RESUMO
Beyond its essential roles in ensuring faithful chromosome segregation and genomic stability, the human Smc5/6 complex acts as an antiviral factor. It binds to and impedes the transcription of extrachromosomal DNA templates; an ability which is lost upon integration of the DNA into the chromosome. How the complex distinguishes among different DNA templates is unknown. Here we show that, in human cells, Smc5/6 preferentially binds to circular rather than linear extrachromosomal DNA. We further demonstrate that the transcriptional process, per se, and particularly the accumulation of DNA secondary structures known to be substrates for topoisomerases, is responsible for Smc5/6 recruitment. More specifically, we find that in vivo Smc5/6 binds to positively supercoiled DNA. Those findings, in conjunction with our genome-wide Smc5/6 binding analysis showing that Smc5/6 localizes at few but highly transcribed chromosome loci, not only unveil a previously unforeseen role of Smc5/6 in DNA topology management during transcription but highlight the significance of sensing DNA topology as an antiviral defense mechanism.
Assuntos
Proteínas de Ciclo Celular , DNA Super-Helicoidal , Transcrição Gênica , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , DNA Super-Helicoidal/metabolismo , DNA Super-Helicoidal/genética , Ligação Proteica , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , DNA/metabolismo , DNA/genética , Conformação de Ácido Nucleico , DNA Circular/metabolismo , DNA Circular/genéticaRESUMO
Background: Clinically, the persistence of HBV cccDNA is the major obstacle in anti-HBV therapy. However, the underlying mechanism of HBV cccDNA is poorly understood. The transcriptional factor STAT3 is able to activate HBV replication in liver. Approach & Results: RNA-Seq analysis demonstrated that cucurbitacin I targeting STAT3 was associated with virus replication in liver. HBV-infected human liver chimeric mouse model and HBV hydrodynamic injection mouse model were established. Then, we validated that cucurbitacin I effectively limited the stability of HBV cccDNA and HBV replication in cells, in which cucurbitacin I enhanced the sensitivity of pegylated interferon α (PEG-IFN α) against HBV via combination in vitro and in vivo. Mechanistically, we identified that cucurbitacin I increased the levels of APOBEC3B to control HBV cccDNA by inhibiting p-STAT3 in cells, resulting in the inhibition of HBV replication. Moreover, RNA-Seq data showed that E3 ubiquitin ligase DTX4 might be involved in the events. Then, we observed that HBV particles could upregulate DTX4 by increasing the levels of p-STAT3 in vitro and in vivo. The p-STAT3-elevated DTX4/male-specific lethal 2 (MSL2) independently and synergistically enhanced the stability of HBV cccDNA by facilitating the ubiquitination degradation of APOBEC3B in cells, leading to the HBV replication. Conclusions: p-STAT3-elevated DTX4 confers the stability of HBV cccDNA and HBV replication by facilitating the ubiquitination degradation of APOBEC3B. Cucurbitacin â effectively enhances the sensitivity of PEG-IFN α in anti-HBV therapy by inhibiting the p-STAT3/DTX4/MSL2/APOBEC3B signalling. Our finding provides new insights into the mechanism of HBV cccDNA. The p-STAT3 and DTX4/MSL2 might serve as the therapeutical targets of HBV cccDNA.
