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1.
Immunol Rev ; 305(1): 29-42, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34927255

RESUMO

B lymphocytes develop from uncommitted precursors into immunoglobulin (antibody)-producing B cells, a major arm of adaptive immunity. Progression of early progenitors to antibody-expressing cells in the bone marrow is orchestrated by the temporal regulation of different gene programs at discrete developmental stages. A major question concerns how B cells control the accessibility of these genes to transcription factors. Research has implicated nucleosome remodeling ATPases as mediators of chromatin accessibility. Here, we describe studies of chromodomain helicase DNA-binding 4 (CHD4; also known as Mi-2ß) in early B cell development. CHD4 comprises multiple domains that function in nucleosome mobilization and histone binding. CHD4 is a key component of Nucleosome Remodeling and Deacetylase, or NuRD (Mi-2) complexes, which assemble with other proteins that mediate transcriptional repression. We review data demonstrating that CHD4 is necessary for B lineage identity: early B lineage progression, proliferation in response to interleukin-7, responses to DNA damage, and cell survival in vivo. CHD4-NuRD is also required for the Ig heavy-chain repertoire by promoting utilization of distal variable (VH ) gene segments in V(D)J recombination. In conclusion, the regulation of chromatin accessibility by CHD4 is essential for production of antibodies by B cells, which in turn mediate humoral immune responses to pathogens and disease.


Assuntos
Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Recombinação V(D)J , Linfócitos B/metabolismo , DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo
2.
PLoS Genet ; 18(6): e1010275, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35696436

RESUMO

Timely completion of eukaryotic genome duplication requires coordinated DNA replication initiation at multiple origins. Replication begins with the loading of the Mini-Chromosome Maintenance (MCM) complex, proceeds by the activation of the Cdc45-MCM-GINS (CMG) helicase, and ends with CMG removal after chromosomes are fully replicated. Post-translational modifications on the MCM and associated factors ensure an orderly transit of these steps. Although the mechanisms of CMG activation and removal are partially understood, regulated MCM loading is not, leaving an incomplete understanding of how DNA replication begins. Here we describe a site-specific modification of Mcm3 by the Small Ubiquitin-like MOdifier (SUMO). Mutations that prevent this modification reduce the MCM loaded at replication origins and lower CMG levels, resulting in impaired cell growth, delayed chromosomal replication, and the accumulation of gross chromosomal rearrangements (GCRs). These findings demonstrate the existence of a SUMO-dependent regulation of origin-bound MCM and show that this pathway is needed to prevent genome rearrangements.


Assuntos
Replicação do DNA , Sumoilação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/genética , Replicação do DNA/genética , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Origem de Replicação/genética , Sumoilação/genética
3.
Nan Fang Yi Ke Da Xue Xue Bao ; 42(5): 681-689, 2022 May 20.
Artigo em Chinês | MEDLINE | ID: mdl-35673911

RESUMO

OBJECTIVE: To assess the value of m7G-lncRNAs in predicting the prognosis and microenvironment of colorectal cancer (CRC). METHODS: We screened m7G-lncRNAs from TCGA to construct an m7G-lncRNAs risk model using multivariate Cox analysis, which was validated using ROC and C-index curves. Calibration and nomogram were used to predict the prognosis of CRC patients. Point-bar charts and K-M survival curves were used to assess the correlation of risk scores with the patients' clinical staging and prognosis. CIBERSORT and ESTIMATE were used to explore the association between the tumor microenvironment and immune cell infiltration in patients in high and low risk groups and the correlation of risk scores with microsatellite instability, stem cell index and immune checkpoint expression. A protein-protein interaction network was constructed, and the key targets regulated by m7G-lncRNAs were identified and validated in paired samples of CRC and adjacent tissues by immunoblotting. RESULTS: We identified a total of 1722 m7G-lncRNAs from TCGA database, from which 12 lncRNAs were screened to construct the risk model. The AUCs of the risk model for predicting survival outcomes at 1, 3 and 5 years were 0.727, 0.747 and 0.794, respectively. The AUC of the nomogram for predicting prognosis was 0.794, and the predicted results were consistent with actual survival outcomes of the patients. The patients in the high-risk group showed more advanced tumor stages and a greater likelihood of high microsatellite instability than those in the low-risk group (P < 0.05). The tumor stemness index was negatively correlated with the risk score (r=-0.19; P=7.3e-05). Patients in the high-risk group had higher stromal cell scores (P=0.0028) and higher total scores (P=0.007) with lowered expressions of activated mast cells (r=-0.11; P=0.045) and resting CD4+ T cells (r=-0.14; P=0.01) and increased expressions of most immune checkpoints (P < 0.05). ATXN2 (P= 0.006) and G3BP1 (P=0.007) were identified as the key targets regulated by m7G-lncRNAs, and their expressions were both higher in CRC than in adjacent tissues. CONCLUSION: The risk model based on 12 m7G-lncRNAs has important prognostic value for CRC and can reflect the microenvironment and the efficacy of immunotherapy in the patients.


