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1.
Nat Commun ; 10(1): 1415, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926791

RESUMO

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.


Assuntos
Elementos de DNA Transponíveis/genética , Testes Genéticos/métodos , Linfoma de Células B/genética , Animais , Sistemas CRISPR-Cas/genética , Células Clonais , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Genes Supressores de Tumor , Estudos de Associação Genética , Humanos , Perda de Heterozigosidade , Linfoma de Células B/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/metabolismo , Reprodutibilidade dos Testes
2.
Life Sci Alliance ; 2(2)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30877233

RESUMO

Adoptive transfer of TCR transgenic T cells holds great promise for treating various cancers. So far, mainly semi-randomly integrating vectors have been used to genetically modify T cells. These carry the risk of insertional mutagenesis, and the sole addition of an exogenous TCR potentially results in the mispairing of TCR chains with endogenous ones. Established approaches using nonviral vectors, such as transposons, already reduce the risk of insertional mutagenesis but have not accomplished site-specific integration. Here, we used CRISPR-Cas9 RNPs and adeno-associated virus 6 for gene targeting to deliver an engineered TCR gene specifically to the TCR alpha constant locus, thus placing it under endogenous transcriptional control. Our data demonstrate that this approach replaces the endogenous TCR, functionally redirects the edited T cells' specificity in vitro, and facilitates potent tumor rejection in an in vivo xenograft model.

3.
Nat Commun ; 9(1): 3839, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30242258

RESUMO

The Wnt signalling pathway, one of the core de-regulated pathways in chronic lymphocytic leukaemia (CLL), is activated in only a subset of patients through somatic mutations. Here we describe alternative, microenvironment-dependent mechanisms of Wnt activation in malignant B cells. We show that tumour cells specifically induce Notch2 activity in mesenchymal stromal cells (MSCs) required for the transcription of the complement factor C1q. MSC-derived C1q in turn inhibits Gsk3-ß mediated degradation of ß-catenin in CLL cells. Additionally, stromal Notch2 activity regulates N-cadherin expression in CLL cells, which interacts with and further stabilises ß-catenin. Together, these stroma Notch2-dependent mechanisms induce strong activation of canonical Wnt signalling in CLL cells. Pharmacological inhibition of the Wnt pathway impairs microenvironment-mediated survival of tumour cells. Similarly, inhibition of Notch signalling diminishes survival of stroma-protected CLL cells in vitro and disease engraftment in vivo. Notch2 activation in the microenvironment is a pre-requisite for the activation of canonical Wnt signalling in tumour cells.

4.
J Mol Cell Biol ; 6(4): 312-23, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24872507

RESUMO

Different from canonical ubiquitin-like proteins, Hub1 does not form covalent conjugates with substrates but binds proteins non-covalently. In Saccharomyces cerevisiae, Hub1 associates with spliceosomes and mediates alternative splicing of SRC1, without affecting pre-mRNA splicing generally. Human Hub1 is highly similar to its yeast homolog, but its cellular function remains largely unexplored. Here, we show that human Hub1 binds to the spliceosomal protein Snu66 as in yeast; however, unlike its S. cerevisiae homolog, human Hub1 is essential for viability. Prolonged in vivo depletion of human Hub1 leads to various cellular defects, including splicing speckle abnormalities, partial nuclear retention of mRNAs, mitotic catastrophe, and consequently cell death by apoptosis. Early consequences of Hub1 depletion are severe splicing defects, however, only for specific splice sites leading to exon skipping and intron retention. Thus, the ubiquitin-like protein Hub1 is not a canonical spliceosomal factor needed generally for splicing, but rather a modulator of spliceosome performance and facilitator of alternative splicing.


Assuntos
Processamento Alternativo/genética , Precursores de RNA/genética , RNA Mensageiro/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Ubiquitinas/metabolismo , Sobrevivência Celular , Células Cultivadas , Éxons/genética , Humanos , Íntrons/genética , RNA Interferente Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genética , Spliceossomos/patologia , Ubiquitinas/antagonistas & inibidores , Ubiquitinas/genética
5.
Nat Cell Biol ; 15(5): 526-32, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23624404

RESUMO

Cdc48 (also known as p97), a conserved chaperone-like ATPase, plays a strategic role in the ubiquitin system. Empowered by ATP-driven conformational changes, Cdc48 acts as a segregase by dislodging ubiquitylated proteins from their environment. Ufd1, a known co-factor of Cdc48, also binds SUMO (ref. 6), but whether SUMOylated proteins are subject to the segregase activity of Cdc48 as well and what these substrates are remains unknown. Here we show that Cdc48 with its co-factor Ufd1 is SUMO-targeted to proteins involved in DNA double-strand break repair. Cdc48 associates with SUMOylated Rad52, a factor that assembles the Rad51 recombinase on chromatin. By acting on the Rad52-Rad51 complex, Cdc48 curbs their physical interaction and displaces the proteins from DNA. Genetically interfering with SUMO-targeting or segregase activity leads to an increase in spontaneous recombination rates, accompanied by aberrant in vivo Rad51 foci formation in yeast and mammalian cells. Our data thus suggest that SUMO-targeted Cdc48 restricts the recombinase Rad51 by counterbalancing the activity of Rad52. We propose that Cdc48, through its ability to associate with co-factors that have affinities for ubiquitin and SUMO, connects the two modification pathways for protein degradation or other regulatory purposes.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Mapeamento de Interação de Proteínas/métodos , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Animais , Western Blotting , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , DNA Fúngico/genética , DNA Fúngico/metabolismo , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Humanos , Imunoprecipitação , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteólise , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Técnicas do Sistema de Duplo-Híbrido , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteína com Valosina
6.
Nature ; 474(7350): 173-8, 2011 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-21614000

RESUMO

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1-HIND interaction, cannot use certain non-canonical 5' splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier.


Assuntos
Processamento Alternativo , Regulação Fúngica da Expressão Gênica , Ligases/metabolismo , Sítios de Splice de RNA/genética , RNA Fúngico/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Deleção de Genes , Humanos , Ligases/deficiência , Ligases/genética , Proteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/genética , Ligação Proteica , Conformação Proteica , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/deficiência , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U5/deficiência , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/deficiência , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/química , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Spliceossomos/química , Spliceossomos/metabolismo , Complexos Ubiquitina-Proteína Ligase/deficiência , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinas
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