Assuntos
Citidina Desaminase , Vírus da Hepatite B , Fígado , Antígenos de Histocompatibilidade Menor , Fator de Transcrição STAT3 , Ubiquitina-Proteína Ligases , Replicação Viral , Fator de Transcrição STAT3/metabolismo , Humanos , Animais , Vírus da Hepatite B/efeitos dos fármacos , Camundongos , Replicação Viral/efeitos dos fármacos , Antígenos de Histocompatibilidade Menor/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Fígado/virologia , Fígado/metabolismo , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Masculino , Ubiquitina-Proteína Ligases/metabolismo , DNA Circular/metabolismo , DNA Circular/genética , Ubiquitinação/efeitos dos fármacos , DNA Viral/metabolismo , DNA Viral/genética , Hepatite B/metabolismo , Hepatite B/virologia , Hepatite B/tratamento farmacológico , Antivirais/farmacologia , Triterpenos/farmacologia , Triterpenos/metabolismo , Células Hep G2 , Modelos Animais de Doenças , Interferon-alfa/metabolismo , Interferon-alfa/farmacologiaRESUMO
Slimming grass carp is a commercial variety with good body form and meat quality, which is cultured by starving common grass carp in a clean flowing water environment. Compared to common grass carp, slimming grass carp has a far higher economic value. Until now, no molecular study has concentrated on the regulation mechanism of the muscle characteristics of slimming grass carp. This study first reported the gene expression profile of the muscle characteristics of slimming grass carp based on the level of extrachromosomal circular DNAs (eccDNAs). EccDNAs are double-stranded circular DNAs derived from genomic DNAs and play crucial roles in the functional regulation of a wide range of biological processes, none of which have been shown to occur in fish. Here, muscle eccDNAs from slimming grass carp and common grass carp were both generally sequenced, and the information, as well as the expression profile of eccDNAs, were compared and analysed. The findings reveal that 82,238 and 25,857 eccDNAs were detected from slimming grass carp and common grass carp, respectively. The length distribution of eccDNAs was in the range of 1~1000 bp, with two peaks at about 200 bp and 400 bp. When the expression profiles of eccDNAs between slimming grass carp and common grass carp were compared, 3523 up-regulated and 175 down-regulated eccDNAs were found. Enrichment analysis showed that these eccDNA genes were correlated with cellular structure and response, cell immunology, enzyme activity, etc. Certain differentially expressed eccDNAs involved in muscle characteristics were detected, which include myosin heavy chain, myosin light chain, muscle segment homeobox C, calsequestrin, calmodulin, etc., among which the majority of genes were linked to muscle structure and contraction. This indicates that during the process of cultivating from common grass carp to slimming grass carp, the treatment primarily affected muscle structure and contraction, making the meat quality of slimming grass carp different from that of common grass carp. This result provides molecular evidence and new insights by which to elucidate the regulating mechanism of muscle phenotypic characterisation in slimming grass carp and other fish.
Assuntos
Carpas , DNA Circular , Carpas/genética , Carpas/metabolismo , Animais , DNA Circular/genética , Músculos/metabolismoRESUMO
Rationale: Extrachromosomal circular DNA is a hallmark of cancer, but its role in shaping the genome heterogeneity of urothelial bladder carcinoma (UBC) remains poorly understood. Here, we comprehensively analyzed the features of extrachromosomal circular DNA in 80 UBC patients. Methods: We performed whole-genome/exome sequencing (WGS/WES), Circle-Seq, single-molecule real-time (SMRT) long-read sequencing of circular DNA, and RNA sequencing (RNA-Seq) on 80 pairs of tumor and AT samples. We used our newly developed circular DNA analysis software, Circle-Map++ to detect small extrachromosomal circular DNA from Circle-Seq data. Results: We observed a high load and significant heterogeneity of extrachromosomal circular DNAs in UBC, including numerous single-locus and complex chimeric circular DNAs originating from different chromosomes. This includes highly chimeric circular DNAs carrying seven oncogenes and circles from nine chromosomes. We also found that large tumor-specific extrachromosomal circular DNAs could influence genome-wide gene expression, and are detectable in time-matched urinary sediments. Additionally, we found that the extrachromosomal circular DNA correlates with hypermutation, copy number variation, oncogene amplification, and clinical outcome. Conclusions: Overall, our study provides a comprehensive extrachromosomal circular DNA map of UBC, along with valuable data resources and bioinformatics tools for future cancer and extrachromosomal circular DNA research.