Assuntos
Neoplasias do Colo , RNA Longo não Codificante , Biomarcadores Tumorais/metabolismo , DNA Helicases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Instabilidade de Microssatélites , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Prognóstico , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Longo não Codificante/metabolismo , Microambiente Tumoral
4.
Gene ; 834: 146638, 2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35680019

RESUMO

BACKGROUND: The association between SWI/SNF genomic alterations and responses to immune checkpoint inhibitors (ICIs) remains conflicting. This meta-analysis was performed to systematically assess the impact of SWI/SNF genomic alterations on response to ICIs in cancer. METHODS: Relevant studies were searched in multiple databases updated to April 29, 2021. Outcomes of interest included prevalence of SWI/SNF alterations, overall survival (OS), progression-free survival (PFS) and time to treatment failure (TTF). For survival data, the hazard ratio (HR) was adopted, and the effect size was described as 95% confidence intervals (CI). RESULTS: 15 studies involving 10,849 patients were included, with the overall frequency of 18.5% in SWI/SNF alterations. Across different cancer types, the mutational frequency of PBRM1 (32.0%) was the highest, followed by ARID1A (18.1%), SMARCA4 (15.6%), SMARCA2 (10.3%), ARID2 (8.1%), SMARCC2 (6.4%) and SMARCB1 (5.0%). Overall analysis showed that SWI/SNF alterations were not associated with improved OS (HR: 0.822, 95 %CI: 0.583-1.158, p = 0.262), PFS (HR: 0.608, 95 %CI: 0.434-1.067, p = 0.094) and TTF (HR: 0.923, 95 %CI: 0.757-1.125, p = 0.427) in patients treated with ICIs. In subgroup analysis, PBRM1 mutations were observed to be linked with improved OS (HR: 0.650, 95 %CI: 0.440-0.960, p = 0.030), PFS (HR: 0.539, 95 %CI: 0.314-0.926, p = 0.025) and TTF (HR: 0.490, 95 %CI: 0.271-0.885, p = 0.018) in RCC patients receiving ICIs. CONCLUSIONS: The overall prevalence of SWI/SNF alterations was 18.5% across different cancer types. Except for PBRM1 mutations in RCC, SWI/SNF alterations may be uncorrelated with improved clinical outcomes in cancer.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Genômica , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Proteínas Nucleares/genética , Prevalência , Fatores de Transcrição/genética
5.
Cell ; 185(12): 2132-2147.e26, 2022 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-35688134

RESUMO

RNA quality control relies on co-factors and adaptors to identify and prepare substrates for degradation by ribonucleases such as the 3' to 5' ribonucleolytic RNA exosome. Here, we determined cryogenic electron microscopy structures of human nuclear exosome targeting (NEXT) complexes bound to RNA that reveal mechanistic insights to substrate recognition and early steps that precede RNA handover to the exosome. The structures illuminate ZCCHC8 as a scaffold, mediating homodimerization while embracing the MTR4 helicase and flexibly anchoring RBM7 to the helicase core. All three subunits collaborate to bind the RNA, with RBM7 and ZCCHC8 surveying sequences upstream of the 3' end to facilitate RNA capture by MTR4. ZCCHC8 obscures MTR4 surfaces important for RNA binding and extrusion as well as MPP6-dependent recruitment and docking onto the RNA exosome core, interactions that contribute to RNA surveillance by coordinating RNA capture, translocation, and extrusion from the helicase to the exosome for decay.


Assuntos
Exossomos , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA/metabolismo , Estabilidade de RNA
6.
Front Biosci (Landmark Ed) ; 27(6): 173, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35748249

RESUMO

BACKGROUND: Epirubicin (EPI) is an important anticancer drug that is well-known for its cardiotoxic side effect. Studying epigenetic modification such as DNA methylation can help to understand the EPI-related toxic mechanisms in cardiac tissue. In this study, we analyzed the DNA methylation profile in a relevant human cell model and inspected the expression of differentially methylated genes at the transcriptome level to understand how changes in DNA methylation could affect gene expression in relation to EPI-induced cardiotoxicity. METHODS: Human cardiac microtissues were exposed to either therapeutic or toxic (IC20) EPI doses during 2 weeks. The DNA and RNA were collected from microtissues in triplicates at 2, 8, 24, 72, 168, 240, and 336 hours of exposure. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) analysis was used to detect DNA methylation levels in EPI-treated and control samples. The MeDIP-seq data were analyzed and processed using the QSEA package with a recently published workflow. RNA sequencing (RNA-seq) was used to measure global gene expression in the same samples. RESULTS: After processing the MeDIP-seq data, we detected 35, 37, 15 candidate genes which show strong methylated alterations between all EPI-treated, EPI therapeutic and EPI toxic dose-treated samples compared to control, respectively. For several genes, gene expressions changed compatibly reflecting the DNA methylation regulation. CONCLUSIONS: The observed DNA methylation modifications provide further insights into the EPI-induced cardiotoxicity. Multiple differentially methylated genes under EPI treatment, such as SMARCA4, PKN1, RGS12, DPP9, NCOR2, SDHA, POLR2A, and AGPAT3, have been implicated in different cardiac dysfunction mechanisms. Together with other differentially methylated genes, these genes can be candidates for further investigations of EPI-related toxic mechanisms. Data Repository: The data has been generated by the HeCaToS project (http://www.ebi.ac.uk/biostudies) under accession numbers S-HECA433 and S-HECA434 for the MeDIP-seq data and S-HECA11 for the RNA-seq data. The R code is available on Github (https://github.com/NhanNguyen000/MeDIP).


Assuntos
Cardiotoxicidade , Metilação de DNA , Cardiotoxicidade/genética , DNA , DNA Helicases , Epirubicina/toxicidade , Humanos , Proteínas Nucleares , Análise de Sequência de DNA , Fatores de Transcrição
7.
J Virol ; 96(12): e0041222, 2022 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-35652658

RESUMO

SARS-CoV-2 is the causative agent of the ongoing pandemic of coronavirus disease 2019 (COVID-19) and poses a significant threat to global health. N protein (NP), which is a major pathogenic protein among betacoronaviruses, binds to the viral RNA genome to allow viral genome packaging and viral particle release. Recent studies showed that NP antagonizes interferon (IFN) induction and mediates phase separation. Using live SARS-CoV-2 viruses, this study provides solid evidence showing that SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming G3BP1-mediated antiviral innate immunity. G3BP1 conditional knockout mice (g3bp1fl/fL, Sftpc-Cre) exhibit significantly higher lung viral loads after SARS-CoV-2 infection than wild-type mice. Our findings contribute to the growing body of knowledge regarding the pathogenicity of NPSARS-CoV-2 and provide insight into new therapeutics targeting NPSARS-CoV-2. IMPORTANCE In this study, by in vitro assay and live SARS-CoV-2 virus infection, we provide solid evidence that the SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming antiviral innate immunity mediated by G3BP1 in A549 cell lines and G3BP1 conditional knockout mice (g3bp1-cKO) mice, which provide in-depth evidence showing the mechanism underlying NP-related SARS-CoV-2 pathogenesis through G3BPs.


Assuntos
COVID-19 , Proteínas do Nucleocapsídeo de Coronavírus , Proteínas de Ligação a Poli-ADP-Ribose , SARS-CoV-2 , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , COVID-19/imunologia , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , DNA Helicases/metabolismo , Interações entre Hospedeiro e Microrganismos/imunologia , Camundongos , Fosfoproteínas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/genética
8.
J Nat Prod ; 85(6): 1503-1513, 2022 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-35687347

