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1.
Rinsho Ketsueki ; 60(7): 800-809, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31391370

RESUMO

In myeloid neoplasms, deletions of the long arm of chromosome 5 del(5q) and 7 (-7/del(7q) ) are common karyotypic abnormalities. The concurrence of del(5q) and -7/del(7q) accounts for poor prognosis in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Comprehensive analysis of copy number abnormalities and genetic mutations related to del(5q) and -7/del(7q) revealed previously cryptic pathophysiology, leading to frequent hemizygous/homozygous mutations and haploinsufficiency. In addition, detailed somatic mutations on chr5q were detected using whole-exome sequencing. CSNK1A1 and G3BP1 are located within the common deleted regions (CDRs) (5q31.1-5q33.1), and another driver gene DDX41 is present in the more telomeric region (5q35.3). All the genes mentioned above exhibited haploinsufficiency because of deletions, and low expression of G3BP1 and DDX41 correlated with poor survival. The related mutational events outside of chr5q, TP53 mutation is most frequently observed in del(5q) cases. Regarding -7/del(7q), 3 CDRs were located in 7q22, 7q34, and 7q35-36. Somatic mutations of the corresponding genes to each CDR (CUX1: 7q22, LUC7L2: 7q34, EZH2: 7q35-36) were identified, indicating that the loss of function or haploinsufficiency might result in the downstream pathological consequences. These recent findings have remarkably offered insights into genetic and clinical consequences in MDS/AML cases with del(5q) and -7/del(7q).


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Cromossomos Humanos Par 7/genética , Leucemia Mieloide Aguda/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética
2.
Life Sci ; 232: 116679, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31340168

RESUMO

AIMS: Amplified in liver cancer 1 gene (ALC1), a recently identified oncogene, was reported to be overexpressed in esophageal cancer cell lines and identified as a target oncogene in esophageal cancer pathogenesis. However, little literature is available to illustrate its significance in cisplatin resistance of esophageal squamous cell carcinoma (ESCC) cells. The aim of the current study was to investigate the effect of ALC1 on cisplatin cytotoxicity of ESCC cells and to study the potential mechanisms. MAIN METHODS: ALC1 at mRNA and protein levels were detected by qRT-PCR and western blot, respectively. Cell viability was evaluated using CCK-8 assay. Apoptosis was assessed using caspase-3/7 activity assay and flow cytometry analysis. Glycolysis level was evaluated by measuring glucose consumption and lactate production. The protein levels of p-protein kinase B (Akt) and Akt were determined by western blot. KEY FINDINGS: ALC1 was highly expressed in ESCC cells compared with human normal esophageal epithelial Het-1A cells. ALC1 knockdown suppressed the viability, induced apoptosis and enhanced cisplatin cytotoxicity in ESCC cells. In addition, ALC1 knockdown inhibited glycolysis and inactivated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in ESCC cells. Mechanistically, activation of the PI3K/Akt pathway by 740Y-P blocked the effects of ALC1 knockdown on cisplatin cytotoxicity and glycolysis in ESCC cells. In contrast, inhibition of the PI3K/Akt pathway by LY294002 or glycolysis by 2-deoxyglucose resisted the effect of ALC1 overexpression on cisplatin cytotoxicity in ESCC cells. SIGNIFICANCE: ALC1 knockdown enhanced cisplatin cytotoxicity of ESCC cells by inhibition of glycolysis through inactivation of the PI3K/Akt pathway.


Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/patologia , Cisplatino/farmacologia , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Neoplasias Esofágicas/patologia , Técnicas de Silenciamento de Genes , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Apoptose , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/metabolismo , Ativação Enzimática , Neoplasias Esofágicas/enzimologia , Neoplasias Esofágicas/metabolismo , Glicólise , Humanos , Células Tumorais Cultivadas
3.
Acta Cytol ; 63(5): 438-444, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31230044

RESUMO

OBJECTIVE: Evidence shows that the switch/sucrose nonfermenting chromatin remodeling complex plays a critical role in DNA repair, cancer progression and dedifferentiation. BRG1 is one of its key catalytic subunits. While the loss of BRG1 expression by immunocytochemistry has been identified in a subset of malignancies arising in various sites with undifferentiated/rhabdoid morphology and poor prognosis, the underlying basis for its loss is unclear. METHODS: A retrospective search was conducted in our cytopathology archive for undifferentiated malignant tumors with rhabdoid phenotype and BRG1 loss. Clinical information was obtained from electronic medical records. Next-generation sequencing was performed following macro-dissection of paraffin-embedded cellblock tissue. RESULTS: Three cases were identified; all presented with widely metastatic disease with no previously diagnosed primary malignancy, and subsequently died within 6 months of initial presentation. Cytologically, the aspirates showed dyshesive and undifferentiated cells with rhabdoid features. Extensive immunocytochemical workup demonstrated immunoreactivity with vimentin only and could not establish a specific lineage. BRG1 expression was absent, while INI1 expression was retained. Two cases harbored deleterious mutations in BRG1/SMARCA4. Pathogenic mutations in TP53 were identified in all tumors. CONCLUSIONS: BRG1 deficiency reflects underlying mutation in SMARCA4 gene in some but not all cases, suggesting that additional mechanisms may be causing BRG1 silencing. Pathogenic mutations in TP53 in all tumors are consistent with their highly aggressive nature. Recognizing the cytomorphology of this group of neoplasms and confirming their BRG1-deficient status by immunocytochemistry not only has prognostic implications, but may also impart potentially therapeutic value in the near future.


Assuntos
Biomarcadores Tumorais/genética , Diferenciação Celular , DNA Helicases/genética , Neoplasias Pulmonares/genética , Mutação , Proteínas Nucleares/genética , Tumor Rabdoide/genética , Neoplasias da Glândula Submandibular/genética , Fatores de Transcrição/genética , Idoso , Biomarcadores Tumorais/deficiência , Biópsia por Agulha Fina , DNA Helicases/deficiência , Análise Mutacional de DNA , Evolução Fatal , Feminino , Predisposição Genética para Doença , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/deficiência , Fenótipo , Valor Preditivo dos Testes , Estudos Retrospectivos , Tumor Rabdoide/enzimologia , Tumor Rabdoide/patologia , Tumor Rabdoide/terapia , Neoplasias da Glândula Submandibular/enzimologia , Neoplasias da Glândula Submandibular/patologia , Neoplasias da Glândula Submandibular/terapia , Fatores de Transcrição/deficiência , Resultado do Tratamento , Proteína Supressora de Tumor p53/genética
4.
Nat Commun ; 10(1): 2885, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253769

RESUMO

Nucleotide excision repair (NER) is the major DNA repair pathway that removes UV-induced and bulky DNA lesions. There is currently no structure of NER intermediates, which form around the large multisubunit transcription factor IIH (TFIIH). Here we report the cryo-EM structure of an NER intermediate containing TFIIH and the NER factor XPA. Compared to its transcription conformation, the TFIIH structure is rearranged such that its ATPase subunits XPB and XPD bind double- and single-stranded DNA, consistent with their translocase and helicase activities, respectively. XPA releases the inhibitory kinase module of TFIIH, displaces a 'plug' element from the DNA-binding pore in XPD, and together with the NER factor XPG stimulates XPD activity. Our results explain how TFIIH is switched from a transcription to a repair factor, and provide the basis for a mechanistic analysis of the NER pathway.


Assuntos
Reparo do DNA , Fator de Transcrição TFIIH/metabolismo , Adenosina Trifosfatases , Animais , Linhagem Celular , Clonagem Molecular , Microscopia Crioeletrônica , DNA/química , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Escherichia coli , Regulação da Expressão Gênica , Humanos , Insetos , Modelos Químicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo
5.
BMC Med Genet ; 20(1): 93, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31146700

RESUMO

BACKGROUND: CHARGE syndrome is characterized by coloboma, heart defects, choanal atresia, growth retardation, genitourinary malformation and ear abnormalities. The chromodomain helicase DNA-binding protein 7 (CHD7) gene is the major cause of CHARGE syndrome and is inherited in an autosomal dominant manner. Currently, the phenotype spectrum of CHARGE syndrome in neonatal population remain elusive. We aimed to investigate the phenotype spectrum of neonatal patients suspected to have CHARGE syndrome with pathogenic or likely pathogenic variants in the CHD7 gene. METHODS: We pooled next-generation sequencing data from the Neonatal Birth Defects Cohort (NBDC, ClinicalTrials.gov Identifier: NCT02551081) in Children's Hospital of Fudan University. The pathogenicity of novel variants was analyzed by bioinformatic and genetic analyses. Clinical information collection, Sanger sequencing and follow-up interviews were performed when possible. Cranial MRI of these patients was performed, the volumes of different regions of the brain were analyzed. RESULTS: A total of 12 unrelated patients in our cohort were found with CHD7 variants. Eight patients received a firm clinical diagnosis of CHARGE syndrome (Bergmann criteria, Blake criteria, Verloes criteria and Hale criteria). Three patients did not match any diagnostic criteria, and no patients matched the Verloes criteria. Phenotype spectrum analysis found that feeding difficulty was the dominant feature among this neonatal cohort. Six novel variants in the CHD7 gene (Glu2408*, Lys651*, c.5607 + 1G > T, Leu373Val, Lys2005Asnfs*37 and Gln1991*) were identified, expanding the variant database of the CHD7 gene. Cranial MRI analysis revealed significant volume loss in cingulate gyrus, occipital lobe, and cerebellum and volume gain in the left medial and inferior temporal gyri anterior white matter parts. CONCLUSIONS: Based on a relatively unbiased neonatal cohort, we concluded that CHARGE syndrome and CHD7 gene variants should be suspected in newborns who have feeding difficulty, and one or more malformations. TRIAL REGISTRATION: Neonatal Birth Defects Cohort (NBDC, ClinicalTrials.gov identifier: NCT02551081 ).


Assuntos
Síndrome CHARGE/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Transtornos da Alimentação e da Ingestão de Alimentos/genética , Predisposição Genética para Doença/genética , Mutação , Grupo com Ancestrais do Continente Asiático/genética , Síndrome CHARGE/diagnóstico , Síndrome CHARGE/etnologia , China , Estudos de Coortes , Transtornos da Alimentação e da Ingestão de Alimentos/diagnóstico , Transtornos da Alimentação e da Ingestão de Alimentos/etnologia , Feminino , Predisposição Genética para Doença/etnologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Recém-Nascido , Masculino , Triagem Neonatal/métodos , Fenótipo , Curva ROC
6.
Nat Commun ; 10(1): 2253, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138795

RESUMO

Telomerase negative immortal cancer cells elongate telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. While sustained telomeric replicative stress is required to maintain ALT, it might also lead to cell death when excessive. Here, we show that the ATPase/translocase activity of FANCM keeps telomeric replicative stress in check specifically in ALT cells. When FANCM is depleted in ALT cells, telomeres become dysfunctional, and cells stop proliferating and die. FANCM depletion also increases ALT-associated marks and de novo synthesis of telomeric DNA. Depletion of the BLM helicase reduces the telomeric replication stress and cell proliferation defects induced by FANCM inactivation. Finally, FANCM unwinds telomeric R-loops in vitro and suppresses their accumulation in cells. Overexpression of RNaseH1 completely abolishes the replication stress remaining in cells codepleted for FANCM and BLM. Thus, FANCM allows controlled ALT activity and ALT cell proliferation by limiting the toxicity of uncontrolled BLM and telomeric R-loops.


Assuntos
DNA Helicases/genética , Replicação do DNA/genética , RecQ Helicases/genética , Homeostase do Telômero/genética , Telômero/metabolismo , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , DNA Helicases/metabolismo , Células HEK293 , Células HeLa , Humanos , RecQ Helicases/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo
7.
Genes Dev ; 33(13-14): 763-781, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31123064

RESUMO

Coordinated induction, but also repression, of genes are key to normal differentiation. Although the role of lineage-specific transcription regulators has been studied extensively, their functional integration with chromatin remodelers, one of the key enzymatic machineries that control chromatin accessibility, remains ill-defined. Here we investigate the role of Mi-2ß, a SNF-2-like nucleosome remodeler and key component of the nucleosome remodeling and histone deacetylase (NuRD) complex in early B cells. Inactivation of Mi-2ß arrested differentiation at the large pre-B-cell stage and caused derepression of cell adhesion and cell migration signaling factors by increasing chromatin access at poised enhancers and chromosome architectural elements. Mi-2ß also supported IL-7R signaling, survival, and proliferation by repressing negative effectors of this pathway. Importantly, overexpression of Bcl2, a mitochondrial prosurvival gene and target of IL-7R signaling, partly rescued the differentiation block caused by Mi-2ß loss. Mi-2ß stably associated with chromatin sites that harbor binding motifs for IKAROS and EBF1 and physically associated with these transcription factors both on and off chromatin. Notably, Mi-2ß shared loss-of-function cellular and molecular phenotypes with IKAROS and EBF1, albeit in a distinct fashion. Thus, the nucleosome remodeler Mi-2ß promotes pre-B-cell differentiation by providing repression capabilities to distinct lineage-specific transcription factor-based regulatory networks.


Assuntos
Linfócitos B/citologia , Diferenciação Celular/genética , Cromatina/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Linhagem da Célula , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Camundongos , Fatores de Transcrição
8.
Molecules ; 24(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067825

RESUMO

G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo.


Assuntos
Proteínas de Ligação a DNA/química , Quadruplex G , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Genoma/genética , Conformação de Ácido Nucleico , Ligação Proteica , RecQ Helicases/química , RecQ Helicases/genética , Proteínas Ribossômicas/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Termodinâmica
9.
Arch Virol ; 164(8): 2091-2106, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31139938

RESUMO

Beak and feather disease virus (BFDV) belongs to the family Circoviridae. A rolling-circle replication strategy based on a replication-associated protein (Rep) has been proposed for BFDV. The Rep gene of BFDV was expressed and purified, and it was shown to cleave short oligonucleotides containing the conserved nonanucleotide sequence found in the replication origin of circoviruses. This endonuclease activity was most efficient in the presence of the divalent metal ions Mg2+ and Mn2+. Rep proteins containing mutation in the ATPase/GTPase motifs and the 14FTLNN18, 61KKRLS65, 89YCSK92, and 170GKS172 motifs lacked endonuclease activity. The endonuclease activity was not affected by ATPase inhibitors, with the exception of N-ethylmaleimide (NEM), or by GTPase inhibitors, but it was decreased by treatment with the endonuclease inhibitor L-742001. Both the ATPase and GTPase activities were decreased by site-directed mutagenesis and deletion of the ATPase/GTPase and endonuclease motifs. The Rep protein was able to bind a double-stranded DNA fragment of P36 (dsP36) containing the stem-loop structure of the replication origin of BFDV. All of the Rep mutant proteins showed reduced ability to bind this fragment, suggesting that all the ATPase/GTPase and endonuclease motifs are involved in the binding. Other than NEM, all ATPase, GTPase, and endonuclease inhibitors inhibited the binding of the Rep protein to the dsP36 fragment. This is the first report describing the endonuclease activity of the Rep protein of BFDV.


Assuntos
Circovirus/genética , Replicação do DNA/genética , Endonucleases/genética , Replicação Viral/genética , Adenosina Trifosfatases/genética , Infecções por Circoviridae/virologia , DNA Helicases/genética , DNA Viral/genética , GTP Fosfo-Hidrolases/genética , Origem de Replicação/genética , Transativadores/genética
10.
Nat Commun ; 10(1): 1545, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30948716

RESUMO

Extrinsic transcription termination typically involves remodeling of RNA polymerase by an accessory helicase. In yeast this is accomplished by the Sen1 helicase homologous to human senataxin (SETX). To gain insight into these processes we develop a DNA scaffold construct compatible with magnetic-trapping assays and from which S. cerevisiae RNA polymerase II (Pol II), as well as E. coli RNA polymerase (ecRNAP), can efficiently initiate transcription without transcription factors, elongate, and undergo extrinsic termination. By stalling Pol II TECs on the construct we can monitor Sen1-induced termination in real-time, revealing the formation of an intermediate in which the Pol II transcription bubble appears half-rewound. This intermediate requires ~40 sec to form and lasts ~20 sec prior to final dissociation of the stalled Pol II. The experiments enabled by the scaffold construct permit detailed statistical and kinetic analysis of Pol II interactions with a range of cofactors in a multi-round, high-throughput fashion.


