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1.
Nature ; 557(7703): 57-61, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29670289

RESUMO

SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.


Assuntos
Replicação do DNA , Interferon Tipo I/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Citosol/metabolismo , DNA de Cadeia Simples/metabolismo , Células HEK293 , Células HeLa , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Interferon Tipo I/imunologia , Proteína Homóloga a MRE11/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , RecQ Helicases/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/deficiência
2.
Cell Microbiol ; 22(5): e13159, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31909863

RESUMO

Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR-Cas9 has been applied successfully to these parasites providing a robust tool to study non-essential gene functions. Here, we have developed a versatile inducible system combining Di-Cre recombinase and CRISPR-Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre-recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di-Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed "universal" template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR-based (molecular cloning-free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania.


Assuntos
Técnicas de Inativação de Genes/métodos , Leishmania/genética , Sistemas CRISPR-Cas , Linhagem Celular , Edição de Genes , Integrases , Proteínas Proto-Oncogênicas c-crk/genética , Recombinação Genética , Transfecção
3.
BMC Genomics ; 16: 297, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25888089

RESUMO

BACKGROUND: Trypanosomatid parasites possess a single mitochondrion which is classically involved in the energetic metabolism of the cell, but also, in a much more original way, through its single and complex DNA (termed kinetoplast), in the correct progress of cell division. In order to identify proteins potentially involved in the cell cycle, we performed RNAi knockdowns of 101 genes encoding mitochondrial proteins using procyclic cells of Trypanosoma brucei. RESULTS: A major cell growth reduction was observed in 10 cases and a moderate reduction in 29 other cases. These data are overall in agreement with those previously obtained by a case-by-case approach performed on chromosome 1 genes, and quantitatively with those obtained by "high-throughput phenotyping using parallel sequencing of RNA interference targets" (RIT-seq). Nevertheless, a detailed analysis revealed many qualitative discrepancies with the RIT-seq-based approach. Moreover, for 37 out of 39 mutants for which a moderate or severe growth defect was observed here, we noted abnormalities in the cell cycle progress, leading to increased proportions of abnormal cell cycle stages, such as cells containing more than 2 kinetoplasts (K) and/or more than 2 nuclei (N), and modified proportions of the normal phenotypes (1N1K, 1N2K and 2N2K). CONCLUSIONS: These data, together with the observation of other abnormal phenotypes, show that all the corresponding mitochondrial proteins are involved, directly or indirectly, in the correct progress or, less likely, in the regulation, of the cell cycle in T. brucei. They also show how post-genomics analyses performed on a case-by-case basis may yield discrepancies with global approaches.


Assuntos
Proteínas Mitocondriais/metabolismo , Proteínas de Protozoários/metabolismo , Interferência de RNA , Trypanosoma brucei brucei/metabolismo , Divisão Celular/fisiologia , Citocinese/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Oligonucleotídeos/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento
4.
Appl Environ Microbiol ; 78(2): 481-92, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22081576

RESUMO

Degradation of the quorum-sensing (QS) signals known as N-acylhomoserine lactones (AHL) by soil bacteria may be useful as a beneficial trait for protecting crops, such as potato plants, against the worldwide pathogen Pectobacterium. In this work, analytical chemistry and microbial and molecular approaches were combined to explore and compare biostimulation of native and introduced AHL-degrading Rhodococcus erythropolis populations in the rhizosphere of potato plants cultivated in farm greenhouses under hydroponic conditions. We first identified gamma-heptalactone (GHL) as a novel biostimulating agent that efficiently promotes plant root colonization by AHL-degrading R. erythropolis population. We also characterized an AHL-degrading biocontrol R. erythropolis isolate, R138, which was introduced in the potato rhizosphere. Moreover, root colonization by AHL-degrading bacteria receiving different combinations of GHL and R138 treatments was compared by using a cultivation-based approach (percentage of AHL-degrading bacteria), pyrosequencing of PCR-amplified rrs loci (total bacterial community), and quantitative PCR (qPCR) of the qsdA gene, which encodes an AHL lactonase in R. erythropolis. Higher densities of the AHL-degrading R. erythropolis population in the rhizosphere were observed when GHL treatment was associated with biocontrol strain R138. Under this condition, the introduced R. erythropolis population displaced the native R. erythropolis population. Finally, chemical analyses revealed that GHL, gamma-caprolactone (GCL), and their by-products, gamma-hydroxyheptanoic acid and gamma-hydroxycaproic acid, rapidly disappeared from the rhizosphere and did not accumulate in plant tissues. This integrative study highlights biostimulation as a potential innovative approach for improving root colonization by beneficial bacteria.


Assuntos
Acil-Butirolactonas/metabolismo , Percepção de Quorum , Rhodococcus/isolamento & purificação , Rhodococcus/fisiologia , Técnicas Bacteriológicas , Técnicas de Química Analítica , Raízes de Plantas/microbiologia , Rizosfera , Rhodococcus/genética , Rhodococcus/crescimento & desenvolvimento , Análise de Sequência de DNA , Solanum tuberosum/microbiologia
5.
Nat Commun ; 11(1): 3940, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769985

RESUMO

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.


Assuntos
Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , Estruturas R-Loop/genética , Regiões Terminadoras Genéticas/genética , Transcrição Gênica , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/genética , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Fosforilação , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo
6.
Oncogene ; 39(13): 2676-2691, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32020053

RESUMO

Chromatin organization is essential for appropriate interpretation of the genetic information. Here, we demonstrated that the chromatin-associated proteins HP1 are dispensable for hepatocytes survival but are essential within hepatocytes to prevent liver tumor development in mice with HP1ß being pivotal in these functions. Yet, we found that the loss of HP1 per se is not sufficient to induce cell transformation but renders cells more resistant to specific stress such as the expression of oncogenes and thus in fine, more prone to cell transformation. Molecular characterization of HP1-Triple KO premalignant livers and BMEL cells revealed that HP1 are essential for the maintenance of heterochromatin organization and for the regulation of specific genes with most of them having well characterized functions in liver functions and homeostasis. We further showed that some specific retrotransposons get reactivated upon loss of HP1, correlating with overexpression of genes in their neighborhood. Interestingly, we found that, although HP1-dependent genes are characterized by enrichment H3K9me3, this mark does not require HP1 for its maintenance and is not sufficient to maintain gene repression in absence of HP1. Finally, we demonstrated that the loss of TRIM28 association with HP1 recapitulated several phenotypes induced by the loss of HP1 including the reactivation of some retrotransposons and the increased incidence of liver cancer development. Altogether, our findings indicate that HP1 proteins act as guardians of liver homeostasis to prevent tumor development by modulating multiple chromatin-associated events within both the heterochromatic and euchromatic compartments, partly through regulation of the corepressor TRIM28 activity.


Assuntos
Transformação Celular Neoplásica/genética , Proteínas Cromossômicas não Histona/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Animais , Linhagem Celular , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Modelos Animais de Doenças , Feminino , Hepatócitos , Heterocromatina/metabolismo , Humanos , Fígado/citologia , Fígado/patologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Knockout , Ligação Proteica/genética , RNA-Seq , Retroelementos/genética , Proteína 28 com Motivo Tripartido/metabolismo
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