Assuntos
Variações do Número de Cópias de DNA , DNA Circular , Neoplasias da Bexiga Urinária , Neoplasias da Bexiga Urinária/genética , Humanos , DNA Circular/genética , Variações do Número de Cópias de DNA/genética , Sequenciamento Completo do Genoma/métodos , Heterogeneidade Genética , Masculino , Feminino , Sequenciamento do Exoma/métodos , Idoso , Mutação/genéticaRESUMO
The discovery of Replication Competent Circular DNA molecules in mammalian cells and tissues is being linked to debilitating diseases, such as multiple sclerosis (MS), bovine spongiform encephalopathy (BSE), and colorectal cancer (CRC). These circular DNA molecules, otherwise known as bovine meat and milk factors (BMMFs) and Slow Progressive Hidden INfections of variable (X) latency (SPHINX), bear significant (80%) sequence similarity with the plasmids of Acinetobacter baumannii strains. Nanostructures, such as bacterial outer membrane vesicles (OMVs) serve as vehicles for transporting biomolecular cargo and have the potential to facilitate interkingdom lateral mobility of DNA. Strengthening the proposed hypothesis, this study demonstrates that OMVs derived from A. baumannii DS002 carrying four plasmids and genome (pTS236) of phage, AbDs1, successfully reached different parts of the body, including the central nervous system, following the injection of fluorescein isothiocyanate (FITC)-labeled OMVs into experimental mice. Out of the four OMV-associated plasmids, three (pTS4586, pTS9900, and pTS134338) were identified within the lumen, and the fourth one (pTS11291) was found on the surface of OMVs. In addition to the indigenous plasmids, the phage-encoded protein, Orf96, anchored on the surface of the OMVs by establishing a strong interaction with the OMV-associated porin, OmpA. Intriguingly, a subset of labeled OMVs, when incubated with Neuro2A cells, translocated across the membrane and reached to the cytoplasmic space of the cells. Collectively, the experimental evidence presented herein underscores the promising potential of OMVs as vehicles for delivering molecular cargo containing plasmids and phage genomes to diverse mammalian tissues and cells. IMPORTANCE: Several independent studies have demonstrated the existence of replication competent circular DNA molecules of bacterial and viral origin in mammalian cells and tissues. However, studies about their origin and lateral mobility to mammalian cells are scarce. Our work describes the existence of circular DNA, similar to that of DNA molecules identified in mammalian cells, OMVs derived from soil isolate of A. baumannii DS002. Furthermore, the work also provides visual evidence that demonstrates the passage of labeled OMVs to different organs of experimental mice within hours after intravenously administering OMVs into experimental mice. Some of the labeled OMVs have even crossed the membrane of Neuro2A, suggesting the existence of interkingdom horizontal mobility between bacteria and mammals.
Assuntos
Acinetobacter baumannii , DNA Circular , Transferência Genética Horizontal , Plasmídeos , Animais , Camundongos , Plasmídeos/genética , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Bovinos , DNA Circular/genética , DNA Circular/metabolismo , Membrana Externa Bacteriana/metabolismo , Feminino , Bacteriófagos/genética , Bacteriófagos/fisiologia , Carne/microbiologia , Leite/microbiologia , Infecções por Acinetobacter/microbiologia , Vesículas Extracelulares/metabolismo , Camundongos Endogâmicos BALB C , DNA Bacteriano/genéticaRESUMO
The efficient preparation of single-stranded DNA (ssDNA) rings, as a macromolecular construction approach with topological features, has aroused much interest due to the ssDNA rings' numerous applications in biotechnology and DNA nanotechnology. However, an extra splint is essential for enzymatic circularization, and by-products of multimers are usually present at high concentrations. Here, we proposed a simple and robust strategy using permuted precursor (linear ssDNA) for circularization by forming an intramolecular dynamic nick using a part of the linear ssDNA substrate itself as the template. After the simulation of the secondary structure for desired circular ssDNA, the linear ssDNA substrate is designed to have its ends on the duplex part (≥5 bp). By using this permuted substrate with 5'-phosphate, the splint-free circularization is simply carried out by T4 DNA ligase. Very interestingly, formation of only several base pairs (2-4) flanking the nick is enough for ligation, although they form only instantaneously under ligation conditions. More significantly, the 5-bp intramolecular duplex part commonly exists in genomes or functional DNA, demonstrating the high generality of our approach. Our findings are also helpful for understanding the mechanism of enzymatic DNA ligation from the viewpoint of substrate binding.
