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1.
bioRxiv ; 2024 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-39005280

RESUMO

Huntington's disease (HD) is an inherited and ultimately fatal neurodegenerative disorder caused by an expanded polyglutamine-encoding CAG repeat within exon 1 of the huntingtin (HTT) gene, which produces a mutant protein that destroys striatal and cortical neurons. Importantly, a critical event in the pathogenesis of HD is the proteolytic cleavage of the mutant HTT protein by caspase-6, which generates fragments of the N-terminal domain of the protein that form highly toxic aggregates. Given the role that proteolysis of the mutant HTT protein plays in HD, strategies for preventing this process hold potential for treating the disorder. By screening 141 CRISPR base editor variants targeting splice elements in the HTT gene, we identified platforms capable of producing HTT protein isoforms resistant to caspase-6-mediated proteolysis via editing of the splice acceptor sequence for exon 13. When delivered to the striatum of a rodent HD model, these base editors induced efficient exon skipping and decreased the formation of the N-terminal fragments, which in turn reduced HTT protein aggregation and attenuated striatal and cortical atrophy. Collectively, these results illustrate the potential for CRISPR base editing to decrease the toxicity of the mutant HTT protein for HD.

2.
Gene ; 752: 144758, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32422235

RESUMO

Drugs targeting chromatin-modifying enzymes have entered clinical trials for myeloid malignancies, including INCB059872, a selective irreversible inhibitor of Lysine-Specific Demethylase 1 (LSD1). While initial studies of LSD1 inhibitors suggested these compounds may be used to induce differentiation of acute myeloid leukemia (AML), the mechanisms underlying this effect and dose-limiting toxicities are not well understood. Here, we used precision nuclear run-on sequencing (PRO-seq) and ChIP-seq in AML cell lines to probe for the earliest regulatory events associated with INCB059872 treatment. The changes in nascent transcription could be traced back to a loss of CoREST activity and activation of GFI1-regulated genes. INCB059872 is in phase I clinical trials, and we evaluated a pre-treatment bone marrow sample of a patient who showed a clinical response to INCB059872 while being treated with azacitidine. We used single-cell RNA-sequencing (scRNA-seq) to show that INCB059872 caused a shift in gene expression that was again associated with GFI1/GFI1B regulation. Finally, we treated mice with INCB059872 and performed scRNA-seq of lineage-negative bone marrow cells, which showed that INCB059872 triggered accumulation of megakaryocyte early progenitor cells with gene expression hallmarks of stem cells. Accumulation of these stem/progenitor cells may contribute to the thrombocytopenia observed in patients treated with LSD1 inhibitors.


Assuntos
Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA-Seq , Análise de Célula Única/métodos , Células-Tronco/metabolismo , Células THP-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequenciamento do Exoma/métodos
3.
Cell Rep ; 26(11): 2916-2928.e13, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30865883

RESUMO

The chromatin-associated protein WDR5 is a promising target for pharmacological inhibition in cancer. Drug discovery efforts center on the blockade of the "WIN site" of WDR5, a well-defined pocket that is amenable to small molecule inhibition. Various cancer contexts have been proposed to be targets for WIN site inhibitors, but a lack of understanding of WDR5 target genes and of the primary effects of WIN site inhibitors hampers their utility. Here, by the discovery of potent WIN site inhibitors, we demonstrate that the WIN site links WDR5 to chromatin at a small cohort of loci, including a specific subset of ribosome protein genes. WIN site inhibitors rapidly displace WDR5 from chromatin and decrease the expression of associated genes, causing translational inhibition, nucleolar stress, and p53 induction. Our studies define a mode by which WDR5 engages chromatin and forecast that WIN site blockade could have utility against multiple cancer types.


Assuntos
Cromatina/metabolismo , Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/química , Masculino , Ligação Proteica/efeitos dos fármacos
4.
Nucleic Acids Res ; 47(8): 3921-3936, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30805632

RESUMO

The t(8;21) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML). We found that t(8;21) AML were extremely sensitive to THZ1, which triggered apoptosis after only 4 h. We used precision nuclear run-on transcription sequencing (PROseq) to define the global effects of THZ1 and other CDK inhibitors on RNA polymerase II dynamics. Inhibition of CDK7 using THZ1 caused wide-spread loss of promoter-proximal paused RNA polymerase. This loss of 5' pausing was associated with accumulation of polymerases in the body of a large number of genes. However, there were modest effects on genes regulated by 'super-enhancers'. At the 3' ends of genes, treatment with THZ1 suppressed RNA polymerase 'read through' at the end of the last exon, which resembled a phenotype associated with a mutant RNA polymerase with slower elongation rates. Consistent with this hypothesis, polyA site-sequencing (PolyA-seq) did not detect differences in poly A sites after THZ1 treatment. PROseq analysis after short treatments with THZ1 suggested that these 3' effects were due to altered CDK7 activity at the 5' end of long genes, and were likely to be due to slower rates of elongation.


Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/genética , Regulação Leucêmica da Expressão Gênica , Fenilenodiaminas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , RNA Polimerase II/genética , Região 3'-Flanqueadora , Região 5'-Flanqueadora/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Óxidos N-Cíclicos , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Flavonoides/farmacologia , Humanos , Indolizinas , Células Mieloides/metabolismo , Células Mieloides/patologia , Piperazinas/farmacologia , Piperidinas/farmacologia , Piperidonas/farmacologia , Piridinas/farmacologia , Compostos de Piridínio/farmacologia , Pirróis/farmacologia , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , Translocação Genética , Quinase Ativadora de Quinase Dependente de Ciclina
5.
Nucleic Acids Res ; 45(13): e121, 2017 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-28460090

RESUMO

The genome-wide identification of microRNA transcription start sites (miRNA TSSs) is essential for understanding how miRNAs are regulated in development and disease. In this study, we developed mirSTP (mirna transcription Start sites Tracking Program), a probabilistic model for identifying active miRNA TSSs from nascent transcriptomes generated by global run-on sequencing (GRO-seq) and precision run-on sequencing (PRO-seq). MirSTP takes advantage of characteristic bidirectional transcription signatures at active TSSs in GRO/PRO-seq data, and provides accurate TSS prediction for human intergenic miRNAs at a high resolution. MirSTP performed better than existing generalized and experiment specific methods, in terms of the enrichment of various promoter-associated marks. MirSTP analysis of 27 human cell lines in 183 GRO-seq and 28 PRO-seq experiments identified TSSs for 480 intergenic miRNAs, indicating a wide usage of alternative TSSs. By integrating predicted miRNA TSSs with matched ENCODE transcription factor (TF) ChIP-seq data, we connected miRNAs into the transcriptional circuitry, which provides a valuable source for understanding the complex interplay between TF and miRNA. With mirSTP, we not only predicted TSSs for 72 miRNAs, but also identified 12 primary miRNAs with significant RNA polymerase pausing alterations after JQ1 treatment; each miRNA was further validated through BRD4 binding to its predicted promoter. MirSTP is available at http://bioinfo.vanderbilt.edu/mirSTP/.


Assuntos
MicroRNAs/genética , Regiões Promotoras Genéticas , Análise de Sequência de RNA/métodos , Algoritmos , Linhagem Celular , DNA Intergênico/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Humanos , MicroRNAs/metabolismo , Modelos Estatísticos , RNA Nuclear/genética , RNA Nuclear/metabolismo , Análise de Sequência de RNA/estatística & dados numéricos , Software , Sítio de Iniciação de Transcrição
6.
Cell Rep ; 16(7): 2003-16, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27498870

RESUMO

Bromodomain and extra-terminal domain (BET) family inhibitors offer an approach to treating hematological malignancies. We used precision nuclear run-on transcription sequencing (PRO-seq) to create high-resolution maps of active RNA polymerases across the genome in t(8;21) acute myeloid leukemia (AML), as these polymerases are exceptionally sensitive to BET inhibitors. PRO-seq identified over 1,400 genes showing impaired release of promoter-proximal paused RNA polymerases, including the stem cell factor receptor tyrosine kinase KIT that is mutated in t(8;21) AML. PRO-seq also identified an enhancer 3' to KIT. Chromosome conformation capture confirmed contacts between this enhancer and the KIT promoter, while CRISPRi-mediated repression of this enhancer impaired cell growth. PRO-seq also identified microRNAs, including MIR29C and MIR29B2, that target the anti-apoptotic factor MCL1 and were repressed by BET inhibitors. MCL1 protein was upregulated, and inhibition of BET proteins sensitized t(8:21)-containing cells to MCL1 inhibition, suggesting a potential mechanism of resistance to BET-inhibitor-induced cell death.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Leucêmica da Expressão Gênica , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Translocação Genética , Antineoplásicos/farmacologia , Azepinas/farmacologia , Linhagem Celular Tumoral , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , RNA Polimerases Dirigidas por DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Elementos Facilitadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Família Multigênica , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transcrição Gênica , Triazóis/farmacologia
7.
G3 (Bethesda) ; 2(11): 1459-72, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23173097

