Non-Mitochondrial Aconitase-2 Mediates the Transcription of Nuclear-Encoded Electron Transport Chain Genes in Fission Yeast.

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.

Viral remodeling of the 4D nucleome.

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.

Establishment of a Dual-Vector System for Gene Delivery Utilizing Prototype Foamy Virus.

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.

Chromatin accessibility analysis and architectural profiling of human kidneys reveal key cell types and a regulator of diabetic kidney disease.

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.

Characterization of a new CCCTC-binding factor binding site as a dual regulator of Epstein-Barr virus latent infection.

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.

Transcriptional Interplay between Malassezia restricta and Staphylococcus Species Co-Existing in the Skin Environment.

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.

4C Analysis of EBV-Host DNA Interactome.

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.

Topological implications of DNA tumor viral episomes.

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].

Deep convolutional neural network-based skeletal classification of cephalometric image compared with automated-tracing software.

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.

Terahertz Spectral Properties of PEO-Based Anti-Adhesion Films Cross-Linked by Electron Beam Irradiation.

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.

Non-mitochondrial aconitase regulates the expression of iron-uptake genes by controlling the RNA turnover process in fission yeast.

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.

Potential roles of condensin in genome organization and beyond in fission yeast.

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.

Epigenetic specifications of host chromosome docking sites for latent Epstein-Barr virus.

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.

Involvement of condensin in cellular senescence through gene regulation and compartmental reorganization.

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.

Architectural alterations of the fission yeast genome during the cell cycle.

Eukaryotic genomes are highly ordered through various mechanisms, including topologically associating domain (TAD) organization. We employed an in situ Hi-C approach to follow the 3D organization of the fission yeast genome during the cell cycle. We demonstrate that during mitosis, large domains of 300 kb-1 Mb are formed by condensin. This mitotic domain organization does not suddenly dissolve, but gradually diminishes until the next mitosis. By contrast, small domains of 30-40 kb that are formed by cohesin are relatively stable across the cell cycle. Condensin and cohesin mediate long- and short-range contacts, respectively, by bridging their binding sites, thereby forming the large and small domains. These domains are inversely regulated during the cell cycle but assemble independently. Our study describes the chromosomal oscillation between the formation and decay phases of the large and small domains, and we predict that the condensin-mediated domains serve as chromosomal compaction units.

Transcription factors mediate condensin recruitment and global chromosomal organization in fission yeast.

It is becoming clear that structural-maintenance-of-chromosomes (SMC) complexes such as condensin and cohesin are involved in three-dimensional genome organization, yet their exact roles in functional organization remain unclear. We used chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) to comprehensively identify genome-wide associations mediated by condensin and cohesin in fission yeast. We found that although cohesin and condensin often bind to the same loci, they direct different association networks and generate small and larger chromatin domains, respectively. Cohesin mediates associations between loci positioned within 100 kb of each other; condensin can drive longer-range associations. Moreover, condensin, but not cohesin, connects cell cycle-regulated genes bound by mitotic transcription factors. This study describes the different functions of condensin and cohesin in genome organization and how specific transcription factors function in condensin loading, cell cycle-dependent genome organization and mitotic chromosome organization to support faithful chromosome segregation.

The iron uptake repressor Fep1 in the fission yeast binds Fe-S cluster through conserved cysteines.

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.

Interaction between TBP and Condensin Drives the Organization and Faithful Segregation of Mitotic Chromosomes.

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.

Size measurement of the thyroid gland on a magnified pinhole thyroid scan using an ultrasonic device measuring distance from the pinhole to the thyroid gland.

PURPOSE: Pinhole has been used for magnification of gamma camera images and is valuable for imaging of small organs, such as thyroid; however, size of the organ cannot be measured on the image due to variable degree of magnification by distance between the pinhole and the organ. The aim of this study was to develop a true size measuring system (TSM system) on magnified pinhole thyroid scan using an ultrasonic sensor. METHODS: An ultrasonic device capable of measuring the distance from the pinhole to the skin overlying the thyroid gland was manufactured using a ~40 kHz piezoelectric-transducer-based sensor, and its accuracy was tested. An interface program was developed and fused with the ultrasonic device for development of the TSM system. Accuracy of the TSM system for measuring size was tested with phantom images and 35 thyroid scans. RESULTS: The ultrasonic device accurately measured the distance from the pinhole to the skin over the thyroid gland and the measured values were highly reproducible (6 cm; 6.02 ± 0.04 cm, 8 cm; 8.00 ± 0.05 cm, 10 cm; 10.00 ± 0.05 cm). Distance on the phantom image corrected by the TSM system was almost the same as the true distance. Size of the thyroid on the pinhole image was larger (+67.3 to 103.1 %) than the true thyroid size on the parallel-hole image and the magnification decreased by increase of the distance between the pinhole and the skin over the thyroid gland. However, size of the thyroid obtained using the TSM system was almost equal (-2.1 to +3.6 %) to the true thyroid size on the parallel-hole image. CONCLUSIONS: We developed the TSM system for magnified pinhole images using a distance measuring ultrasonic sensor. Size of the thyroid on the magnified pinhole image obtained using the system was almost the same as the true thyroid size. The TSM system can be applied to obtain accurate size of the thyroid gland or lesions in the thyroid gland on pinhole thyroid scan.

Centromeric motion facilitates the mobility of interphase genomic regions in fission yeast.

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.