Assuntos
DNA Ligases , DNA Circular , DNA de Cadeia Simples , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/química , DNA Ligases/metabolismo , DNA Ligases/química , DNA Circular/química , DNA Circular/metabolismo , Conformação de Ácido NucleicoRESUMO
Extrachromosomal DNA (ecDNA) are circular DNA structures associated with cancer and drug resistance. One specific type, double minute (DM) chromosomes, has been studied since the 1960s using imaging techniques like cytogenetics and fluorescence microscopy. Specialized techniques such as atomic force microscopy (AFM) and scanning electron microscopy (SEM) offer micro to nano-scale visualization, but current sample preparation methods may not optimally preserve ecDNA structure. Our study introduces a systematic protocol using SEM for high-resolution ecDNA visualization. We have optimized the end-to-end procedure, providing a standardized approach to explore the circular architecture of ecDNA and address the urgent need for better understanding in cancer research.
Despite advances in extrachromosomal DNA (ecDNA) detection, current methods struggle to reveal ecDNA's architecture within cells. Specialized techniques like scanning electron microscopy (SEM) provide the needed resolution, but existing sample preparation may not preserve ecDNA well. Our study introduces a systematic method using SEM, optimizing procedures for preparing and visualizing metaphase spread samples. This offers a standardized approach to study ecDNA's circular architecture, addressing a pressing need in cancer research.
Assuntos
DNA Circular , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Varredura/métodos , Humanos , DNA Circular/química , DNA Circular/genética , DNA Circular/ultraestrutura , DNA/genética , DNA/análise , DNA/química , DNA/ultraestruturaRESUMO
The persistent presence of covalently closed circular DNA (cccDNA) in hepatocyte nuclei poses a significant obstacle to achieving a comprehensive cure for hepatitis B virus (HBV). Current applications of CRISPR/Cas9 for targeting and eliminating cccDNA have been confined to in vitro studies due to challenges in stable cccDNA expression in animal models and the limited non-immunogenicity of delivery systems. This study addresses these limitations by introducing a novel non-viral gene delivery system utilizing Gemini Surfactant (GS). The developed system creates stable and targeted CRISPR/Cas9 nanodrugs with a negatively charged surface through modification with red blood cell membranes (RBCM) or hepatocyte membranes (HCM), resulting in GS-pDNA@Cas9-CMs complexes. These GS-pDNA complexes demonstrated complete formation at a 4:1 w/w ratio. The in vitro transfection efficiency of GS-pDNA-HCM reached 54.61%, showing homotypic targeting and excellent safety. Additionally, the study identified the most effective single-guide RNA (sgRNA) from six sequences delivered by GS-pDNA@Cas9-HCM. Using GS-pDNA@Cas9-HCM, a significant reduction of 96.47% in in vitro HBV cccDNA and a 52.34% reduction in in vivo HBV cccDNA were observed, along with a notable decrease in other HBV-related markers. The investigation of GS complex uptake by AML-12 cells under varied time and temperature conditions revealed clathrin-mediated endocytosis (CME) for GS-pDNA and caveolin-mediated endocytosis (CVME) for GS-pDNA-HCM and GS-pDNA-RBCM. In summary, this research presents biomimetic gene-editing nanovectors based on GS (GS-pDNA@Cas9-CMs) and explores their precise and targeted clearance of cccDNA using CRISPR/Cas9, demonstrating good biocompatibility both in vitro and in vivo. This innovative approach provides a promising therapeutic strategy for advancing the cure of HBV.
