macrophages, and identify miR-223 as a negative regulator of the AHR/ARNT pathway through ARNT targeting.

Artrite Reumatoide/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Citocinas/metabolismo , Macrófagos/metabolismo , MicroRNAs/genética , Receptores Notch/genética , Idoso , Artrite Reumatoide/patologia , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Técnicas de Cocultura , Citocinas/genética , Feminino , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/genética , Osteoartrite/patologia , Transdução de Sinais
PLoS Comput Biol ; 11(12): e1004647, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26642228


Sequencing projects have identified large numbers of rare stop-gain and frameshift variants in the human genome. As most of these are observed in the heterozygous state, they test a gene's tolerance to haploinsufficiency and dominant loss of function. We analyzed the distribution of truncating variants across 16,260 autosomal protein coding genes in 11,546 individuals. We observed 39,893 truncating variants affecting 12,062 genes, which significantly differed from an expectation of 12,916 genes under a model of neutral de novo mutation (p<10-4). Extrapolating this to increasing numbers of sequenced individuals, we estimate that 10.8% of human genes do not tolerate heterozygous truncating variants. An additional 10 to 15% of truncated genes may be rescued by incomplete penetrance or compensatory mutations, or because the truncating variants are of limited functional impact. The study of protein truncating variants delineates the essential genome and, more generally, identifies rare heterozygous variants as an unexplored source of diversity of phenotypic traits and diseases.

Mapeamento Cromossômico/métodos , Códon sem Sentido/genética , Variação Genética/genética , Genoma Humano/genética , Proteínas/genética , Sequência de Bases , Humanos , Dados de Sequência Molecular
Oncoimmunology ; 2(9): e25669, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24319638


Inflammatory mediators can play a dual role in oncogenesis and tumor progression. CX3CL1, a chemokine previously implicated in natural killer cell- and CD8+ T cell-mediated antitumor immune responses, has now been identified as a promoter of ERBB2-expressing breast carcinomas as it cross-activates members of the epidermal growth factor receptor family.

Oncotarget ; 4(12): 2288-301, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24317954


Beyond their ability to inhibit cholesterol biosynthesis, the statins have pleiotropic effects that include anti-inflammatory and immunomodulatory activities. Statins could have clinical utility, alone or in combination with other chemotherapeutics, in the treatment of cancer. The mechanisms that underlie the anti-tumor activity of the statins are nonetheless poorly defined. No studies have analyzed how they alter the tumor-associated leukocyte infiltrate, a central factor that influences tumor stroma and cancer evolution. Here we used HER2/neu transgenic (Tg-neu) mice to analyze the effect of lovastatin (Lov) on the inflammatory reaction of spontaneous mammary tumors. Lov treatment of tumor-bearing Tg-neu mice did not alter growth of established tumors, but significantly reduced the number of new oncogenic lesions in these mice. Moreover, Lov inhibited the growth of newly implanted Tg-neu tumors in immunocompetent but not in immunodeficient mice. We found that Lov enhanced tumor infiltration by effector T cells, and reduced the number of immunosuppressive and pro-angiogenic M2-like tumor-associated macrophages (TAM). Concomitantly, the drug improved the structure and function of the tumor vasculature, measured as enhanced tumor oxygenation and penetration of cytotoxic drugs. Microarray analysis identified a Lov-elicited genetic program in Tg-neu tumors that might explain these effects; we observed Lov-induced downregulation of placental growth factor, which triggers aberrant angiogenesis and M2-like TAM polarization. Our results identify a role for lovastatin in the shaping and re-education of the inflammatory infiltrate in tumors, with functional consequences in angiogenesis and antitumor immunity.

Anticolesterolemiantes/farmacologia , Lovastatina/farmacologia , Macrófagos/efeitos dos fármacos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/imunologia , Linfócitos T/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Polaridade Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Feminino , Lovastatina/administração & dosagem , Macrófagos/imunologia , Macrófagos/patologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Linfócitos T/imunologia , Linfócitos T/metabolismo
Cancer Res ; 73(14): 4461-73, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23720051


Chemokines are relevant molecules in shaping the tumor microenvironment, although their contributions to tumorigenesis are not fully understood. We studied the influence of the chemokine CX3CL1/fractalkine in de novo breast cancer formation using HER2/neu transgenic mice. CX3CL1 expression was downmodulated in HER2/neu tumors, yet, paradoxically, adenovirus-mediated CX3CL1 expression in the tumor milieu enhanced mammary tumor numbers in a dose-dependent manner. Increased tumor multiplicity was not a consequence of CX3CL1-induced metastatic dissemination of the primary tumor, although CX3CL1 induced epithelial-to-mesenchymal transition in breast cancer cells in vitro. Instead, CX3CL1 triggered cell proliferation by induction of ErbB receptors through the proteolytic shedding of an ErbB ligand. This effect was important insofar as mammary tumorigenesis was delayed and tumor multiplicity was reduced by genetic deletion of CX3CL1 in HER2/neu mice, but not in polyoma middle T-antigen oncomice. Our findings support the conclusion that CX3CL1 acts as a positive modifier of breast cancer in concert with ErbB receptors.

Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/metabolismo , Ativação Transcricional , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática , Células MCF-7 , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Animais , Neoplasias Mamárias Experimentais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Transdução de Sinais
Mol Endocrinol ; 25(3): 385-93, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21239609


Covalent histone modifications clearly play an essential role in ligand-dependent transcriptional regulation by nuclear receptors. One of the predominant mechanisms used by nuclear receptors to activate or repress target-gene transcription is the recruitment of coregulatory factors capable of covalently modify the amino terminal ends of histones. Here we show that the thyroid hormone (T3) produces a rapid increase in histone H3Ser10 phosphorylation (H3Ser10ph) concomitant to the rapid displacement of the heterochromatin protein 1ß (HP1ß) to the nuclear periphery. Moreover, we found that T3-mediated pituitary gene transcription is associated with an increase in H3Ser10ph. Interestingly, the Aurora kinase B inhibitor ZM443979 abolishes the effect of T3 on H3Ser10ph, blocks HP1ß delocalization, and significantly reduces ligand-dependent transactivation. Similar effects were shown when Aurora kinase B expression was abrogated in small interfering RNA assays. In an effort to understand the underlying mechanism by which T3 increases H3Ser10ph, we demonstrate that liganded thyroid hormone receptor directly interacts with Aurora kinase B, increasing its kinase activity. Moreover, using chromatin immunoprecipitation assays, we have shown that Aurora kinase B participates of a mechanism that displaces HP1ß from promoter region, thus preparing the chromatin for the transcriptional activation of T3 regulated genes. Our findings reveal a novel role for Aurora kinase B during transcriptional initiation in GO/G1, apart from its well-known mitotic activity.

Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Genética/efeitos dos fármacos , Tri-Iodotironina/farmacologia , Aurora Quinase B , Aurora Quinases , Western Blotting , Ciclo Celular , Linhagem Celular , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Imunofluorescência , Humanos , Imunoprecipitação , Fosforilação/efeitos dos fármacos , Hipófise/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Genética/genética