Assuntos
DNA Helicases/fisiologia , Escherichia coli/genética , RNA Helicases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Terminação da Transcrição Genética/fisiologia , Transcrição Genética , DNA Helicases/genética , DNA Helicases/metabolismo , Escherichia coli/metabolismo , Modelos Moleculares , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase II/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
Nat Biotechnol ; 37(6): 651-656, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31011178

RESUMO

Nanopore DNA sequencing is limited by low base-calling accuracy. Improved base-calling accuracy has so far relied on specialized base-calling algorithms, different nanopores and motor enzymes, or biochemical methods to re-read DNA molecules. Two primary error modes hamper sequencing accuracy: enzyme mis-steps and sequences with indistinguishable signals. We vary the driving voltage from 100 to 200 mV, with a frequency of 200 Hz, across a Mycobacterium smegmatis porin A (MspA) nanopore, thus changing how the DNA strand moves through the nanopore. A DNA helicase moves the DNA through the nanopore in discrete steps, and the variable voltage moves the DNA continuously between these steps. The electronic signal produced with variable voltage is used to overcome the primary error modes in base calling. We found that single-passage de novo base-calling accuracy of 62.7 ± 0.5% with a constant driving voltage improves to 79.3 ± 0.3% with a variable driving voltage. The variable-voltage sequencing mode is complementary to other methods to boost the accuracy of nanopore sequencing and could be incorporated into any enzyme-actuated nanopore sequencing device.


Assuntos
DNA Helicases/genética , DNA/genética , Nanoporos , Porinas/genética , Algoritmos , DNA/isolamento & purificação , DNA Helicases/química , Mycobacterium smegmatis/genética , Porinas/química , Análise de Sequência de DNA/métodos
12.
Cancer Res ; 79(7): 1297-1298, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30936075

RESUMO

DNA replication stress is prevalent in human cancers, but absent in normal cells, suggesting that proteins involved in the cellular response to DNA replication stress could be potential therapeutic targets. SMARCAL1 and ZRANB3 are annealing helicases that mediate the repair of collapsed DNA replication forks. In a study in this issue of Cancer Research, Puccetti and colleagues report that mice lacking either SMARCAL1 or ZRANB3 activity have delayed development of MYC-induced B-cell lymphomas. Thus, inhibiting the response to DNA replication stress could benefit patients with cancer.See related article by Puccetti et al., p. 1612.


Assuntos
DNA Helicases/genética , Replicação do DNA , Animais , Humanos , Camundongos
13.
J Cancer Res Ther ; 15(2): 350-357, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30964110

RESUMO

Background: We assessed the frequency of epigenetic lesions, including lymphoid-specific helicase (LSH), 5-hydroxymethylcytosine (5-hmC) and E2F1, and the possible correlations among molecular findings, phenotype, clinical features, and outcome. Methods: We investigated 181 paraffin-embedded B-cell lymphoma samples using immunohistochemistry and in situ hybridization. Results: The levels of Ki67, LSH, 5-hmC, and E2F1 were all increased in germinal center B-cell lymphomas when compared with those in normal lymph nodes, and LSH was highly expressed in diffuse large B-cell lymphomas (DLBCLs) and Burkitt lymphomas (BLs) that were positive for Epstein-Barr virus (EBV) infection, indicating that LSH is linked to EBV infection in DLBCL and BL. Interestingly, LSH was mainly localized in the germinal centers of lymph nodes whereas 5-hmC staining localized to areas surrounding the germinal centers. Conclusions: These findings indicate a critical role for LSH as a biomarker and therapeutic target in follicular germinal center B-cell lymphoma.


Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/genética , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/virologia , Centro Germinativo/patologia , Herpesvirus Humano 4 , Linfoma de Células B/etiologia , Linfoma de Células B/patologia , Biomarcadores , Diagnóstico Diferencial , Humanos , Imuno-Histoquímica
14.
Nat Commun ; 10(1): 1270, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894540

RESUMO

Gfi1b is a transcriptional repressor expressed in hematopoietic stem cells (HSCs) and megakaryocytes (MKs). Gfi1b deficiency leads to expansion of both cell types and abrogates the ability of MKs to respond to integrin. Here we show that Gfi1b forms complexes with ß-catenin, its co-factors Pontin52, CHD8, TLE3 and CtBP1 and regulates Wnt/ß-catenin-dependent gene expression. In reporter assays, Gfi1b can activate TCF-dependent transcription and Wnt3a treatment enhances this activation. This requires interaction between Gfi1b and LSD1 and suggests that a tripartite ß-catenin/Gfi1b/LSD1 complex exists, which regulates Wnt/ß-catenin target genes. Consistently, numerous canonical Wnt/ß-catenin target genes, co-occupied by Gfi1b, ß-catenin and LSD1, have their expression deregulated in Gfi1b-deficient cells. When Gfi1b-deficient cells are treated with Wnt3a, their normal cellularity is restored and Gfi1b-deficient MKs regained their ability to spread on integrin substrates. This indicates that Gfi1b controls both the cellularity and functional integrity of HSCs and MKs by regulating Wnt/ß-catenin signaling pathway.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Megacariócitos/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Via de Sinalização Wnt , Proteína Wnt3A/genética , beta Catenina/genética , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Ontologia Genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Células K562 , Megacariócitos/citologia , Camundongos , Camundongos Knockout , Anotação de Sequência Molecular , Cultura Primária de Células , Proteínas Proto-Oncogênicas/deficiência , Proteínas Repressoras/deficiência , Tamoxifeno , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo
15.
Plant Mol Biol ; 100(1-2): 133-149, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30843130

RESUMO

KEY MESSAGE: The OsPLS2 locus was isolated and cloned by map-based cloning that encodes a Upf1-like helicase. Disruption of OsPLS2 accelerated light-dependent leaf senescence in the rice mutant of ospls2. Leaf senescence is a very complex physiological process controlled by both genetic and environmental factors, however its underlying molecular mechanisms remain elusive. In this study, we report a novel Oryza sativa premature leaf senescence mutant (ospls2). Through map-based cloning, a G-to-A substitution was determined at the 1st nucleotide of the 13th intron in the OsPLS2 gene that encodes a Upf1-like helicase. This mutation prompts aberrant splicing of OsPLS2 messenger and consequent disruption of its full-length protein translation, suggesting a negative role of OsPLS2 in regulating leaf senescence. Wild-type rice accordingly displayed a progressive drop of OsPSL2 protein levels with age-dependent leaf senescence. Shading and light filtration studies showed that the ospls2 phenotype, which was characteristic of photo-oxidative stress and reactive oxygen species (ROS) accumulation, was an effect of irritation by light. When continuously exposed to far-red light, exogenous H2O2 and/or abscisic acid (ABA), the ospls2 mutant sustained hypersensitive leaf senescence. In consistence, light and ROS signal pathways in ospls2 were activated by down-regulation of phytochrome genes, and up-regulation of PHYTOCHROME-INTERACTING FACTORS (PIFs) and WRKY genes, all promoting leaf senescence. Together, these data indicated that OsPLS2 played an essential role in leaf senescence and its disruption triggered light-dependent leaf senescence in rice.