Assuntos
Sistemas CRISPR-Cas , Técnicas de Transferência de Genes , Vírus da Hepatite B , Hepatite B , Nanopartículas , Hepatite B/terapia , Vírus da Hepatite B/genética , Humanos , Nanopartículas/química , Animais , Células Hep G2 , Terapia Genética/métodos , Materiais Biomiméticos/química , DNA Circular , Plasmídeos/genética , Plasmídeos/administração & dosagem , Hepatócitos/metabolismo , Biomimética/métodos , Tensoativos/química , DNA Viral/genética , CamundongosRESUMO
Extrachromosomal circular DNA (eccDNA) derived from linear chromosomes, are showed typical nucleosomal ladder pattern in agarose gel which as a known feature of apoptosis and demonstrated to be immunogenicity. In systemic lupus erythematosus (SLE) patients, elevated levels of cell-free DNA (cfDNA) can be found in either linear forms or circular forms, while circular ones are much less common and harder to detect. The molecular characteristics and function of circular forms in plasma SLE patients remains elusive. Herein, we characterized the hallmarks of plasma eccDNA in SLE patients, including the lower normalized number and GC content of eccDNA in SLE plasma than in the healthy, and SLE eccDNA number positively correlated with C3 and negatively with anti-dsDNA antibodies. The differential eccGenes (eccDNAs carrying the protein coding gene sequence) of SLE was significantly enriched in apoptosis-related pathways. The artificially synthesized eccDNA with sequences of the PRF1 exon region could promote transcriptional expression of PRF1, IFNA and IFIT3 and inhibit early-stage apoptosis. Plasma eccDNA can serve as a novel autoantigen in the pathogenesis of SLE.
Assuntos
Apoptose , DNA Circular , Lúpus Eritematoso Sistêmico , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Humanos , DNA Circular/genética , Feminino , Adulto , Masculino , Pessoa de Meia-Idade , Ácidos Nucleicos Livres/genética , Ácidos Nucleicos Livres/sangue , Anticorpos Antinucleares/sangue , Estudo de Associação Genômica AmplaRESUMO
BACKGROUND: Gestational diabetes mellitus (GDM) significantly impacts maternal and infant health both immediately and over the long term, yet effective early diagnostic biomarkers are currently lacking. Thus, it is essential to identify early diagnostic biomarkers for GDM risk screening. Extrachromosomal circular DNA (eccDNA), being more stable than linear DNA and involved in disease pathologies, is a viable biomarker candidate for diverse conditions. In this study, eccDNA biomarkers identified for early diagnosis and assessment of GDM risk were explored. METHODS: Using Circle-seq, we identified plasma eccDNA profiles in five pregnant women who later developed GDM and five matched healthy controls at 11-13 weeks of gestation. These profiles were subsequently analyzed through bioinformatics and validated through outward PCR combined with Sanger sequencing. Furthermore, candidate eccDNA was validated by quantitative PCR (qPCR) in a larger cohort of 70 women who developed GDM and 70 normal glucose-tolerant (NGT) subjects. A ROC curve assessed the eccDNA's diagnostic potential for GDM. RESULTS: 2217 eccDNAs were differentially detected between future GDM patients and controls, with 1289 increased and 928 decreased in abundance. KEGG analysis linked eccDNA genes mainly to GDM-related pathways such as Rap1, MAPK, and PI3K-Akt, and Insulin resistance, among others. Validation confirmed a significant decrease in eccDNA PRDM16circle in the plasma of 70 women who developed GDM compared to 70 NGT women, consistent with the eccDNA-seq results. PRDM16circle showed significant diagnostic value in 11-13 weeks of gestation (AUC = 0.941, p < 0.001). CONCLUSIONS: Our study first demonstrats that eccDNAs are aberrantly produced in women who develop GDM, including PRDM16circle, which can predict GDM at an early stage of pregnancy, indicating its potential as a biomarker. TRIAL REGISTRATION: ChiCTR2300075971, http://www.chictr.org.cn . Registered 20 September 2023.