RESUMO

Aurora kinases (Aurora A, B, and C) are a family of serine/threonine kinases that play critical roles during mitotic initiation and progression. Aurora A and B kinases are ubiquitously expressed, and their overexpression and/or amplification in many cancers have been associated with poor prognosis. Several inhibitors that target Aurora kinases A, B, or both have been developed during the past decade with efficacy in different in vitro and in vivo models for a variety of cancers. Recent studies have also identified Aurora A as a synthetic lethal target for different tumor suppressors, including RB1, SMARCA4, and ARID1A, which signifies the need for Aurora-A-selective inhibitors. Here, we report the screening of a small library of quinones (nine naphthoquinones, one orthoquinone, and one anthraquinone) in a biochemical assay for Aurora A kinase that resulted in the identification of several quinones as inhibitors. IC50 determination against Aurora A and B kinases revealed the inhibition of both kinases with selectivity toward Aurora A. Two of the compounds, natural quinone naphthazarin (1) and a pseudo anthraquinone, 2-(chloromethyl)quinizarin (11), potently inhibited the proliferation of various cancer cell lines with IC50 values ranging from 0.16 ± 0.15 to 1.7 ± 0.06 and 0.15 ± 0.04 to 6.3 ± 1.8 µM, respectively. Treatment of cancer cells with these compounds for 24 h resulted in abrogated mitosis and apoptotic cell death. Direct binding of both the compounds with Aurora A kinase was also confirmed through STD NMR analysis. Docking studies predicted the binding of both compounds to the ATP binding pocket of Aurora A kinase. We have, therefore, identified quinones as Aurora kinase inhibitors that can serve as a lead for future drug discovery endeavors.


Assuntos
Aurora Quinase A , Neoplasias , Antraquinonas , Aurora Quinase B , DNA Helicases , Humanos , Proteínas Nucleares , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinonas/farmacologia , Fatores de Transcrição
9.
Genes (Basel) ; 13(6)2022 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-35741741

RESUMO

In wheat, a series of dwarf and semi-dwarf plant varieties have been developed and utilized worldwide since the 1960s and caused the 'Green Revolution'. To date, 25 reduced-height (Rht) genes have been identified, but only several genes for plant height (PH) have been isolated previously. In this study, we identified a candidate gene, ATP-dependent DNA helicase (TaDHL-7B), for PH via QTL mapping and genome-wide association study (GWAS) methods. We knocked out this gene using the CRISPR/Cas9 system in variety 'Fielder'. Two homozygous mutant genotypes, AAbbDD (-5 bp) and AAbbDD (-1 bp), were obtained in the T2 generation. The PH values of AAbbDD (-5 bp) and AAbbDD (-1 bp) were significantly reduced compared with the wild-type (WT, 'Fielder'), indicating that TaDHL-7B is a novel Rht gene that controls the PH. This is the first time that a PH gene of wheat has been isolated with a non-hormone pathway, providing a new insight into the genetic control of PH. The TaDHL gene reduced the PH without a yield penalty. It could be used to improve the lodging resistance and yield in wheat breeding programs.


Assuntos
Melhoramento Vegetal , Triticum , DNA Helicases/genética , Genes de Plantas , Estudo de Associação Genômica Ampla , Triticum/genética
10.
Int J Mol Sci ; 23(12)2022 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-35743069

RESUMO

As an ATP-dependent DNA helicase, human ChlR1/DDX11 (Chl1 in yeast) can unwind both DNA:RNA and DNA:DNA substrates in vitro. Studies have demonstrated that ChlR1 plays a vital role in preserving genome stability by participating in DNA repair and sister chromatid cohesion, whereas the ways in which the biochemical features of ChlR1 function in DNA metabolism are not well understood. Here, we illustrate that Chl1 localizes to double-strand DNA break (DSB) sites and restrains DNA:RNA hybrid accumulation at these loci. Mutation of Chl1 strongly impairs DSB repair capacity by homologous recombination (HR) and nonhomologous end-joining (NHEJ) pathways, and deleting RNase H further reduces DNA repair efficiency, which indicates that the enzymatic activities of Chl1 are needed in Schizosaccharomyces pombe. In addition, we found that the Rpc37 subunit of RNA polymerase III (RNA Pol III) interacts directly with Chl1 and that deletion of Chl1 has no influence on the localization of Rpc37 at DSB site, implying the role of Rpc37 in the recruitment of Chl1 to this site.


Assuntos
DNA Helicases , Schizosaccharomyces , Moléculas de Adesão Celular/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Instabilidade Genômica , Humanos , RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo
11.
Breast Cancer Res ; 24(1): 38, 2022 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-35659359

RESUMO

BACKGROUND: The effect of extracellular microenvironment (hypoxia and pH) has been regarded as a key hallmark in cancer progression. The study aims to investigate the effects of carbonic anhydrase IX (CAIX), a key hypoxia-inducible marker, in triple-negative breast cancer (TNBC) in correlation with clinicopathological parameters and predicting survival outcomes. METHODS: A total of 323 TNBC cases diagnosed at the Department of Anatomical Pathology, Singapore General Hospital from 2003 to 2013 were used. Immunohistochemical staining (IHC) was performed using CAIX antibody and digital mRNA quantification was performed using NanoString assays. CAIX membranous expression was correlated with clinicopathological parameters using Chi-squared test or Fisher's exact tests. Disease-free survival (DFS) and overall-survival (OS) were estimated using Kaplan-Meier analysis and compared between groups with the log-rank test. RESULTS: Forty percent of TNBCs were observed to express CAIX protein and demonstrated significant association with larger tumour size (P = 0.002), higher histological grade (P < 0.001), and significantly worse disease-free survival (DFS) and overall survival (OS) (after adjustment: HR = 2.99, 95% CI = 1.78-5.02, P < 0.001 and HR = 2.56, 95% CI = 1.41-4.65, P = 0.002, respectively). Gene ontology enrichment analysis revealed six significantly enriched cellular functions (secretion, cellular component disassembly, regulation of protein complex assembly, glycolytic process, cellular macromolecular complex assembly, positive regulation of cellular component biogenesis) associated with genes differentially expressed (CAIX, SETX, WAS, HK2, DDIT4, TUBA4α, ARL1). Three genes (WAS, SETX and DDIT4) were related to DNA repair, indicating that DNA stability may be influenced by hypoxia in TNBC. CONCLUSIONS: Our results demonstrate that CAIX appears to be a significant hypoxia-inducible molecular marker and increased CAIX protein levels are independently associated with poor survival in TNBC. Identification of CAIX-linked seven gene-signature and its relationship with enriched cellular functions further support the implication and influence of hypoxia-mediated CAIX expression in TNBC tumour microenvironment.


Assuntos
Neoplasias da Mama , Anidrases Carbônicas , Neoplasias de Mama Triplo Negativas , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Anidrase Carbônica IX/genética , Anidrase Carbônica IX/metabolismo , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , DNA Helicases , Feminino , Humanos , Hipóxia/genética , Enzimas Multifuncionais , Prognóstico , RNA Helicases , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/genética
12.
Sci Immunol ; 7(72): eabn2888, 2022 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-35658009

RESUMO

The SKIV2L RNA exosome is an evolutionarily conserved RNA degradation complex in the eukaryotes. Mutations in the SKIV2L gene are associated with a severe inherited disorder, trichohepatoenteric syndrome (THES), with multisystem involvement but unknown disease mechanism. Here, we reported a THES patient with SKIV2L mutations showing severe primary B cell immunodeficiency, hypogammaglobulinemia, and kappa-restricted plasma cell dyscrasia but normal T cell and NK cell function. To corroborate these findings, we made B cell-specific Skiv2l knockout mice (Skiv2lfl/flCd79a-Cre), which lacked both conventional B-2 and innate-like B-1 B cells in the periphery and secondary lymphoid organs. This was linked to a requirement of SKIV2L RNA exosome activity in the bone marrow during early B cell development at the pro-B cell to large pre-B cell transition. Mechanistically, Skiv2l-deficient pro-B cells exhibited cell cycle arrest and DNA damage. Furthermore, loss of Skiv2l led to substantial out-of-frame V(D)J rearrangement of immunoglobulin heavy chain and severely reduced surface expression of µH, both of which are crucial for pre-BCR signaling and proliferative burst during early B cell development. Together, our data demonstrated a crucial role for SKIV2L RNA exosome in early B cell development in both human and mice by ensuring proper V(D)J recombination and Igh expression, which serves as the molecular basis for immunodeficiency associated with THES.


Assuntos
Diarreia Infantil , Doenças do Cabelo , Animais , DNA Helicases , Diarreia Infantil/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Facies , Retardo do Crescimento Fetal , Doenças do Cabelo/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Mamíferos/metabolismo , Camundongos
13.
Methods Mol Biol ; 2528: 305-316, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35704200

RESUMO

R-loops, three-stranded RNA-DNA hybrids that arise mostly during transcription, could cause genomic instability via distinct routes. Detection of genomic RNA-DNA hybrids via immunofluorescence and RNA-DNA hybrid immunoprecipitation techniques have facilitated the discovery of many cellular factors that maintain R-loop homeostasis. One of multiple R-loop avoidance mechanisms is mediated by several nucleic acid motor proteins that utilize the energy from ATP hydrolysis to dissociate the R-loop structure. The biochemical activity of such motor proteins can be interrogated using synthetic R-loop substrates. Here, we describe methods to generate R-loop and RNA-DNA substrates for studying the activity of R-loop processing motor proteins such as human DHX9 and S. cerevisiae Pif1. Such studies provide valuable information regarding the directionality, nucleic acid strand preference, and processivity of these enzymes. Moreover, these substrates and companion biochemical assays provide the requisite tool for understanding the action of physiologically relevant regulators of these motor proteins.


Assuntos
Estruturas R-Loop , Proteínas de Saccharomyces cerevisiae , DNA/química , DNA Helicases/metabolismo , Humanos , Imunoprecipitação , RNA/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Nat Commun ; 13(1): 3485, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35710802

RESUMO

The chromatin remodeller ATRX interacts with the histone chaperone DAXX to deposit the histone variant H3.3 at sites of nucleosome turnover. ATRX is known to bind repetitive, heterochromatic regions of the genome including telomeres, ribosomal DNA and pericentric repeats, many of which are putative G-quadruplex forming sequences (PQS). At these sites ATRX plays an ancillary role in a wide range of nuclear processes facilitating replication, chromatin modification and transcription. Here, using an improved protocol for chromatin immunoprecipitation, we show that ATRX also binds active regulatory elements in euchromatin. Mutations in ATRX lead to perturbation of gene expression associated with a reduction in chromatin accessibility, histone modification, transcription factor binding and deposition of H3.3 at the sequences to which it normally binds. In erythroid cells where downregulation of α-globin expression is a hallmark of ATR-X syndrome, perturbation of chromatin accessibility and gene expression occurs in only a subset of cells. The stochastic nature of this process suggests that ATRX acts as a general facilitator of cell specific transcriptional and epigenetic programmes, both in heterochromatin and euchromatin.


Assuntos
Cromatina , Heterocromatina , DNA Helicases/genética , DNA Helicases/metabolismo , Eucromatina/genética , Heterocromatina/genética , Histonas/metabolismo , Retardo Mental Ligado ao Cromossomo X , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo , Talassemia alfa
15.
Sci Rep ; 12(1): 10276, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35715539

RESUMO

Sepsis is caused by an uncontrolled inflammatory response, whose underlying mechanisms are not fully understood. It is well known that the majority of human genes can be expressed as alternative isoforms. While isoform switching is implicated in many diseases and is particularly prominent in cancer, it has never been reported in the context of sepsis. Patients presented to the emergency department of three tertiary care hospitals from January 2020 to December 2020 were enrolled. Clinical variables and genome-wide transcriptome of peripheral blood mononuclear cells (PBMC) were obtained. Isoform switching analysis were performed to identify significant isoform switches and relevant biological consequences. A total of 48 subjects with sepsis, involving 42 survivors and 6 non-survivors, admitted to the emergency department of three tertiary care hospitals were enrolled in this study. PBMCs were extracted for RNA sequencing (RNA-seq). Patients (n = 4) with mild stroke or acute coronary syndrome without infection were enrolled in this study as controls. The most frequent functional changes resulting from isoform switching were changes affecting the open reading frame, protein domains and intron retention. Many genes without differences in gene expression showed significant isoform switching. Many genes with significant isoform switches ([Formula: see text]> 0.1) were associated with higher mortality risk, including PIGS, CASP3, LITAF, HBB and RUVBL2. The study for the first time described the landscape of isoform switching in sepsis, including differentially expressed isoform fractions between patients with and without sepsis and survivors and nonsurvivors. The biological consequences of isoform switching, including protein domain loss, signal peptide gain, and intron retention, were identified.