Assuntos
DNA Helicases/genética , Genes de Plantas , Luz , Oryza/crescimento & desenvolvimento , Oryza/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Ácido Abscísico/metabolismo , Sequência de Aminoácidos , Antioxidantes/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação/genética , Oryza/enzimologia , Oryza/efeitos da radiação , Fenótipo , Fotossíntese/genética , Folhas de Planta/genética , Folhas de Planta/efeitos da radiação , Folhas de Planta/ultraestrutura , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo
16.
Mol Genet Genomic Med ; 7(5): e639, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30924321

RESUMO

BACKGROUND: Warsaw Breakage Syndrome (WABS) is an ultra rare cohesinopathy caused by biallelic mutation of DDX11 gene. It is clinically characterized by pre and postnatal growth delay, microcephaly, hearing loss with cochlear hypoplasia, skin color abnormalities, and dysmorphisms. METHODS: Mutational screening and functional analyses (protein expression and 3D-modeling) were performed in order to investigate the presence and pathogenicity of DDX11 variant identified in our patients. RESULTS: We report the clinical history of two sisters affected by WABS with a pathological mytomicin C test carrying compound heterozygous mutations (c.2507T > C / c.907_920del) of the DDX11 gene. The pathogenicity of this variant was confirmed in the light of a bioinformatic study and protein three-dimensional modeling, as well as expression analysis. CONCLUSION: These findings further extend the clinical and molecular knowledge about the WABS showing a possible mild phenotype without major malformations or intellectual disability.


Assuntos
Anormalidades Múltiplas/genética , Manchas Café com Leite/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Perda Auditiva Neurossensorial/genética , Fenótipo , Anormalidades Múltiplas/patologia , Manchas Café com Leite/patologia , Linhagem Celular , Células Cultivadas , Criança , Pré-Escolar , Feminino , Perda Auditiva Neurossensorial/patologia , Humanos , Mutação , Síndrome
17.
PLoS Genet ; 15(3): e1008057, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30875366

RESUMO

Identifying small molecules that inhibit protein synthesis by selectively stalling the ribosome constitutes a new strategy for therapeutic development. Compounds that inhibit the translation of PCSK9, a major regulator of low-density lipoprotein cholesterol, have been identified that reduce LDL cholesterol in preclinical models and that affect the translation of only a few off-target proteins. Although some of these compounds hold potential for future therapeutic development, it is not known how they impact the physiology of cells or ribosome quality control pathways. Here we used a genome-wide CRISPRi screen to identify proteins and pathways that modulate cell growth in the presence of high doses of a selective PCSK9 translational inhibitor, PF-06378503 (PF8503). The two most potent genetic modifiers of cell fitness in the presence of PF8503, the ubiquitin binding protein ASCC2 and helicase ASCC3, bind to the ribosome and protect cells from toxic effects of high concentrations of the compound. Surprisingly, translation quality control proteins Pelota (PELO) and HBS1L sensitize cells to PF8503 treatment. In genetic interaction experiments, ASCC3 acts together with ASCC2, and functions downstream of HBS1L. Taken together, these results identify new connections between ribosome quality control pathways, and provide new insights into the selectivity of compounds that stall human translation that will aid the development of next-generation selective translation stalling compounds to treat disease.


Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/metabolismo , Sequência de Aminoácidos , Sistemas CRISPR-Cas , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , DNA Helicases/metabolismo , Endonucleases/metabolismo , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Técnicas de Silenciamento de Genes , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Células K562 , Modelos Biológicos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pró-Proteína Convertase 9/antagonistas & inibidores , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Ribossomos/efeitos dos fármacos , Inibidores de Serino Proteinase/farmacologia
18.
World J Microbiol Biotechnol ; 35(4): 53, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30900038

RESUMO

The oxidative stress response of the highly resistant actinomycete Dietzia cinnamea P4 after treatment with hydrogen peroxide (H2O2) was assessed in order to depict the possible mechanisms underlying its intrinsic high resistance to DNA damaging agents. We used transcriptional profiling to monitor the magnitude and kinetics of changes in the mRNA levels after exposure to different concentrations of H2O2 at 10 min and 1 h following the addition of the stressor. Catalase and superoxide dismutase genes were induced in different ways, according to the condition applied. Moreover, alkyl hydroperoxide reductase ahpCF, thiol peroxidase, thioredoxin and glutathione genes were upregulated in the presence of H2O2. Expression of peroxidase genes was not detected during the experiment. Overall results point to an actinomycete strain endowed with a set of enzymatic defenses against oxidative stress and with the main genes belonging to a functional SOS system (lexA, recA, uvrD), including suppression of lexA repressor, concomitantly to recA and uvrD gene upregulation upon H2O2 challenge.


