RESUMO
Distinct viral gene expression characterizes Epstein-Barr virus (EBV) infection in EBV-producing marmoset B-cell (B95-8) and EBV-associated gastric carcinoma (SNU719) cell lines. CCCTC-binding factor (CTCF) is a structural chromatin factor that coordinates chromatin interactions in the EBV genome. Chromatin immunoprecipitation followed by sequencing against CTCF revealed 16 CTCF binding sites in the B95-8 and SNU719 EBV genomes. The biological function of one CTCF binding site (S13 locus) located on the BamHI A right transcript (BART) miRNA promoter was elucidated experimentally. Microscale thermophoresis assay showed that CTCF binds more readily to the stable form than the mutant form of the S13 locus. EBV BART miRNA clusters encode 22 miRNAs, whose roles are implicated in EBV-related cancer pathogenesis. The B95-8 EBV genome lacks a 11.8-kb EcoRI C fragment, whereas the SNU719 EBV genome is full-length. ChIP-PCR assay revealed that CTCF, RNA polymerase II, H3K4me3 histone, and H3K9me3 histone were more enriched at S13 and S16 (167-kb) loci in B95-8 than in the SNU719 EBV genome. 4C-Seq and 3C-PCR assays using B95-8 and SNU719 cells showed that the S13 locus was associated with overall EBV genomic loci including 3-kb and 167-kb region in both EBV genomes. We generated mutations in the S13 locus in bacmids with or without the 11.8-kb BART transcript unit (BART(+/-)). The S13 mutation upregulated BART miRNA expression, weakened EBV latency, and reduced EBV infectivity in the presence of EcoRI C fragment. Another 3C-PCR assay using four types of BART(+/-)·S13(wild-type(Wt)/mutant(Mt)) HEK293-EBV cells revealed that the S13 mutation decreased DNA associations between the 167-kb region and 3-kb in the EBV genome. Based on these results, CTCF bound to the S13 locus along with the 11.8-kb EcoRI C fragment is suggested to form an EBV 3-dimensional DNA loop for coordinated EBV BART miRNA expression and infectivity.
Assuntos
Infecções por Vírus Epstein-Barr , Infecção Latente , MicroRNAs , Humanos , Infecções por Vírus Epstein-Barr/genética , Fator de Ligação a CCCTC/genética , Herpesvirus Humano 4/genética , Histonas/genética , Células HEK293 , MicroRNAs/genética , Cromatina , Sítios de LigaçãoRESUMO
Diabetes is the leading cause of kidney disease that progresses to kidney failure. However, the key molecular and cellular pathways involved in diabetic kidney disease (DKD) pathogenesis are largely unknown. Here, we performed a comparative analysis of adult human kidneys by examining cell type-specific chromatin accessibility by single-nucleus ATAC-seq (snATAC-seq) and analyzing three-dimensional chromatin architecture via high-throughput chromosome conformation capture (Hi-C method) of paired samples. We mapped the cell type-specific and DKD-specific open chromatin landscape and found that genetic variants associated with kidney diseases were significantly enriched in the proximal tubule- (PT) and injured PT-specific open chromatin regions in samples from patients with DKD. BACH1 was identified as a core transcription factor of injured PT cells; its binding target genes were highly associated with fibrosis and inflammation, which were also key features of injured PT cells. Additionally, Hi-C analysis revealed global chromatin architectural changes in DKD, accompanied by changes in local open chromatin patterns. Combining the snATAC-seq and Hi-C data identified direct target genes of BACH1, and indicated that BACH1 binding regions showed increased chromatin contact frequency with promoters of their target genes in DKD. Thus, our multi-omics analysis revealed BACH1 target genes in injured PTs and highlighted the role of BACH1 as a novel regulator of tubular inflammation and fibrosis.
Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Adulto , Humanos , Cromatina/genética , Nefropatias Diabéticas/genética , Cromossomos , Rim , Fibrose , Inflamação , Diabetes Mellitus/genéticaRESUMO
Genome/chromosome organization is highly ordered and controls various nuclear events, although the molecular mechanisms underlying the functional organization remain largely unknown. Here, we show that the TATA box-binding protein (TBP) interacts with the Cnd2 kleisin subunit of condensin to mediate interphase and mitotic chromosomal organization in fission yeast. TBP recruits condensin onto RNA polymerase III-transcribed (Pol III) genes and highly transcribed Pol II genes; condensin in turn associates these genes with centromeres. Inhibition of the Cnd2-TBP interaction disrupts condensin localization across the genome and the proper assembly of mitotic chromosomes, leading to severe defects in chromosome segregation and eventually causing cellular lethality. We propose that the Cnd2-TBP interaction coordinates transcription with chromosomal architecture by linking dispersed gene loci with centromeres. This chromosome arrangement can contribute to the efficient transmission of physical force at the kinetochore to chromosomal arms, thereby supporting the fidelity of chromosome segregation.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/química , Centrômero/genética , Centrômero/metabolismo , Segregação de Cromossomos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Fúngicos , Mitose , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/química , Proteína de Ligação a TATA-Box/químicaRESUMO
Iron homeostasis is tightly regulated since iron is an essential but toxic element in the cell. The GATA-type transcription factor Fep1 and its orthologs contribute to iron homeostasis in many fungi by repressing genes for iron uptake when intracellular iron is high. Even though the function and interaction partners of Fep1 have been elucidated extensively In Schizosaccharomyces pombe, the mechanism behind iron-sensing by Fep1 remains elusive. It has been reported that Fep1 interacts with Fe-S-containing monothiol glutaredoxin Grx4 and Grx4-Fra2 complex. In this study, we demonstrate that Fep1 also binds iron, in the form of Fe-S cluster. Spectroscopic and biochemical analyses of as isolated and reconstituted Fep1 suggest that the dimeric Fep1 binds Fe-S clusters. The mutation study revealed that the cluster-binding depended on the conserved cysteines located between the two zinc fingers in the DNA binding domain. EPR analyses revealed [Fe-S]-specific peaks indicative of mixed presence of [2Fe-2S], [3Fe-4S], or [4Fe-4S]. The finding that Fep1 is an Fe-S protein fits nicely with the model that the Fe-S-trafficking Grx4 senses intracellular iron environment and modulates the activity of Fep1.
Assuntos
Cisteína/química , Fatores de Transcrição GATA/química , Proteínas Ferro-Enxofre/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/química , Sítios de Ligação , Sequência Conservada , Ferro , Ligação ProteicaRESUMO
Dispersed genetic elements, such as retrotransposons and Pol-III-transcribed genes, including tRNA and 5S rRNA, cluster and associate with centromeres in fission yeast through the function of condensin. However, the dynamics of these condensin-mediated genomic associations remains unknown. We have examined the 3D motions of genomic loci including the centromere, telomere, rDNA repeat locus, and the loci carrying Pol-III-transcribed genes or long-terminal repeat (LTR) retrotransposons in live cells at as short as 1.5-second intervals. Treatment with carbendazim (CBZ), a microtubule-destabilizing agent, not only prevents centromeric motion, but also reduces the mobility of the other genomic loci during interphase. Further analyses demonstrate that condensin-mediated associations between centromeres and the genomic loci are clonal, infrequent and transient. However, when associated, centromeres and the genomic loci migrate together in a coordinated fashion. In addition, a condensin mutation that disrupts associations between centromeres and the genomic loci results in a concomitant decrease in the mobility of the loci. Our study suggests that highly mobile centromeres pulled by microtubules in cytoplasm serve as 'genome mobility elements' by facilitating physical relocations of associating genomic regions.
Assuntos
Centrômero/genética , Interfase/genética , Mitose/genética , Schizosaccharomyces/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/ultraestrutura , Benzimidazóis/farmacologia , Carbamatos/farmacologia , DNA Ribossômico/genética , DNA Ribossômico/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Genoma Fúngico , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Mitose/efeitos dos fármacos , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , RNA Ribossômico 5S/genética , RNA Ribossômico 5S/ultraestrutura , RNA de Transferência/genética , RNA de Transferência/ultraestrutura , Retroelementos/genética , Schizosaccharomyces/citologia , Telômero/genética , Telômero/ultraestruturaRESUMO
The dynamic spatial organization of genomes across time, referred to as the four-dimensional nucleome (4DN), is a key component of gene regulation and biological fate. Viral infections can lead to a reconfiguration of viral and host genomes, impacting gene expression, replication, latency, and oncogenic transformation. This review provides a summary of recent research employing three-dimensional genomic methods such as Hi-C, 4C, ChIA-PET, and HiChIP in virology. We review how viruses induce changes in gene loop formation between regulatory elements, modify chromatin accessibility, and trigger shifts between A and B compartments in the host genome. We highlight the central role of cellular chromatin organizing factors, such as CTCF and cohesin, that reshape the 3D structure of both viral and cellular genomes. We consider how viral episomes, viral proteins, and viral integration sites can alter the host epigenome and how host cell type and conditions determine viral epigenomes. This review consolidates current knowledge of the diverse host-viral interactions that impact the 4DN.