Assuntos
DNA Circular , Diabetes Gestacional , Idade Gestacional , Valor Preditivo dos Testes , Humanos , Diabetes Gestacional/diagnóstico , Diabetes Gestacional/sangue , Diabetes Gestacional/genética , Feminino , Gravidez , Adulto , Estudos de Casos e Controles , Medição de Risco , Fatores de Risco , DNA Circular/sangue , DNA Circular/genética , Primeiro Trimestre da Gravidez/sangue , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Biomarcadores/sangue , Reprodutibilidade dos Testes , Diagnóstico PrecoceRESUMO
Cyclic nucleic acids are biologically stable against nucleic acid exonucleases due to the absence of 5' and 3' termini. Studies of cyclic nucleic acids mainly focus on cyclic single-stranded nucleic acids. Cyclic single-stranded nucleic acids are further divided into circular RNA (circRNA) and circular single-stranded DNA (cssDNA). The synthesis methods of circRNA include lasso-driven cyclization, intron-paired cyclization, intron cyclization, intron complementary pairing-driven cyclization, RNA-binding protein-driven cyclization, and artificial synthesis depending on the source. Its main role is to participate in gene expression and the treatment of some diseases. Circular single-stranded DNA is mainly synthesized by chemical ligation, template-directed enzyme ligation, and new techniques for the efficient preparation of DNA single loops and topologies based on CircLigase. It is mainly used in rolling circle amplification (RCA) technology and in the bioprotection of circular aptamers and second messengers. This review focuses on the types, synthesis methods, and applications of cyclic single-stranded nucleic acids, providing a reference for further research on cyclic single-stranded nucleic acids.
Assuntos
DNA de Cadeia Simples , RNA Circular , RNA Circular/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , DNA Circular/genética , DNA Circular/química , Ciclização , Técnicas de Amplificação de Ácido Nucleico/métodos , HumanosRESUMO
The holoparasitic plant Lophophytum mirabile exhibits remarkable levels of mitochondrial horizontal gene transfer (HGT). Gathering comparative data from other individuals and host plants can provide insights into the HGT process. We sequenced the mitochondrial genome (mtDNA) from individuals of two species of Lophophytum and from mimosoid hosts. We applied a stringent phylogenomic approach to elucidate the origin of the whole mtDNAs, estimate the timing of the transfers, and understand the molecular mechanisms involved. Ancestral and recent HGT events replaced and enlarged the multichromosomal mtDNA of Lophophytum spp., with the foreign DNA ascending to 74%. A total of 14 foreign mitochondrial chromosomes originated from continuous regions in the host mtDNA flanked by short direct repeats. These foreign tracts are circularized by microhomology-mediated repair pathways and replicate independently until they are lost or they eventually recombine with other chromosomes. The foreign noncoding chromosomes are variably present in the population and likely evolve by genetic drift. We present the 'circle-mediated HGT' model in which foreign mitochondrial DNA tracts become circular and are maintained as plasmid-like molecules. This model challenges the conventional belief that foreign DNA must be integrated into the recipient genome for successful HGT.
Assuntos
DNA Circular , DNA Mitocondrial , Transferência Genética Horizontal , Filogenia , DNA Mitocondrial/genética , DNA Circular/genética , Reparo do DNA/genética , Genoma Mitocondrial/genéticaRESUMO
Hepatitis B virus (HBV) is an obligate human hepatotropic DNA virus causing both transient and chronic infection. The livers of chronic hepatitis B patients have a high risk of developing liver fibrosis, cirrhosis, and hepatocellular carcinoma. The nuclear episomal viral DNA intermediate, covalently closed circular DNA (cccDNA), forms a highly stable complex with host and viral proteins to serve as a transcription template and support HBV infection chronicity. Thus, characterization of the composition and dynamics of cccDNA nucleoprotein complexes providing cccDNA stability and gene regulation is of high importance for both basic and medical research. The presented method for chromatin immunoprecipitation coupled with qPCR (ChIP-qPCR) allows to assess provisional physical interaction of the protein of interest (POI) with cccDNA using POI-specific antibody, the level of enrichment of a POI on cccDNA versus control/background is characterized quantitatively using qPCR.