RESUMO

The retinoblastoma (RB) tumor suppressor protein is a transcriptional cofactor with essential roles in cell cycle and development. Physical and functional targets of RB and its paralogs p107/p130 have been studied largely in cultured cells, but the full biological context of this family of proteins' activities will likely be revealed only in whole organismal studies. To identify direct targets of the major Drosophila RB counterpart in a developmental context, we carried out ChIP-Seq analysis of Rbf1 in the embryo. The association of the protein with promoters is developmentally controlled; early promoter access is globally inhibited, whereas later in development Rbf1 is found to associate with promoter-proximal regions of approximately 2000 genes. In addition to conserved cell-cycle-related genes, a wholly unexpected finding was that Rbf1 targets many components of the insulin, Hippo, JAK/STAT, Notch, and other conserved signaling pathways. Rbf1 may thus directly affect output of these essential growth-control and differentiation pathways by regulation of expression of receptors, kinases and downstream effectors. Rbf1 was also found to target multiple levels of its own regulatory hierarchy. Bioinformatic analysis indicates that different classes of genes exhibit distinct constellations of motifs associated with the Rbf1-bound regions, suggesting that the context of Rbf1 recruitment may vary within the Rbf1 regulon. Many of these targeted genes are bound by Rbf1 homologs in human cells, indicating that a conserved role of RB proteins may be to adjust the set point of interlinked signaling networks essential for growth and development.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma de Inseto , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Genes Controladores do Desenvolvimento , Genes cdc , Homeostase/genética , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Ligação Proteica , Regulon , Proteína do Retinoblastoma/genética , Fatores de Transcrição/genética
8.
Mol Biol Cell ; 21(22): 3890-901, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20861300

RESUMO

The retinoblastoma (RB) transcriptional corepressor and related family of pocket proteins play central roles in cell cycle control and development, and the regulatory networks governed by these factors are frequently inactivated during tumorigenesis. During normal growth, these proteins are subject to tight control through at least two mechanisms. First, during cell cycle progression, repressor potential is down-regulated by Cdk-dependent phosphorylation, resulting in repressor dissociation from E2F family transcription factors. Second, RB proteins are subject to proteasome-mediated destruction during development. To better understand the mechanism for RB family protein instability, we characterized Rbf1 turnover in Drosophila and the protein motifs required for its destabilization. We show that specific point mutations in a conserved C-terminal instability element strongly stabilize Rbf1, but strikingly, these mutations also cripple repression activity. Rbf1 is destabilized specifically in actively proliferating tissues of the larva, indicating that controlled degradation of Rbf1 is linked to developmental signals. The positive linkage between Rbf1 activity and its destruction indicates that repressor function is governed in a manner similar to that described by the degron theory of transcriptional activation. Analogous mutations in the mammalian RB family member p107 similarly induce abnormal accumulation, indicating substantial conservation of this regulatory pathway.


Assuntos
Proteínas de Drosophila/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Western Blotting , Linhagem Celular , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fatores de Transcrição E2F/metabolismo , Olho/crescimento & desenvolvimento , Olho/metabolismo , Feminino , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Leupeptinas/farmacologia , Masculino , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estabilidade Proteica , Sequências Reguladoras de Ácido Nucleico/genética , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Proteína p107 Retinoblastoma-Like/genética , Proteína p107 Retinoblastoma-Like/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética
9.
J Bacteriol ; 187(20): 6909-16, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16199560

RESUMO

Vibrio vulnificus is a causative agent of serious food-borne diseases in humans related to the consumption of raw seafood. It secretes a metalloprotease that is associated with skin lesions and serious hemorrhagic complications. In this study, we purified and characterized an extracellular metalloprotease (designated as vEP) having prothrombin activation and fibrinolytic activities from V. vulnificus ATCC 29307. vEP could cleave various blood clotting-associated proteins such as prothrombin, plasminogen, fibrinogen, and factor Xa, and the cleavage could be stimulated by addition of 1 mM Mn2+ in the reaction. The cleavage of prothrombin produced active thrombin capable of converting fibrinogen to fibrin. The formation of active thrombin appeared to be transient, with further cleavage resulting in a loss of activity. The cleavage of plasminogen, however, did not produce an active plasmin. vEP could cleave all three major chains of fibrinogen without forming a clot. It could cleave fibrin polymer formed by thrombin as well as the cross-linked fibrin formed by factor XIIIa. In addition, vEP could also cleave plasma proteins such as bovine serum albumin and gamma globulin, and its broad specificity is reflected in the cleavage sites, which include Asp207-Phe208 and Thr272-Ala273 bonds in prothrombin and a Tyr80-Leu81 bond in plasminogen. Taken together, the data suggest that vEP is a broad-specificity protease that could function as a prothrombin activator and a fibrinolytic enzyme to interfere with blood homeostasis as part of the mechanism associated with the pathogenicity of V. vulnificus in humans and thereby facilitate the development of systemic infection.


Assuntos
Fibrinólise/fisiologia , Metaloproteases/metabolismo , Protrombina/metabolismo , Vibrioses/microbiologia , Vibrio vulnificus/enzimologia , Cátions/farmacologia , Inibidores Enzimáticos/farmacologia , Fator XIIIa/metabolismo , Homeostase , Humanos , Técnicas In Vitro , Metaloproteases/antagonistas & inibidores , Metaloproteases/isolamento & purificação , Plasminogênio/metabolismo , Soroalbumina Bovina/metabolismo , Especificidade por Substrato , gama-Globulinas/metabolismo
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