Assuntos
Genoma Viral , Humanos , Animais , Interações Hospedeiro-Patógeno , Vírus/metabolismo , Vírus/genética , Cromatina/metabolismo , Viroses/virologia , Viroses/metabolismoRESUMO
Foamy viruses (FVs) are generally recognized as non-pathogenic, often causing asymptomatic or mild symptoms in infections. Leveraging these unique characteristics, FV vectors hold significant promise for applications in gene therapy. This study introduces a novel platform technology using a pseudo-virus with single-round infectivity. In contrast to previous vector approaches, we developed a technique employing only two vectors, pcHFV lacking Env and pCMV-Env, to introduce the desired genes into target cells. Our investigation demonstrated the efficacy of the prototype foamy virus (PFV) dual-vector system in producing viruses and delivering transgenes into host cells. To optimize viral production, we incorporated the codon-optimized Env (optEnv) gene in pCMV-Env and the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) at the 3' end of the transgene in the transfer vector. Consequently, the use of optEnv led to a significant enhancement in transgene expression in host cells. Additionally, the WPRE exhibited an enhancing effect. Furthermore, the introduced EGFP transgene was present in host cells for a month. In an effort to expand transgene capacity, we further streamlined the viral vector, anticipating the delivery of approximately 4.3 kbp of genes through our PFV dual-vector system. This study underscores the potential of PFVs as an alternative to lentiviruses or other retroviruses in the realm of gene therapy.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Spumavirus , Transgenes , Spumavirus/genética , Vetores Genéticos/genética , Humanos , Terapia Genética/métodos , Animais , Células HEK293 , Proteínas de Fluorescência Verde/genética , Linhagem CelularRESUMO
Aconitase-2 (Aco2) is present in the mitochondria, cytosol, and nucleus of fission yeast. To explore its function beyond the well-known role in the mitochondrial tricarboxylic acid (TCA) cycle, we conducted genome-wide profiling using the aco2ΔNLS mutant, which lacks a nuclear localization signal (NLS). The RNA sequencing (RNA-seq) data showed a general downregulation of electron transport chain (ETC) genes in the aco2ΔNLS mutant, except for those in the complex II, leading to a growth defect in respiratory-prone media. Complementation analysis with non-catalytic Aco2 [aco2ΔNLS + aco2(3CS)], where three cysteines were substituted with serine, restored normal growth and typical ETC gene expression. This suggests that Aco2's catalytic activity is not essential for its role in ETC gene regulation. Our mRNA decay assay indicated that the decrease in ETC gene expression was due to transcriptional regulation rather than changes in mRNA stability. Additionally, we investigated the Php complex's role in ETC gene regulation and found that ETC genes, except those within complex II, were downregulated in php3Δ and php5Δ strains, similar to the aco2ΔNLS mutant. These findings highlight a novel role for nuclear aconitase in ETC gene regulation and suggest a potential connection between the Php complex and Aco2.
RESUMO
EBV persist as multicopy episomes in latently infected cells and alter transcriptional program of host systems. Knowledge of EBV tethering site helps us understand how EBV attaches to and regulates the host chromosome. Here, we introduce a step-by-step protocol for 4C-seq analysis, including cell fixation, 4C-DNA construction, and sequencing library preparation performed with EBV-positive Burkitt's lymphoma cells. The method can be applied in a variety of studies and cell-types to identify target loci associated with bait positions, such as viral episomes.
Assuntos
Linfoma de Burkitt , Herpesvirus Humano 4 , Humanos , Herpesvirus Humano 4/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , DNA , PlasmídeosRESUMO
Malassezia and Staphylococcus are the most dominant genera in human skin microbiome. To explore the inter-kingdom interactions between the two genera, we examined the transcriptional changes in Malassezia and Staphylococcus species induced upon co-culturing. RNA-seq analyses revealed that genes encoding ribosomal proteins were upregulated, while those encoding aspartyl proteases were downregulated in M. restricta after co-culturing with Staphylococcus species. We identified MRET_3770 as a major secretory aspartyl protease coding gene in M. restricta through pepstatin-A affinity chromatography followed by mass spectrometry and found that the expression of MRET_3770 was significantly repressed upon co-culturing with Staphylococcus species or by incubation in media with reduced pH. Moreover, biofilm formation by Staphylococcus aureus was inhibited in the spent medium of M. restricta, suggesting that biomolecules secreted by M. restricta such as secretory aspartyl proteases may degrade the biofilm structure. We also examined the transcriptional changes in S. aureus co-cultured with M. restricta and found co-cultured S. aureus showed increased expression of genes encoding ribosomal proteins and downregulation of those involved in riboflavin metabolism. These transcriptome data of co-cultured fungal and bacterial species demonstrate a dynamic interplay between the two co-existing genera.