Assuntos
Leucócitos Mononucleares , Sepse , ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Transporte/genética , Estudos de Coortes , DNA Helicases/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Leucócitos Mononucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sepse/genética
16.
Int Rev Psychiatry ; 34(2): 154-167, 2022 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-35699097

RESUMO

Chromatin remodelling is an important process in neural development and is related to autism spectrum disorder (ASD) and schizophrenia (SCZ) aetiology. To further elucidate the involvement of chromatin remodelling genes in the genetic aetiology of ASD and SCZ in the Japanese population, we performed a case-control study. Targeted sequencing was conducted on coding regions of four BAF chromatin remodelling complex genes: SMARCA2, SMARCA4, SMARCC2, and ARID1B in 185 ASD, 432 SCZ patients, and 517 controls. 27 rare non-synonymous variants were identified in ASD and SCZ patients, including 25 missense, one in-frame deletion in SMRACA4, and one frame-shift variant in SMARCC2. Association analysis was conducted to investigate the burden of rare variants in BAF genes in ASD and SCZ patients. Significant enrichment of rare missense variants in BAF genes, but not synonymous variants, was found in ASD compared to controls. Rare pathogenic variants indicated by in silico tools were significantly enriched in ASD, but not statistically significant in SCZ. Pathogenic-predicted variants were located in disordered binding regions and may confer risk for ASD and SCZ by disrupting protein-protein interactions. Our study supports the involvement of rare missense variants of BAF genes in ASD and SCZ susceptibility.


Assuntos
Transtorno do Espectro Autista , Montagem e Desmontagem da Cromatina , Transtorno do Espectro Autista/genética , Estudos de Casos e Controles , Montagem e Desmontagem da Cromatina/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença , Humanos , Japão , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Esquizofrenia , Fatores de Transcrição/genética
17.
Oncol Rep ; 48(1)2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35656882

RESUMO

Excision repair cross­complementation group 6 like (ERCC6L) has been reported to be upregulated in a variety of malignant tumors and plays a critical oncogenic role. However, the role and molecular mechanism of ERCC6L in lung adenocarcinoma (LUAD) remain unclear, and were therefore investigated in the present study. Clinical data of patients with LUAD were obtained and bioinformatics analysis was performed to investigate the expression characteristics, prognostic value, and biological function of ERCC6L. In addition, cell function experiments were performed to detect the effect of ERCC6L silencing on the biological behavior of LUAD cells. The results revealed that ERCC6L expression was significantly higher in LUAD tissues vs. normal lung tissues and closely associated with nodal invasion, advanced clinical stage and survival in LUAD. Overexpression of ERCC6L was an independent prognostic biomarker of overall survival, progression­free interval, and disease­specific survival in patients with LUAD. DNA amplification and low methylation levels of ERCC6L suggested regulation at both the genetic and epigenetic levels. The most significant positive genes co­expressed with ERCC6L were mainly enriched in the cell cycle signaling pathway. The major functions of ERCC6L in LUAD cells were positively correlated with the cell cycle, DNA damage, DNA repair, proliferation, invasion and epithelial­mesenchymal transition (EMT). Knockdown of ERCC6L inhibited the proliferative, migratory and invasive abilities of A549 and PC9 cells. It also promoted cell apoptosis, and led to cell cycle arrest in the S phase. ERCC6L may regulate the EMT process through the Wnt/ß­catenin and Wnt/Notch 3 signaling pathways, thus regulating the tumorigenesis and progression of LUAD. The overexpression of ERCC6L may be a biological indicator for the diagnosis and prognosis of LUAD. ERCC6L may be a novel molecular target for the treatment of lung cancer.


Assuntos
Adenocarcinoma de Pulmão , DNA Helicases , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/patologia , Proliferação de Células/genética , DNA , DNA Helicases/genética , Humanos , Neoplasias Pulmonares/patologia , Fenótipo , Prognóstico
18.
Oncogene ; 41(23): 3239-3250, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35508542

