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Cell Discov ; 5: 40, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31636953


Isotope-labeling-based mass spectrometry (MS) is widely used in quantitative proteomic studies. With this technique, the relative abundance of thousands of proteins can be efficiently profiled in parallel, greatly facilitating the detection of proteins differentially expressed across samples. However, this task remains computationally challenging. Here we present a new approach, termed Model-based Analysis of Proteomic data (MAP), for this task. Unlike many existing methods, MAP does not require technical replicates to model technical and systematic errors, and instead utilizes a novel step-by-step regression analysis to directly assess the significance of observed protein abundance changes. We applied MAP to compare the proteomic profiles of undifferentiated and differentiated mouse embryonic stem cells (mESCs), and found it has superior performance compared with existing tools in detecting proteins differentially expressed during mESC differentiation. A web-based application of MAP is provided for online data processing at

Nat Microbiol ; 4(5): 813-825, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30833724


Human immunodeficiency virus (HIV) actively modulates the protein stability of host cells to optimize viral replication. To systematically examine this modulation in HIV infection, we used isobaric tag-based mass spectrometry to quantify changes in the abundance of over 14,000 proteins during HIV-1 infection of human primary CD4+ T cells. We identified P-selectin glycoprotein ligand 1 (PSGL-1) as an HIV-1 restriction factor downregulated by HIV-1 Vpu, which binds to PSGL-1 and induces its ubiquitination and degradation through the ubiquitin ligase SCFß-TrCP2. PSGL-1 is induced by interferon-γ in activated CD4+ T cells to inhibit HIV-1 reverse transcription and potently block viral infectivity by incorporating in progeny virions. This infectivity block is antagonized by Vpu via PSGL-1 degradation. We further show that PSGL-1 knockdown can significantly abolish the anti-HIV activity of interferon-γ in primary CD4+ T cells. Our study identifies an HIV restriction factor and a key mediator of interferon-γ's anti-HIV activity.

Linfócitos T CD4-Positivos/virologia , Infecções por HIV/metabolismo , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Glicoproteínas de Membrana/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/genética , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , HIV-1/genética , Proteínas do Vírus da Imunodeficiência Humana/genética , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Glicoproteínas de Membrana/genética , Proteólise , Proteômica , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitinação , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
Stem Cell Reports ; 11(1): 22-31, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29861165


Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) offer a promising cell resource for disease modeling and transplantation. However, differentiated HLCs exhibit an immature phenotype and comprise a heterogeneous population. Thus, a better understanding of HLC differentiation will improve the likelihood of future application. Here, by taking advantage of CRISPR-Cas9-based genome-wide screening technology and a high-throughput hPSC screening platform with a reporter readout, we identified several potential genetic regulators of HLC differentiation. By using a chemical screening approach within our platform, we also identified compounds that can further promote HLC differentiation and preserve the characteristics of in vitro cultured primary hepatocytes. Remarkably, both screenings identified histone deacetylase 3 (HDAC3) as a key regulator in hepatic differentiation. Mechanistically, HDAC3 formed a complex with liver transcriptional factors, e.g., HNF4, and co-regulated the transcriptional program during hepatic differentiation. This study highlights a broadly useful approach for studying and optimizing hPSC differentiation.

Diferenciação Celular , Hepatócitos/citologia , Hepatócitos/metabolismo , Histona Desacetilases/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Sistemas CRISPR-Cas , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Citometria de Fluxo , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Genes Reporter , Genes abl , Fator 4 Nuclear de Hepatócito/metabolismo , Histona Desacetilases/genética , Humanos , Modelos Biológicos , Fenilenodiaminas/farmacologia
Curr Protoc Mol Biol ; 123(1): e64, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29927077


Cis-regulatory elements (CREs) play a pivotal role in spatiotemporal control of tissue-specific gene expression, yet the molecular composition of the vast majority of CREs in native chromatin remains unknown. In this article, we describe the clustered regularly interspaced short palindromic repeats (CRISPR) affinity purification in situ of regulatory elements (CAPTURE) approach to simultaneously identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated nuclease-deficient Cas9 (dCas9) protein and programmable single guide RNAs (sgRNAs), this approach allows for high-resolution and locus-specific isolation of protein complexes and long-range chromatin looping associated with single copy CREs in mammalian cells. Unbiased analysis of the compositional structure of developmentally regulated or disease-associated CREs identifies new features of transcriptional regulation. Hence, CAPTURE provides a versatile platform to study genomic locus-regulating chromatin composition in a mammalian genome. © 2018 by John Wiley & Sons, Inc.

Proteína 9 Associada à CRISPR/química , Cromatina/química , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genômica/métodos , Linhagem Celular , Humanos , Elementos Reguladores de Transcrição
Cell ; 170(5): 1028-1043.e19, 2017 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-28841410


Cis-regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs in chromatin remains unknown. Here, we describe a CRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated nuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show high-resolution and selective isolation of chromatin interactions at a single-copy genomic locus. Purification of human telomeres using CAPTURE identifies known and new telomeric factors. In situ capture of individual constituents of the enhancer cluster controlling human ß-globin genes establishes evidence for composition-based hierarchical organization. Furthermore, unbiased analysis of chromatin interactions at disease-associated cis-elements and developmentally regulated super-enhancers reveals spatial features that causally control gene transcription. Thus, comprehensive and unbiased analysis of locus-specific regulatory composition provides mechanistic insight into genome structure and function in development and disease.

Sistemas CRISPR-Cas , Endonucleases/metabolismo , Técnicas Genéticas , Elementos Reguladores de Transcrição , Animais , Biotinilação , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Endonucleases/genética , Elementos Facilitadores Genéticos , Humanos , Células K562 , Camundongos , RNA Guia/metabolismo , Telômero/metabolismo , Globinas beta/genética
Nat Cell Biol ; 19(6): 626-638, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28504707


Advances in genomic profiling present new challenges of explaining how changes in DNA and RNA are translated into proteins linking genotype to phenotype. Here we compare the genome-scale proteomic and transcriptomic changes in human primary haematopoietic stem/progenitor cells and erythroid progenitors, and uncover pathways related to mitochondrial biogenesis enhanced through post-transcriptional regulation. Mitochondrial factors including TFAM and PHB2 are selectively regulated through protein translation during erythroid specification. Depletion of TFAM in erythroid cells alters intracellular metabolism, leading to elevated histone acetylation, deregulated gene expression, and defective mitochondria and erythropoiesis. Mechanistically, mTORC1 signalling is enhanced to promote translation of mitochondria-associated transcripts through TOP-like motifs. Genetic and pharmacological perturbation of mitochondria or mTORC1 specifically impairs erythropoiesis in vitro and in vivo. Our studies support a mechanism for post-transcriptional control of erythroid mitochondria and may have direct relevance to haematologic defects associated with mitochondrial diseases and ageing.

Eritropoese , Células-Tronco Hematopoéticas/enzimologia , Mitocôndrias/enzimologia , Complexos Multiproteicos/metabolismo , Biogênese de Organelas , Biossíntese de Proteínas , Serina-Treonina Quinases TOR/metabolismo , Acetilação , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica/métodos , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Knockout , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fenótipo , Proteômica/métodos , RNA/genética , RNA/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção