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1.
Nature ; 590(7847): 655-659, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33473214

RESUMO

Break-induced replication (BIR) repairs one-ended double-strand breaks in DNA similar to those formed by replication collapse or telomere erosion, and it has been implicated in the initiation of genome instability in cancer and other human diseases1,2. Previous studies have defined the enzymes that are required for BIR1-5; however, understanding of initial and extended BIR synthesis, and of how the migrating D-loop proceeds through known replication roadblocks, has been precluded by technical limitations. Here we use a newly developed assay to show that BIR synthesis initiates soon after strand invasion and proceeds more slowly than S-phase replication. Without primase, leading strand synthesis is initiated efficiently, but is unable to proceed beyond 30 kilobases, suggesting that primase is needed for stabilization of the nascent leading strand. DNA synthesis can initiate in the absence of Pif1 or Pol32, but does not proceed efficiently. Interstitial telomeric DNA disrupts and terminates BIR progression, and BIR initiation is suppressed by transcription proportionally to the transcription level. Collisions between BIR and transcription lead to mutagenesis and chromosome rearrangements at levels that exceed instabilities induced by transcription during normal replication. Together, these results provide fundamental insights into the mechanism of BIR and how BIR contributes to genome instability.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Saccharomyces cerevisiae , Cromossomos Fúngicos/genética , DNA Helicases/deficiência , DNA Primase/metabolismo , DNA Fúngico/biossíntese , DNA Polimerase Dirigida por DNA/deficiência , Instabilidade Genômica , Cinética , Mutagênese , Mutação , Fase S , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Telômero/genética , Fatores de Tempo , Transcrição Gênica
2.
Nature ; 581(7807): 209-214, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32405004

RESUMO

Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions-particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions-are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates-including the nucleolus, Cajal bodies, stress granules and P-bodies-implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates.


Assuntos
Espaço Intracelular/química , Espaço Intracelular/metabolismo , Organelas/química , Organelas/metabolismo , Termodinâmica , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Corpos Enovelados/química , Corpos Enovelados/metabolismo , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/deficiência , Células HeLa , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleofosmina , Transição de Fase , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , RNA Helicases/deficiência , Proteínas com Motivo de Reconhecimento de RNA/deficiência , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA , Ribossomos/química , Ribossomos/metabolismo
3.
Arterioscler Thromb Vasc Biol ; 44(8): 1784-1798, 2024 08.
Artigo em Inglês | MEDLINE | ID: mdl-38868942

RESUMO

BACKGROUND: The chromatin-remodeling enzymes BRG1 (brahma-related gene 1) and CHD4 (chromodomain helicase DNA-binding protein 4) independently regulate the transcription of genes critical for vascular development, but their coordinated impact on vessels in late-stage embryos has not been explored. METHODS: In this study, we genetically deleted endothelial Brg1 and Chd4 in mixed background mice (Brg1fl/fl;Chd4fl/fl;VE-Cadherin-Cre), and littermates that were negative for Cre recombinase were used as controls. Tissues were analyzed by immunostaining, immunoblot, and flow cytometry. Quantitative reverse transcription polymerase chain reaction was used to determine gene expression, and chromatin immunoprecipitation revealed gene targets of BRG1 and CHD4 in cultured endothelial cells. RESULTS: We found Brg1/Chd4 double mutants grew normally but died soon after birth with small and compact lungs. Despite having normal cellular composition, distal air sacs of the mutant lungs displayed diminished ECM (extracellular matrix) components and TGFß (transforming growth factor-ß) signaling, which typically promotes ECM synthesis. Transcripts for collagen- and elastin-related genes and the TGFß ligand Tgfb1 were decreased in mutant lung endothelial cells, but genetic deletion of endothelial Tgfb1 failed to recapitulate the small lungs and ECM defects seen in Brg1/Chd4 mutants. We instead found several ECM genes to be direct targets of BRG1 and CHD4 in cultured endothelial cells. CONCLUSIONS: Collectively, our data highlight essential roles for endothelial chromatin-remodeling enzymes in promoting ECM deposition in the distal lung tissue during the saccular stage of embryonic lung development.


Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases , Células Endoteliais , Regulação da Expressão Gênica no Desenvolvimento , Pulmão , Proteínas Nucleares , Fatores de Transcrição , Animais , DNA Helicases/metabolismo , DNA Helicases/genética , DNA Helicases/deficiência , Pulmão/embriologia , Pulmão/metabolismo , Pulmão/enzimologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Células Endoteliais/metabolismo , Células Endoteliais/enzimologia , Camundongos Knockout , Transdução de Sinais , Matriz Extracelular/metabolismo , Camundongos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Camundongos Endogâmicos C57BL , Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Fenótipo , Humanos , Organogênese
4.
Cell Mol Life Sci ; 81(1): 339, 2024 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-39120648

RESUMO

Senataxin is an evolutionarily conserved DNA/RNA helicase, whose dysfunctions are linked to neurodegeneration and cancer. A main activity of this protein is the removal of R-loops, which are nucleic acid structures capable to promote DNA damage and replication stress. Here we found that Senataxin deficiency causes the release of damaged DNA into extranuclear bodies, called micronuclei, triggering the massive recruitment of cGAS, the apical sensor of the innate immunity pathway, and the downstream stimulation of interferon genes. Such cGAS-positive micronuclei are characterized by defective membrane envelope and are particularly abundant in cycling cells lacking Senataxin, but not after exposure to a DNA breaking agent or in absence of the tumor suppressor BRCA1 protein, a partner of Senataxin in R-loop removal. Micronuclei with a discontinuous membrane are normally cleared by autophagy, a process that we show is impaired in Senataxin-deficient cells. The formation of Senataxin-dependent inflamed micronuclei is promoted by the persistence of nuclear R-loops stimulated by the DSIF transcription elongation complex and the engagement of EXO1 nuclease activity on nuclear DNA. Coherently, high levels of EXO1 result in poor prognosis in a subset of tumors lacking Senataxin expression. Hence, R-loop homeostasis impairment, together with autophagy failure and unscheduled EXO1 activity, elicits innate immune response through micronuclei formation in cells lacking Senataxin.


Assuntos
Autofagia , Dano ao DNA , DNA Helicases , Inflamação , Enzimas Multifuncionais , Nucleotidiltransferases , Estruturas R-Loop , RNA Helicases , Humanos , Autofagia/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/deficiência , DNA Helicases/metabolismo , DNA Helicases/genética , DNA Helicases/deficiência , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/deficiência , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Imunidade Inata , Inflamação/patologia , Inflamação/metabolismo , Inflamação/genética , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/genética , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Fosfoproteínas , RNA Helicases/metabolismo , RNA Helicases/genética
5.
Int J Gynecol Pathol ; 43(4): 354-361, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38113031

RESUMO

SMARCA4 gene encodes BRG1 , a member of the SWItch/sucrose non-fermentable protein family involved in epigenetic transcriptional regulation of important cellular processes. In the uterine corpus, SMARCA4 / BRG1 deficiency is associated with a novel class of undifferentiated uterine sarcomas, characterized by younger age onset, rhabdoid histology, focal phyllodiform architecture, high-risk pathologic findings, and dismal prognosis. Herein, we report a case of a 34-year-old Asian woman with a SMARCA4 / BRG1 -deficient uterine tumor fulfilling the clinicopathologic features of an undifferentiated uterine sarcoma. However, the tumor exhibited several unique features that have not been previously emphasized, including (1) conspicuous phyllodiform architecture recapitulating conventional adenosarcoma, (2) rhabdoid tumor cells forming cords and keratin-positive cohesive epithelial islands, and (3) cooccurrence with a spatially distinct and discrete endometrial complex atypical hyperplasia from the rest of the proliferation. By immunohistochemistry, the tumor cells were diffusely positive for synaptophysin, whereas BRG1 was lost. Pertinent molecular findings included frameshift mutations in the SMARCA4 gene, mutations in histone modification and chromatin remodeling genes, including KMT2C , ARID1B , KAT6A , and NCOR1 , and mutations in Wnt signaling involving APC and CTNNB1 . Copy number gain in MDM2 and CDK4 were also identified. The tumor mutation burden was intermediate (6.8/MB) and it was microsatellite stable. On balance, our case exhibited morphologic and molecular features that overlap with (1) an undifferentiated uterine sarcoma, (2) an adenosarcoma with sarcomatous overgrowth, and (3) a mixed adenosarcoma and undifferentiated endometrial carcinoma. These hybrid features further expand the molecular-morphologic spectrum of SMARCA4 / BRG1 -deficient uterine neoplasms.


Assuntos
Adenossarcoma , DNA Helicases , Proteínas Nucleares , Fatores de Transcrição , Neoplasias Uterinas , Humanos , Feminino , DNA Helicases/genética , DNA Helicases/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/deficiência , Adulto , Adenossarcoma/patologia , Adenossarcoma/genética , Neoplasias Uterinas/patologia , Neoplasias Uterinas/genética , Imuno-Histoquímica , Carcinoma/patologia , Carcinoma/genética
6.
Nature ; 564(7735): 287-290, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30518856

RESUMO

Insertions of mobile elements1-4, mitochondrial DNA5 and fragments of nuclear chromosomes6 at DNA double-strand breaks (DSBs) threaten genome integrity and are common in cancer7-9. Insertions of chromosome fragments at V(D)J recombination loci can stimulate antibody diversification10. The origin of insertions of chromosomal fragments and the mechanisms that prevent such insertions remain unknown. Here we reveal a yeast mutant, lacking evolutionarily conserved Dna2 nuclease, that shows frequent insertions of sequences between approximately 0.1 and 1.5 kb in length into DSBs, with many insertions involving multiple joined DNA fragments. Sequencing of around 500 DNA inserts reveals that they originate from Ty retrotransposons (8%), ribosomal DNA (rDNA) (15%) and from throughout the genome, with preference for fragile regions such as origins of replication, R-loops, centromeres, telomeres or replication fork barriers. Inserted fragments are not lost from their original loci and therefore represent duplications. These duplications depend on nonhomologous end-joining (NHEJ) and Pol4. We propose a model in which alternative processing of DNA structures arising in Dna2-deficient cells can result in the release of DNA fragments and their capture at DSBs. Similar DNA insertions at DSBs are expected to occur in any cells with linear extrachromosomal DNA fragments.


Assuntos
Quebra Cromossômica , Duplicação Cromossômica , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , DNA Helicases/deficiência , Mutagênese Insercional/genética , Saccharomyces cerevisiae/genética , Centrômero/genética , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Polimerase beta/metabolismo , Replicação do DNA/genética , DNA Ribossômico/genética , Origem de Replicação/genética , Retroelementos/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/genética
7.
Mol Cell ; 61(3): 405-418, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26774285

RESUMO

DNA double-strand break repair by homologous recombination is initiated by the formation of 3' single-stranded DNA (ssDNA) overhangs by a process termed end resection. Although much focus has been given to the decision to initiate resection, little is known of the mechanisms that regulate the ongoing formation of ssDNA tails. Here we report that DNA helicase B (HELB) underpins a feedback inhibition mechanism that curtails resection. HELB is recruited to ssDNA by interacting with RPA and uses its 5'-3' ssDNA translocase activity to inhibit EXO1 and BLM-DNA2, the nucleases catalyzing resection. HELB acts independently of 53BP1 and is exported from the nucleus as cells approach S phase, concomitant with the upregulation of resection. Consistent with its role as a resection antagonist, loss of HELB results in PARP inhibitor resistance in BRCA1-deficient tumor cells. We conclude that mammalian DNA end resection triggers its own inhibition via the recruitment of HELB.


Assuntos
Reparo do DNA por Junção de Extremidades , DNA Helicases/metabolismo , Neoplasias Mamárias Experimentais/enzimologia , Animais , Proteína BRCA1/genética , DNA Helicases/deficiência , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Retroalimentação Fisiológica , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Interferência de RNA , RecQ Helicases/genética , RecQ Helicases/metabolismo , Fase S , Fatores de Tempo , Transfecção , Proteínas Supressoras de Tumor/genética
8.
PLoS Genet ; 17(5): e1008919, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34003859

RESUMO

An essential component of the homologous recombination machinery in eukaryotes, the RAD54 protein is a member of the SWI2/SNF2 family of helicases with dsDNA-dependent ATPase, DNA translocase, DNA supercoiling and chromatin remodelling activities. It is a motor protein that translocates along dsDNA and performs multiple functions in homologous recombination. In particular, RAD54 is an essential cofactor for regulating RAD51 activity. It stabilizes the RAD51 nucleofilament, remodels nucleosomes, and stimulates the homology search and strand invasion activities of RAD51. Accordingly, deletion of RAD54 has dramatic consequences on DNA damage repair in mitotic cells. In contrast, its role in meiotic recombination is less clear. RAD54 is essential for meiotic recombination in Drosophila and C. elegans, but plays minor roles in yeast and mammals. We present here characterization of the roles of RAD54 in meiotic recombination in the model plant Arabidopsis thaliana. Absence of RAD54 has no detectable effect on meiotic recombination in otherwise wild-type plants but RAD54 becomes essential for meiotic DSB repair in absence of DMC1. In Arabidopsis, dmc1 mutants have an achiasmate meiosis, in which RAD51 repairs meiotic DSBs. Lack of RAD54 leads to meiotic chromosomal fragmentation in absence of DMC1. The action of RAD54 in meiotic RAD51 activity is thus mainly downstream of the role of RAD51 in supporting the activity of DMC1. Equivalent analyses show no effect on meiosis of combining dmc1 with the mutants of the RAD51-mediators RAD51B, RAD51D and XRCC2. RAD54 is thus required for repair of meiotic DSBs by RAD51 and the absence of meiotic phenotype in rad54 plants is a consequence of RAD51 playing a RAD54-independent supporting role to DMC1 in meiotic recombination.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Meiose , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/deficiência , DNA Helicases/genética , Proteínas de Ligação a DNA , Genes Essenciais , Meiose/genética , Mutação , Rad51 Recombinase/genética , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Proteínas Repressoras
9.
Pathologica ; 116(3): 163-169, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38979590

RESUMO

The 5th WHO classification of thoracic tumours includes thoracic SMARCA4-deficient undifferentiated tumour (SMARCA4-UT) among the "other epithelial tumours of the lung" chapter. Herein, we present a case of undifferentiated thoracic neoplasm with retention of SMARCA4 expression, lack of NUT fusion protein and loss of SMARCB1/INI1 expression. After presenting the clinical and pathological features of the tumour, we carried out a review of the literature on the same topic. Albeit very rare, we believe this entity should be included in the heterogeneous group of undifferentiated neoplasms of the thorax.


Assuntos
DNA Helicases , Proteína SMARCB1 , Neoplasias Torácicas , Fatores de Transcrição , Humanos , Proteína SMARCB1/deficiência , Proteína SMARCB1/genética , Fatores de Transcrição/genética , Fatores de Transcrição/deficiência , Neoplasias Torácicas/patologia , Neoplasias Torácicas/genética , DNA Helicases/deficiência , DNA Helicases/genética , Proteínas Nucleares/genética , Proteínas Nucleares/deficiência , Masculino , Feminino , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Pessoa de Meia-Idade , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/diagnóstico
10.
J Immunol ; 206(10): 2453-2467, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33941659

RESUMO

The detection of intracellular nucleic acids is a fundamental mechanism of host defense against infections. The dysregulated nucleic acid sensing, however, is a major cause for a number of autoimmune diseases. In this study, we report that GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is critical for both intracellular DNA- and RNA-induced immune responses. We found that in both human and mouse cells, the deletion of G3BP1 led to the dampened cGAS activation by DNA and the insufficient binding of RNA by RIG-I. We further found that resveratrol (RSVL), a natural compound found in grape skin, suppressed both intracellular DNA- and RNA-induced type I IFN production through inhibiting G3BP1. Importantly, using experimental mouse models for Aicardi-Goutières syndrome, an autoimmune disorder found in humans, we demonstrated that RSVL effectively alleviated intracellular nucleic acid-stimulated autoimmune responses. Thus, our study demonstrated a broader role of G3BP1 in sensing different kinds of intracellular nucleic acids and presented RSVL as a potential treatment for autoimmune conditions caused by dysregulated nucleic acid sensing.


Assuntos
Autoimunidade/genética , DNA Helicases/deficiência , DNA Helicases/metabolismo , Espaço Intracelular/metabolismo , Ácidos Nucleicos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/deficiência , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/deficiência , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Transdução de Sinais/genética , Células A549 , Animais , Autoimunidade/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Espaço Intracelular/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/genética , Resveratrol/administração & dosagem , Transdução de Sinais/imunologia , Transfecção
11.
Nature ; 542(7642): 484-488, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28166537

RESUMO

Synthetic lethality and collateral lethality are two well-validated conceptual strategies for identifying therapeutic targets in cancers with tumour-suppressor gene deletions. Here, we explore an approach to identify potential synthetic-lethal interactions by screening mutually exclusive deletion patterns in cancer genomes. We sought to identify 'synthetic-essential' genes: those that are occasionally deleted in some cancers but are almost always retained in the context of a specific tumour-suppressor deficiency. We also posited that such synthetic-essential genes would be therapeutic targets in cancers that harbour specific tumour-suppressor deficiencies. In addition to known synthetic-lethal interactions, this approach uncovered the chromatin helicase DNA-binding factor CHD1 as a putative synthetic-essential gene in PTEN-deficient cancers. In PTEN-deficient prostate and breast cancers, CHD1 depletion profoundly and specifically suppressed cell proliferation, cell survival and tumorigenic potential. Mechanistically, functional PTEN stimulates the GSK3ß-mediated phosphorylation of CHD1 degron domains, which promotes CHD1 degradation via the ß-TrCP-mediated ubiquitination-proteasome pathway. Conversely, PTEN deficiency results in stabilization of CHD1, which in turn engages the trimethyl lysine-4 histone H3 modification to activate transcription of the pro-tumorigenic TNF-NF-κB gene network. This study identifies a novel PTEN pathway in cancer and provides a framework for the discovery of 'trackable' targets in cancers that harbour specific tumour-suppressor deficiencies.


Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes Essenciais/genética , Neoplasias/metabolismo , Neoplasias/patologia , PTEN Fosfo-Hidrolase/deficiência , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , DNA Helicases/química , DNA Helicases/deficiência , DNA Helicases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Masculino , Metilação , Terapia de Alvo Molecular , NF-kappa B/metabolismo , Neoplasias/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteólise , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação , Proteínas Contendo Repetições de beta-Transducina/metabolismo
12.
EMBO J ; 37(14)2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-29773570

RESUMO

DNA2 is a nuclease/helicase that is involved in Okazaki fragment maturation, replication fork processing, and end resection of DNA double-strand breaks. Similar such helicase activity for resolving secondary structures and structure-specific nuclease activity are needed during DNA replication to process the chromosome-specific higher order repeat units present in the centromeres of human chromosomes. Here, we show that DNA2 binds preferentially to centromeric DNA The nuclease and helicase activities of DNA2 are both essential for resolution of DNA structural obstacles to facilitate DNA replication fork movement. Loss of DNA2-mediated clean-up mechanisms impairs centromeric DNA replication and CENP-A deposition, leading to activation of the ATR DNA damage checkpoints at centromeric DNA regions and late-S/G2 cell cycle arrest. Cells that escape arrest show impaired metaphase plate formation and abnormal chromosomal segregation. Furthermore, the DNA2 inhibitor C5 mimics DNA2 knockout and synergistically kills cancer cells when combined with an ATR inhibitor. These findings provide mechanistic insights into how DNA2 supports replication of centromeric DNA and give further insights into new therapeutic strategies.


Assuntos
Centrômero/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , Instabilidade Genômica , Ciclo Celular , Linhagem Celular , Cromossomos Humanos/metabolismo , DNA Helicases/deficiência , Humanos
13.
Oncologist ; 27(6): 501-511, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35278076

RESUMO

BACKGROUND: Thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT) are aggressive neoplasms. Data linking BAF alterations with tumor microenvironment (TME) and efficacy of immune checkpoint inhibitors (ICI) are contradictory. The TME of SMARCA4-UT and their response to ICI are unknown. MATERIALS AND METHODS: Patients diagnosed with SMARCA4-UT in our institution were included. Immunostainings for tertiary lymphoid structures (TLS), immune cell markers, and checkpoints were assessed. Validation was performed using an independent transcriptome dataset including SMARCA4-UT, non-small cell lung cancers (NSCLC) with/without SMARCA4 mutations, and unclassified thoracic sarcomas (UTS). CXCL9 and PD-L1 expressions were assessed in NSCLC and thoracic fibroblast cell lines, with/without SMARCA4 knockdown, treated with/without interferon gamma. RESULTS: Nine patients were identified. All samples but one showed no TLS, consistent with an immune desert TME phenotype. Four patients received ICI as part of their treatment, but the only one who responded, had a tumor with a TLS and immune-rich TME. Unsupervised clustering of the validation cohort using immune cell scores identified 2 clusters associated with cell ontogeny and immunity (cluster 1 enriched for NSCLC independently of SMARCA4 status (n = 9/10; P = .001); cluster 2 enriched for SMARCA4-UT (n = 11/12; P = .005) and UTS (n = 5/5; P = .0005). SMARCA4 loss-of-function experiments revealed interferon-induced upregulation of CXCL9 and PD-L1 expression in the NSCLC cell line with no effect on the thoracic fibroblast cell line. CONCLUSION: SMARCA4-UT mainly have an immune desert TME with limited efficacy to ICI. TME of SMARCA4-driven tumors varies according to the cell of origin questioning the interplay between BAF alterations, cell ontogeny and immunity.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , DNA Helicases , Inibidores de Checkpoint Imunológico , Neoplasias Pulmonares , Proteínas Nucleares , Sarcoma , Neoplasias de Tecidos Moles , Neoplasias Torácicas , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/imunologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , DNA Helicases/deficiência , DNA Helicases/imunologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Proteínas Nucleares/deficiência , Proteínas Nucleares/imunologia , Sarcoma/tratamento farmacológico , Sarcoma/imunologia , Sarcoma/patologia , Neoplasias de Tecidos Moles/tratamento farmacológico , Neoplasias de Tecidos Moles/imunologia , Neoplasias de Tecidos Moles/patologia , Neoplasias Torácicas/tratamento farmacológico , Neoplasias Torácicas/imunologia , Neoplasias Torácicas/patologia , Fatores de Transcrição/imunologia , Microambiente Tumoral/imunologia
14.
J Pathol ; 255(1): 1-15, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33999421

RESUMO

Extracranial rhabdoid tumours (ECRTs) are an aggressive malignancy of infancy and early childhood. The vast majority of cases demonstrate inactivation of SMARCB1 (ECRTSMARCB1 ) on a background of a remarkably stable genome, a low mutational burden, and no other recurrent mutations. Rarely, ECRTs can harbour the alternative inactivation of SMARCA4 (ECRTSMARCA4 ) instead of SMARCB1. However, very few ECRTSMARCA4 cases have been published to date, and a systematic characterization of ECRTSMARCA4 is missing from the literature. In this study, we report the clinical, pathological, and genomic features of additional cases of ECRTSMARCA4 and show that they are comparable to those of ECRTSMARCB1. We also assess whether ECRTSMARCB1 , ECRTSMARCA4 , and small cell carcinomas of the ovary, hypercalcaemic type (SCCOHT) represent distinct or overlapping entities at a molecular level. Using DNA methylation and transcriptomics-based tumour classification approaches, we demonstrate that ECRTSMARCA4 display molecular features intermediate between SCCOHT and ECRTSMARCB1 ; however, ECRTSMARCA4 appear to be more closely related to SCCOHT by DNA methylation. Conversely, both transcriptomics and DNA methylation show a larger gap between SCCOHT and ECRTSMARCB1 , potentially supporting their continuous separate classification. Lastly, we show that ECRTSMARCA4 display concomitant lack of SMARCA4 (BRG1) and SMARCA2 (BRM) expression at the protein level, similar to what is seen in SCCOHT. Overall, these results expand our knowledge on this rare tumour type and explore the similarities and differences among entities from the 'rhabdoid tumour' spectrum. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
DNA Helicases/deficiência , Proteínas Nucleares/deficiência , Tumor Rabdoide/genética , Tumor Rabdoide/patologia , Fatores de Transcrição/deficiência , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Pré-Escolar , DNA Helicases/genética , Feminino , Humanos , Lactente , Masculino , Proteínas Nucleares/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Proteína SMARCB1/deficiência , Proteína SMARCB1/genética , Fatores de Transcrição/genética
15.
Genes Chromosomes Cancer ; 60(3): 190-209, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33252159

RESUMO

Our knowledge regarding the role of genes encoding the chromatin remodeling switch/sucrose non-fermenting (SWI/SNF) complex in the initiation and progression of gynecologic malignancies continues to evolve. This review focuses on gynecologic tumors in which the sole or primary genetic alteration is in SMARCA4 or SMARCB1, two members of the SWI/SNF chromatin remodeling complex. In this review, we present a brief overview of the classical example of such tumors, ovarian small cell carcinoma of hypercalcemic type, and then a detailed review and update of SMARCB1-deficient and SMARCA4-deficient tumors of the uterus and vulva.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/deficiência , Proteínas Nucleares/deficiência , Neoplasias Ovarianas/metabolismo , Proteína SMARCB1/deficiência , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo , Neoplasias Uterinas/metabolismo , Neoplasias Vulvares/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Feminino , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/genética , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Sarcoma/genética , Sarcoma/metabolismo , Fatores de Transcrição/genética , Neoplasias Uterinas/genética , Neoplasias Vulvares/genética
16.
Mol Hum Reprod ; 27(2)2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33337500

RESUMO

Premature loss of ovarian activity before 40 years of age is known as primary ovarian insufficiency (POI) and occurs in ∼1% of women. A more subtle decline in ovarian activity, known as premature ovarian ageing (POA), occurs in ∼10% of women. Despite the high prevalence of POA, very little is known regarding its genetic causation. Senataxin (SETX) is an RNA/DNA helicase involved in repair of oxidative stress-induced DNA damage. Homozygous mutation of SETX leads to the neurodegenerative disorder, ataxia oculomotor apraxia type 2 (AOA2). There have been reports of POI in AOA2 females suggesting a link between SETX and ovarian ageing. Here, we studied female mice lacking either one (Setx+/-) or both (Setx-/-) copies of SETX over a 12- to 14-month period. We find that DNA damage is increased in oocytes from 8-month-old Setx+/- and Setx-/- females compared with Setx+/+ oocytes leading to a marked reduction in all classes of ovarian follicles at least 4 months earlier than typically occurs in female mice. Furthermore, during a 12-month long mating trial, Setx+/- and Setx-/- females produced significantly fewer pups than Setx+/+ females from 7 months of age onwards. These data show that SETX is critical for preventing POA in mice, likely by preserving DNA integrity in oocytes. Intriguingly, heterozygous Setx loss causes an equally severe impact on ovarian ageing as homozygous Setx loss. Because heterozygous SETX disruption is less likely to produce systemic effects, SETX compromise could underpin some cases of insidious POA.


Assuntos
Dano ao DNA , DNA Helicases/deficiência , Infertilidade Feminina/metabolismo , Enzimas Multifuncionais/deficiência , Oócitos/metabolismo , Reserva Ovariana , Insuficiência Ovariana Primária/metabolismo , RNA Helicases/deficiência , Fatores Etários , Animais , Células Cultivadas , DNA Helicases/genética , Feminino , Predisposição Genética para Doença , Heterozigoto , Técnicas de Maturação in Vitro de Oócitos , Infertilidade Feminina/genética , Infertilidade Feminina/patologia , Infertilidade Feminina/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Enzimas Multifuncionais/genética , Oócitos/patologia , Fenótipo , Insuficiência Ovariana Primária/genética , Insuficiência Ovariana Primária/patologia , Insuficiência Ovariana Primária/fisiopatologia , RNA Helicases/genética
17.
Gynecol Oncol ; 160(2): 568-578, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33328126

RESUMO

OBJECTIVE: The development of effective cancer treatments depends on the availability of cell lines that faithfully recapitulate the cancer in question. This study definitively re-assigns the histologic identities of two ovarian cancer cell lines, COV434 (originally described as a granulosa cell tumour) and TOV-112D (originally described as grade 3 endometrioid carcinoma), both of which were recently suggested to represent small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), based on their shared gene expression profiles and sensitivity to EZH2 inhibitors. METHODS: For COV434 and TOV-112D, we re-reviewed the original pathology slides and obtained clinical follow-up on the patients, when available, and performed immunohistochemistry for SMARCA4, SMARCA2 and additional diagnostic markers on the original formalin-fixed, paraffin-embedded (FFPE) clinical material, when available. For COV434, we further performed whole exome sequencing and validated SMARCA4 mutations by Sanger sequencing. We studied the growth of the cell lines at baseline and upon re-expression of SMARCA4 in vitro for both cell lines and evaluated the serum calcium levels in vivo upon injection into immunodeficient mice for COV434 cells. RESULTS: The available morphological, immunohistochemical, genetic, and clinical features indicate COV434 is derived from SCCOHT, and TOV-112D is a dedifferentiated carcinoma. Transplantation of COV434 into mice leads to increased serum calcium level. Re-expression of SMARCA4 in either COV434 and TOV-112D cells suppressed their growth dramatically. CONCLUSIONS: COV434 represents a bona fide SCCOHT cell line. TOV-112D is a dedifferentiated ovarian carcinoma cell line.


Assuntos
Carcinoma Epitelial do Ovário/diagnóstico , Carcinoma de Células Pequenas/diagnóstico , Linhagem Celular Tumoral/patologia , Neoplasias Ovarianas/diagnóstico , Animais , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/patologia , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Desdiferenciação Celular/genética , Linhagem Celular Tumoral/efeitos dos fármacos , DNA Helicases/análise , DNA Helicases/deficiência , DNA Helicases/genética , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Proteínas Nucleares/análise , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fatores de Transcrição/análise , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Proc Natl Acad Sci U S A ; 115(26): 6727-6732, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29891665

RESUMO

Cardiac development relies on proper cardiomyocyte differentiation, including expression and assembly of cell-type-specific actomyosin subunits into a functional cardiac sarcomere. Control of this process involves not only promoting expression of cardiac sarcomere subunits but also repressing expression of noncardiac myofibril paralogs. This level of transcriptional control requires broadly expressed multiprotein machines that modify and remodel the chromatin landscape to restrict transcription machinery access. Prominent among these is the nucleosome remodeling and deacetylase (NuRD) complex, which includes the catalytic core subunit CHD4. Here, we demonstrate that direct CHD4-mediated repression of skeletal and smooth muscle myofibril isoforms is required for normal cardiac sarcomere formation, function, and embryonic survival early in gestation. Through transcriptomic and genome-wide analyses of CHD4 localization, we identified unique CHD4 binding sites in smooth muscle myosin heavy chain, fast skeletal α-actin, and the fast skeletal troponin complex genes. We further demonstrate that in the absence of CHD4, cardiomyocytes in the developing heart form a hybrid muscle cell that contains cardiac, skeletal, and smooth muscle myofibril components. These misexpressed paralogs intercalate into the nascent cardiac sarcomere to disrupt sarcomere formation and cause impaired cardiac function in utero. These results demonstrate the genomic and physiological requirements for CHD4 in mammalian cardiac development.


Assuntos
DNA Helicases/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/fisiologia , Miócitos Cardíacos/fisiologia , Sarcômeros/fisiologia , Animais , DNA Helicases/química , DNA Helicases/deficiência , Feminino , Técnicas de Silenciamento de Genes , Genes Letais , Coração/diagnóstico por imagem , Coração/embriologia , Cardiopatias Congênitas/diagnóstico por imagem , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/patologia , Masculino , Camundongos , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Miofibrilas/metabolismo , Miofibrilas/patologia , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Sarcômeros/ultraestrutura , Transcrição Gênica , Ultrassonografia Pré-Natal
19.
J Cell Mol Med ; 24(19): 11592-11602, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32845093

RESUMO

Brg1 and Hippo signalling pathway are abnormally expressed in many malignant tumours, especially in Hepatocellular carcinoma, but their role in liver regeneration (LR) is unknown. In our research, we investigated the role of Brg1 and Hippo signalling pathway in hepatocyte proliferation and LR. Following 2/3 partial hepatectomy (PH) in liver-specific Brg1 deleted mice (Brg1-/-) (KO) mice and sex-matched wild-type (WT), depletion of Brg1 in mouse embryos caused liver cell growth disorders and significantly decreased expression of miR-187-5p. We identified LATS1 as a target gene of miR-187-5p and the introduction of miR-187-5p decrease the expression of LATS1 and inactivated the Hippo signalling pathway, which facilitated the expression of cell cycle-related proteins, and rescues the inhibitory effect of Brg1 in LR. Taken together, our findings suggested that deletion of Brg1 inhibits hepatocyte proliferation and LR by targeting miR-187-5p dependent on Hippo signalling pathway.


Assuntos
DNA Helicases/metabolismo , Regeneração Hepática/genética , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , DNA Helicases/deficiência , Hepatócitos/metabolismo , Via de Sinalização Hippo , Camundongos Knockout , MicroRNAs/genética , Proteínas Nucleares/deficiência , Especificidade de Órgãos , Fatores de Transcrição/deficiência
20.
Mol Microbiol ; 111(2): 338-353, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30379365

RESUMO

All organisms possess DNA repair pathways that are used to maintain the integrity of their genetic material. Although many DNA repair pathways are well understood, new pathways continue to be discovered. Here, we report an antibiotic specific DNA repair pathway in Bacillus subtilis that is composed of a previously uncharacterized helicase (mrfA) and exonuclease (mrfB). Deletion of mrfA and mrfB results in sensitivity to the DNA damaging agent mitomycin C, but not to any other type of DNA damage tested. We show that MrfAB function independent of canonical nucleotide excision repair, forming a novel excision repair pathway. We demonstrate that MrfB is a metal-dependent exonuclease and that the N-terminus of MrfB is required for interaction with MrfA. We determined that MrfAB failed to unhook interstrand cross-links in vivo, suggesting that MrfAB are specific to the monoadduct or the intrastrand cross-link. A phylogenetic analysis uncovered MrfAB homologs in diverse bacterial phyla, and cross-complementation indicates that MrfAB function is conserved in closely related species. B. subtilis is a soil dwelling organism and mitomycin C is a natural antibiotic produced by the soil bacterium Streptomyces lavendulae. The specificity of MrfAB suggests that these proteins are an adaptation to environments with mitomycin producing bacteria.


Assuntos
Antibacterianos/metabolismo , Bacillus subtilis/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA , DNA Bacteriano/metabolismo , Exodesoxirribonucleases/metabolismo , DNA Helicases/deficiência , DNA Helicases/genética , Exodesoxirribonucleases/deficiência , Exodesoxirribonucleases/genética , Deleção de Genes , Mitomicina/metabolismo , Filogenia , Homologia de Sequência
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