Assuntos
Ácido Aspártico Proteases , Malassezia , Humanos , Malassezia/genética , Staphylococcus , Staphylococcus aureus/genética , Pele/microbiologia , Ácido Aspártico Proteases/genética , Ácido Aspártico Endopeptidases , Proteínas RibossômicasRESUMO
This study aimed to investigate deep convolutional neural network- (DCNN-) based artificial intelligence (AI) model using cephalometric images for the classification of sagittal skeletal relationships and compare the performance of the newly developed DCNN-based AI model with that of the automated-tracing AI software. A total of 1574 cephalometric images were included and classified based on the A-point-Nasion- (N-) point-B-point (ANB) angle (Class I being 0-4°, Class II > 4°, and Class III < 0°). The DCNN-based AI model was developed using training (1334 images) and validation (120 images) sets with a standard classification label for the individual images. A test set of 120 images was used to compare the AI models. The agreement of the DCNN-based AI model or the automated-tracing AI software with a standard classification label was measured using Cohen's kappa coefficient (0.913 for the DCNN-based AI model; 0.775 for the automated-tracing AI software). In terms of their performances, the micro-average values of the DCNN-based AI model (sensitivity, 0.94; specificity, 0.97; precision, 0.94; accuracy, 0.96) were higher than those of the automated-tracing AI software (sensitivity, 0.85; specificity, 0.93; precision, 0.85; accuracy, 0.90). With regard to the sagittal skeletal classification using cephalometric images, the DCNN-based AI model outperformed the automated-tracing AI software.
Assuntos
Inteligência Artificial , Redes Neurais de Computação , SoftwareRESUMO
A persistent DNA tumor virus infection transforms normal cells into cancer cells by either integrating its genome into host chromosomes or retaining it as an extrachromosomal entity called episome. Viruses have evolved mechanisms for attaching episomes to infected host cell chromatin to efficiently segregate the viral genome during mitosis. It has been reported that viral episome can affect the gene expression of the host chromosomes through interactions between viral episomes and epigenetic regulatory host factors. This mini review summarizes our current knowledge of the tethering sites of viral episomes, such as EBV, KSHV, and HBV, on host chromosomes analyzed by three-dimensional genomic tools. [BMB Reports 2022; 55(12): 587-594].
Assuntos
Herpesvirus Humano 8 , Neoplasias , Humanos , Antígenos Virais/genética , Antígenos Virais/metabolismo , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Plasmídeos , Genoma Viral/genética , DNA , Neoplasias/genéticaRESUMO
We investigated the spectral property changes in anti-adhesion films, which were cross-linked and surface-modified through electron beam irradiation, using terahertz time-domain spectroscopy (THz-TDS). Polyethylene oxide (PEO), which is a biocompatible and biodegradable polymer, was the main component of these anti-adhesion films being manufactured for testing. The terahertz characteristics of the films were affected by the porosity generated during the freeze-drying and compression processes of sample preparation, and this was confirmed using optical coherence tomography (OCT) imaging. An anti-adhesion polymer film made without porosity was measured by using the THz-TDS method, and it was confirmed that the refractive index and absorption coefficient were dependent on the crosslinking state. To our knowledge, this is the first experiment on the feasibility of monitoring cross-linking states using terahertz waves. The THz-TDS method has potential as a useful nondestructive technique for polymer inspection and analysis.