RESUMO

Although enzalutamide improves the overall survival of patients with metastatic prostate cancers, enzalutamide resistance (ENZR) will be inevitably developed. Emerging evidence support that alternative oncogenic pathways may bypass the androgen receptor (AR) signaling to promote ENZR progression, however, the underpinning mechanisms remain poorly defined. Here, we report that the expression of RuvB like AAA ATPase 1 (RUVBL1) is upregulated in ENZR cells and xenograft models and prostate tumors in patients. Enzalutamide increases RUVBL1 accumulation in the cytoplasm, which in turn enhances the recruitment of CRAF proto-oncogene serine/threonine kinase protein to plexin A1 (PLXNA1) and the subsequent activation of the downstream MAPK pathway. Co-overexpression of RUVBL1 and PLXNA1 defines a subgroup of prostate cancer (PCa) patients with a poor prognosis. Furthermore, pharmacological inhibition of RUVBL1 by CB-6644 suppresses ENZR cell proliferation and xenograft growth and allows re-sensitization of ENZR cells and xenografts to enzalutamide, indicating that RUVBL1 may act to substitute the AR signaling to promote cancer cell survival and ENZR development. Together, these findings may lead to the identification of RUVBL1 as a potential therapeutic target for ENZR tumors.


Assuntos
Neoplasias de Próstata Resistentes à Castração , ATPases Associadas a Diversas Atividades Celulares/genética , Benzamidas , Proteínas de Transporte , Linhagem Celular Tumoral , Proliferação de Células , DNA Helicases/genética , DNA Helicases/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Masculino , Proteínas do Tecido Nervoso/metabolismo , Nitrilas/uso terapêutico , Feniltioidantoína , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores de Superfície Celular/metabolismo
19.
Cells ; 11(10)2022 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-35626643

RESUMO

DEAD/H-box proteins are the largest family of RNA helicases in mammalian genomes, and they are present in all kingdoms of life. Since their discovery in the late 1980s, DEAD/H-box family proteins have been a major focus of study. They have been found to play central roles in RNA metabolism, gene expression, signal transduction, programmed cell death, and the immune response to bacterial and viral infections. Aberrant functions of DEAD/H-box proteins have been implicated in a wide range of human diseases that include cancer, neurodegeneration, and inherited genetic disorders. In this review, we provide a historical context and discuss the molecular functions of DEAD/H-box proteins, highlighting the recent discoveries linking their dysregulation to human diseases. We will also discuss the state of knowledge regarding two specific DEAD/H-box proteins that have critical roles in immune responses and programmed cell death, DDX3X and DDX58, also known as RIG-I. Given their importance in homeostasis and disease, an improved understanding of DEAD/H-box protein biology and protein-protein interactions will be critical for informing strategies to counteract the pathogenesis associated with several human diseases.


Assuntos
RNA Helicases DEAD-box , RNA , Animais , Morte Celular , Diferenciação Celular , RNA Helicases DEAD-box/metabolismo , DNA Helicases , Humanos , Inflamação , Mamíferos/metabolismo , RNA/metabolismo
20.
JCI Insight ; 7(12)2022 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-35511432

RESUMO

Recent studies highlighted the clinicopathologic importance of the tumor microenvironment (TME) in delineating molecular attributes and therapeutic potentials. However, the overall TME cell infiltration landscape in nonsquamous non-small cell lung cancer (NSCLC) has not been comprehensively characterized. In this study, we used consensus non-negative matrix factorization molecular subtyping to determine TME cell infiltration patterns and identified 3 TME clusters (TME-C1, -C2, -C3) characterized by distinct clinicopathologic features, infiltrating cells, and biological processes. Proteomics analyses revealed that cyclic GMP-AMP-stimulator of interferon genes immune signaling-mediated protein and phosphorylation levels were significantly upregulated in inflammation-related TME-C2 clusters. The score extracted from the TME-related signature (TMEsig-score) divided patients with NSCLC into high- and low-score subgroups, where a high score was associated with favorable prognosis and immune infiltration. The genomic landscape revealed that patients with low TMEsig-score harbored more somatic copy number alterations and higher mutation frequency of driver genes involving STK11, KEAP1, SMARCA4, and others. Drug sensitivity analyses suggested that tumors with high TMEsig-score were responsible for favorable clinical response to immune checkpoint inhibitor treatment. In summary, this study highlights that comprehensive recognizing of the TME cell infiltration landscape will contribute to enhancing our understanding of TME immune regulation and promote effectiveness of precision biotherapy strategies.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , DNA Helicases , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Fator 2 Relacionado a NF-E2 , Proteínas Nucleares , Fatores de Transcrição , Microambiente Tumoral
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