Assuntos
Actinomycetales/efeitos dos fármacos , Actinomycetales/metabolismo , Peróxido de Hidrogênio/efeitos adversos , Estresse Oxidativo , Resposta SOS (Genética)/fisiologia , Actinomycetales/enzimologia , Actinomycetales/genética , Proteínas de Bactérias/genética , Catalase/classificação , Catalase/genética , Dano ao DNA/efeitos dos fármacos , DNA Helicases/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Glutationa/genética , Cinética , Peroxidases/genética , Peroxirredoxinas/genética , Filogenia , RNA Mensageiro/metabolismo , Recombinases Rec A/genética , Resposta SOS (Genética)/genética , Análise de Sequência , Serina Endopeptidases/genética , Superóxido Dismutase/genética , Tiorredoxinas/genética , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos
19.
Gene ; 699: 110-114, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30844479

RESUMO

Tricho-hepatic-enteric syndrome (THES) is a genetically heterogeneous rare syndrome (OMIM: 222470 (THES1) and 614602 (THES2)) that typically presents in the neonatal period with intractable diarrhoea, intra-uterine growth retardation (IUGR), facial dysmorphism, and hair and skin changes. THES is associated with pathogenic variants in either TTC37 or SKIV2L; both are components of the human SKI complex, an RNA exosome cofactor. We report an 8 year old girl who was diagnosed with THES by the Undiagnosed Disease Program-WA with compound heterozygous pathogenic variants in SKIV2L. While THES was considered in the differential diagnosis, the absence of protracted diarrhoea delayed definitive diagnosis. We therefore suggest that SKIV2L testing should be considered in cases otherwise suggestive of THES, but without the characteristic diarrhoea. We expand the phenotypic spectrum while reviewing the current knowledge on SKIV2L.


Assuntos
Diarreia Infantil/diagnóstico , Diarreia Infantil/genética , Diarreia/diagnóstico , Diarreia/genética , Retardo do Crescimento Fetal/diagnóstico , Retardo do Crescimento Fetal/genética , Doenças do Cabelo/diagnóstico , Doenças do Cabelo/genética , DNA Helicases/genética , Facies , Heterozigoto , Humanos
20.
PLoS Genet ; 15(2): e1007992, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30794539

RESUMO

Histone modifications regulate gene expression and chromosomal events, yet how histone-modifying enzymes are targeted is poorly understood. Here we report that a conserved DNA repair protein, SMRC-1, associates with MET-2, the C. elegans histone methyltransferase responsible for H3K9me1 and me2 deposition. We used molecular, genetic, and biochemical methods to investigate the biological role of SMRC-1 and to explore its relationship with MET-2. SMRC-1, like its mammalian ortholog SMARCAL1, provides protection from DNA replication stress. SMRC-1 limits accumulation of DNA damage and promotes germline and embryonic viability. MET-2 and SMRC-1 localize to mitotic and meiotic germline nuclei, and SMRC-1 promotes an increase in MET-2 abundance in mitotic germline nuclei upon replication stress. In the absence of SMRC-1, germline H3K9me2 generally decreases after multiple generations at high culture temperature. Genetic data are consistent with MET-2 and SMRC-1 functioning together to limit replication stress in the germ line and in parallel to promote other germline processes. We hypothesize that loss of SMRC-1 activity causes chronic replication stress, in part because of insufficient recruitment of MET-2 to nuclei.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , DNA Helicases/metabolismo , Instabilidade Genômica , Células Germinativas/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Caenorhabditis elegans/metabolismo , DNA Helicases/genética , Reparo do DNA , Replicação do DNA , Feminino , Histonas/metabolismo , Masculino , Ligação Proteica
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