RESUMO
The fission yeast Schizosaccharomyces pombe contains two CGFS-type monothiol glutaredoxins, Grx4 and Grx5, which are localized primarily in the nucleus and mitochondria, respectively. We observed involvement of Grx4 in regulating iron-responsive gene expression, which is modulated by a repressor Fep1. Lack of Grx4 caused defects not only in growth but also in the expression of both iron-uptake and iron-utilizing genes regardless of iron availability. In order to unravel how Grx4 is involved in Fep1-mediated regulation, interaction between them was investigated. Co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) revealed that Grx4 physically interacts with Fep1 in vivo. BiFC revealed localized nuclear dots produced by interaction of Grx4 with Fep1. Mutation of cysteine-172 in the CGFS motif to serine (C172S) produced effects similarly observed under Grx4 depletion, such as the loss of iron-dependent gene regulation and the absence of nuclear dots in BiFC analysis. These results suggest that the ability of Grx4 to bind iron, most likely Fe-S cofactor, could be critical in interacting with and modulating the activity of Fep1.
Assuntos
Fatores de Transcrição GATA/metabolismo , Glutarredoxinas/metabolismo , Ferro/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Motivos de Aminoácidos , Fatores de Transcrição GATA/genética , Glutarredoxinas/genética , Homeostase , Estrutura Terciária de Proteína , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
The genome is highly organized hierarchically by the function of structural maintenance of chromosomes (SMC) complex proteins such as condensin and cohesin from bacteria to humans. Although the roles of SMC complex proteins have been well characterized, their specialized roles in nuclear processes remain unclear. Condensin and cohesin have distinct binding sites and mediate long-range and short-range genomic associations, respectively, to form cell cycle-specific genome organization. Condensin can be recruited to highly expressed genes as well as dispersed repeat genetic elements, such as Pol III-transcribed genes, LTR retrotransposon, and rDNA repeat. In particular, mitotic transcription factors Ace2 and Ams2 recruit condensin to their target genes, forming centromeric clustering during mitosis. Condensin is potentially involved in various chromosomal processes such as the mobility of chromosomes, chromosome territories, DNA reannealing, and transcription factories. The current knowledge of condensin in fission yeast summarized in this review can help us understand how condensin mediates genome organization and participates in chromosomal processes in other organisms.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromossomos , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Schizosaccharomyces/fisiologia , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Humanos , Mitose , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição/genética , CoesinasRESUMO
Aconitase, a highly conserved protein across all domains of life, functions in converting citrate to isocitrate in the tricarboxylic acid cycle. Cytosolic aconitase is also known to act as an iron regulatory protein in mammals, binding to the RNA hairpin structures known as iron-responsive elements within the untranslated regions of specific RNAs. Aconitase-2 (Aco2) in fission yeast is a fusion protein consisting of an aconitase and a mitochondrial ribosomal protein, bL21, residing not only in mitochondria but also in cytosol and the nucleus. To investigate the role of Aco2 in the nucleus and cytoplasm of fission yeast, we analyzed the transcriptome of aco2ΔN mutant that is deleted of nuclear localization signal (NLS). RNA sequencing revealed that the aco2ΔN mutation caused increase in mRNAs encoding iron uptake transporters, such as Str1, Str3, and Shu1. The half-lives of mRNAs for these genes were found to be significantly longer in the aco2ΔN mutant than the wild-type strain, suggesting the role of Aco2 in mRNA turnover. The three conserved cysteines required for the catalytic activity of aconitase were not necessary for this role. The UV cross-linking RNA immunoprecipitation analysis revealed that Aco2 directly bound to the mRNAs of iron uptake transporters. Aco2-mediated degradation of iron-uptake mRNAs appears to utilize exoribonuclease pathway that involves Rrp6 as evidenced by genetic interactions. These results reveal a novel role of non-mitochondrial aconitase protein in the mRNA turnover in fission yeast to fine-tune iron homeostasis, independent of regulation by transcriptional repressor Fep1.
Assuntos
Aconitato Hidratase/metabolismo , Proteínas de Transporte de Cátions/genética , Regulação Fúngica da Expressão Gênica , Ferro/metabolismo , RNA Fúngico/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas de Transporte de Cátions/metabolismo , Núcleo Celular/enzimologia , Citoplasma/enzimologia , Exorribonucleases/genética , Exorribonucleases/metabolismo , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Genes Fúngicos , Proteínas Reguladoras de Ferro/genética , Proteínas Reguladoras de Ferro/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Regulon , Ribonucleases/genética , Ribonucleases/metabolismo , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The Deltagrx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1(+) gene encoding a putative A-type Fe-S scaffold, in addition to mas5(+) and hsc1(+) genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2(+), but not isu1(+) and ssc1(+), complemented the growth phenotype of Deltagrx5 mutant as isa1(+) did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.