Assuntos
Imunoprecipitação da Cromatina , DNA Circular , DNA Viral , Vírus da Hepatite B , Vírus da Hepatite B/genética , DNA Circular/genética , DNA Circular/metabolismo , Humanos , DNA Viral/genética , Imunoprecipitação da Cromatina/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Hepatite B/virologia , Hepatite B/genéticaAssuntos
Biomarcadores , DNA Circular , Humanos , Biomarcadores/sangue , DNA Circular/sangue , DNA Circular/análise , DNA Circular/genética , Nódulo da Glândula Tireoide/diagnóstico , Nódulo da Glândula Tireoide/sangue , Nódulo da Glândula Tireoide/genética , Feminino , Masculino , Pessoa de Meia-Idade , AdultoRESUMO
We investigate the melting transition of non-supercoiled circular DNA of different lengths, employing Brownian dynamics simulations. In the absence of supercoiling, we find that melting of circular DNA is driven by a large bubble, which agrees with the previous predictions of circular DNA melting in the presence of supercoiling. By analyzing sector-wise changes in average base-pair distance, our study reveals that the melting behavior of circular DNA closely resembles that of linear DNA. Additionally, we find a marked difference in the thermal stability of circular DNA over linear DNA at very short length scales, an effect that diminishes as the length of circular DNA increases. The stability of smaller circular DNA is linked to the occurrence of transient small bubbles, characterized by a lower probability of growth.
Assuntos
DNA Circular , Desnaturação de Ácido Nucleico , DNA Circular/química , Conformação de Ácido Nucleico , Simulação de Dinâmica Molecular , Temperatura de Transição , DNA/química , TermodinâmicaRESUMO
BACKGROUND: Extrachromosomal circular DNA (eccDNA), a kind of circular DNA that originates from chromosomes, carries complete gene information, particularly the oncogenic genes. This study aimed to examine the contributions of FAM84B induced by eccDNA to prostate cancer (PCa) development and the biomolecules involved. METHODS: The presence of eccDNA in PCa cells and the FAM84B transcripts that eccDNA carries were verified by outward and inward PCR. The effect of inhibition of eccDNA synthesis on FAM84B expression in PCa cells was analyzed by knocking down Lig3. The impact of FAM84B on the growth and metastases of PCa cells was verified by Cell Counting Kit-8 (CCK8), EdU, transwell assays, and a xenograft mouse model. Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) and dual-luciferase reporter assays were carried out to examine the effect of FAM84B/MYC on WWP1 transcription, and a co-immunoprecipitation (Co-IP) assay was conducted to verify the modification of CDKN1B by WWP1. The function of this molecular axis in PCa was explored by rescue assays. RESULTS: The inhibited eccDNA synthesis significantly downregulated FAM84B in PCa cells, thereby attenuating the growth and metastasis of PCa. FAM84B promoted the transcription of WWP1 by MYC by activating the expression of MYC coterminous with the 8q24.21 gene desert in a beta catenin-dependent approach. WWP1 transcription promoted by MYC facilitated the ubiquitination and degradation of CDKN1B protein and inversely attenuated the repressive effect of CDKN1B on MYC expression. Exogenous overexpression of CDKN1B blocked FAM84B-activated MYC/WWP1 expression, thereby inhibiting PCa progression. CONCLUSIONS: FAM84B promoted by eccDNA mediates degradation of CDKN1B via MYC/WWP1, thereby accelerating PCa progression.
Assuntos
DNA Circular , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata , Proteínas Proto-Oncogênicas c-myc , Ubiquitina-Proteína Ligases , Masculino , Humanos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Linhagem Celular Tumoral , Animais , DNA Circular/genética , DNA Circular/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Camundongos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proliferação de Células/genética , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Inibidor de Quinase Dependente de Ciclina p27RESUMO
The covalently closed circular DNA (cccDNA) of the hepatitis B virus (HBV) is organized as a minichromosome structure in the nucleus of infected hepatocytes and considered the major obstacle to the discovery of a cure for HBV. Until now, no strategies directly targeting cccDNA have been advanced to clinical stages as much is unknown about the accessibility and activity regulation of the cccDNA minichromosome. We have described the method for evaluation of the cccDNA minichromosome accessibility using micrococcal nuclease-quantitative polymerase chain reaction and high-throughput sequencing, which could be useful tools for cccDNA research and HBV cure studies.
Assuntos
DNA Circular , DNA Viral , Vírus da Hepatite B , Sequenciamento de Nucleotídeos em Larga Escala , Vírus da Hepatite B/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA Circular/genética , Humanos , DNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Nuclease do Micrococo/metabolismo , Nuclease do Micrococo/genéticaRESUMO
Hepatitis B virus (HBV) infects hepatocytes that are in the G0/G1 phase with intact nuclear membrane and organized chromosome architecture. In the nucleus of the infected cells, HBV covalently closed circular (ccc) DNA, an episomal minichromosome, serves as the template for all viral transcripts and the reservoir of persistent infection. Nuclear positioning of cccDNA can be assessed by the spatial distance between viral DNA and host chromosomal DNA through Circular Chromosome Conformation Capture (4C) combined with high-throughput sequencing (4C-seq). The 4C-seq analysis relies on proximity ligation and is commonly used for mapping genomic DNA regions that communicate within a host chromosome. The method has been tailored for studying nuclear localization of HBV episomal cccDNA in relation to the host chromosomes. In this study, we present a step-by-step protocol for 4C-seq analysis of HBV infection, including sample collection and fixation, 4C DNA library preparation, sequence library preparation, and data analysis. Although limited by proximity ligation of DNA fragments, 4C-seq analysis provides useful information of HBV localization in 3D genome, and aids the understanding of viral transcription in light of host chromatin conformation.
Assuntos
DNA Circular , DNA Viral , Vírus da Hepatite B , Sequenciamento de Nucleotídeos em Larga Escala , Vírus da Hepatite B/genética , Humanos , DNA Circular/genética , DNA Circular/metabolismo , DNA Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hepatite B/virologia , Interações Hospedeiro-Patógeno/genética , Cromossomos/genética , Biblioteca Gênica , Cromossomos Humanos/genética , Cromossomos Humanos/virologiaRESUMO
HBV covalently closed circular DNA (cccDNA) plays an important role in the persistence of hepatitis B virus (HBV) infection by serving as the template for transcription of viral RNAs. To cure HBV infection, it is expected that cccDNA needs either to be eliminated or silenced. Hence, precise cccDNA quantification is essential. Sample preparation is crucial to specifically detect cccDNA. Southern blot is regarded as the "gold standard" for specific cccDNA detection but lacks sensitivity. Here, we describe a rapid and reliable modified kit-based, HBV protein-free DNA extraction method as well as a novel enhanced sensitivity Southern blot that uses branched DNA technology to detect HBV DNA in cell culture and liver tissue samples. It is useful for both HBV molecular biology and antiviral research.
Assuntos
Southern Blotting , DNA Circular , DNA Viral , Vírus da Hepatite B , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Humanos , DNA Viral/genética , DNA Viral/isolamento & purificação , DNA Circular/isolamento & purificação , DNA Circular/análise , DNA Circular/genética , Southern Blotting/métodos , Hepatite B/virologia , Hepatite B/diagnóstico , Fígado/virologiaRESUMO
Hepatitis B virus (HBV) is undoubtedly a master in exploiting host resources while evading host defense for its multiplication within a constrained genetic coding capacity. To further unravel these cunning strategies, a clear picture of virus-host interaction with key subcellular and molecular contexts is needed. Here, we describe a FISH protocol modified from the ViewRNA assay that allows direct visualization of HBV RNA, DNA, and cccDNA in cell culture models (e.g., HepAD38, HepG2-NTCP). It can be coupled with immunofluorescence staining of viral or host proteins or other fluorescent tagging systems which could illuminate numerous aspects of virus-host interactions.