Assuntos
DNA Mitocondrial/metabolismo , Glutarredoxinas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Cisteína/metabolismo , Glutarredoxinas/genética , Glicerol/metabolismo , Proteínas Ferro-Enxofre/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Epstein-Barr virus (EBV) genomes persist in latently infected cells as extrachromosomal episomes that attach to host chromosomes through the tethering functions of EBNA1, a viral encoded sequence-specific DNA binding protein. Here we employ circular chromosome conformation capture (4C) analysis to identify genome-wide associations between EBV episomes and host chromosomes. We find that EBV episomes in Burkitt's lymphoma cells preferentially associate with cellular genomic sites containing EBNA1 binding sites enriched with B-cell factors EBF1 and RBP-jK, the repressive histone mark H3K9me3, and AT-rich flanking sequence. These attachment sites correspond to transcriptionally silenced genes with GO enrichment for neuronal function and protein kinase A pathways. Depletion of EBNA1 leads to a transcriptional de-repression of silenced genes and reduction in H3K9me3. EBV attachment sites in lymphoblastoid cells with different latency type show different correlations, suggesting that host chromosome attachment sites are functionally linked to latency type gene expression programs.
Assuntos
Sítios de Ligação Microbiológicos/genética , Sítios de Ligação Microbiológicos/fisiologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Interações entre Hospedeiro e Microrganismos/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/virologia , Linhagem Celular Tumoral , Cromossomos Humanos/genética , Cromossomos Humanos/virologia , Epigênese Genética , Antígenos Nucleares do Vírus Epstein-Barr/fisiologia , Herpesvirus Humano 4/patogenicidade , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Modelos Biológicos , Plasmídeos/genética , Latência Viral/genética , Latência Viral/fisiologiaRESUMO
Thioredoxins usually perform a role as a thiol-disulfide oxidoreductase using their active-site cysteines. The fission yeast Schizosaccharomyces pombe contains two thioredoxins: Trx1 for general stress protection and Trx2 for mitochondrial functions. The Deltatrx2 mutant grows as well as the wild type on complex media containing glucose. However, on nonfermentable carbon source such as glycerol, the mutant did not grow, indicating a defect in mitochondrial function. The mutant also exhibited auxotrophy for arginine and cysteine on minimal medium. In order to find the reason for the unexpected arginine auxotrophy, we searched for multicopy suppressors and found that the arg3(+) gene encoding ornithine carbamoyltransferase (OCTase) in the urea cycle of the arginine biosynthetic pathway rescued the arginine auxotrophy. The levels of arg3(+) transcript, Arg3 protein, and OCTase activity were all decreased in Deltatrx2. Through immunocoprecipitation, we observed a direct interaction between Trx2 and Arg3 in cell extracts. The mutant forms of Trx2 lacking either one or both of the active site cysteines through substitution to serines also rescued the arginine auxotrophy and restored the decreased OCTase activity. They also rescued the growth defect of Deltatrx2 on glycerol medium. This contrasts with the thiol-dependent action of overproduced Trx2 in complementing glutathione reductase. Therefore, Trx2 serves multiple functions in mitochondria, protecting mitochondrial components against thiol-oxidative damage as a thiol-disulfide oxidoreductase, and supporting urea cycle and respiration in mitochondria in a manner independent of active site thiols.
Assuntos
Arginina/biossíntese , Mitocôndrias/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Compostos de Sulfidrila/metabolismo , Tiorredoxinas/metabolismo , Ureia/metabolismo , Cisteína/metabolismo , Mitocôndrias/genética , Mutação , Ornitina Carbamoiltransferase , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Tiorredoxinas/genéticaRESUMO
Senescence is induced by various stimuli such as oncogene expression and telomere shortening, referred to as oncogene-induced senescence (OIS) and replicative senescence (RS), respectively, and accompanied by global transcriptional alterations and 3D genome reorganization. Here, we demonstrate that the human condensin II complex participates in senescence via gene regulation and reorganization of euchromatic A and heterochromatic B compartments. Both OIS and RS are accompanied by A-to-B and B-to-A compartmental transitions, the latter of which occur more frequently and are undergone by 14% (430 Mb) of the human genome. Mechanistically, condensin is enriched in A compartments and implicated in B-to-A transitions. The full activation of senescence genes (SASP genes and p53 targets) requires condensin; its depletion impairs senescence markers. This study describes that condensin reinforces euchromatic A compartments and promotes B-to-A transitions, both of which are coupled to optimal expression of senescence genes, thereby allowing condensin to contribute to senescent processes.