GTP-dependent regulation of heterochromatin fluctuations at subtelomeric regions in Saccharomyces cerevisiae.

In eukaryotes, single cells in a population display different transcriptional profiles. One of the factors regulating this heterogeneity is the chromatin state in each cell. However, the mechanisms of epigenetic chromatin regulation of specific chromosomal regions remain unclear. Therefore, we used single-cell tracking system to analyze IMD2. IMD2 is located at the subtelomeric region of budding yeast, and its expression is epigenetically regulated by heterochromatin fluctuations. Treatment with mycophenolic acid, an inhibitor of de novo GTP biosynthesis, triggered a decrease in GTP, which caused heterochromatin fluctuations at the IMD2 locus. Interestingly, within individually tracked cells, IMD2 expression state underwent repeated switches even though IMD2 is positioned within the heterochromatin region. We also found that 30% of the cells in a population always expressed IMD2. Furthermore, the addition of nicotinamide, a histone deacetylase inhibitor, or guanine, the GTP biosynthesis factor in salvage pathway of GTP biosynthesis, regulated heterogeneity, resulting in IMD2 expression being uniformly induced or suppressed in the population. These results suggest that gene expression heterogeneity in the IMD2 region is regulated by changes in chromatin structure triggered by slight decreases in GTP.

Histone H2A T120 Phosphorylation Promotes Oncogenic Transformation via Upregulation of Cyclin D1.

How deregulation of chromatin modifiers causes malignancies is of general interest. Here, we show that histone H2A T120 is phosphorylated in human cancer cell lines and demonstrate that this phosphorylation is catalyzed by hVRK1. Cyclin D1 was one of ten genes downregulated upon VRK1 knockdown in two different cell lines and showed loss of H2A T120 phosphorylation and increased H2A K119 ubiquitylation of its promoter region, resulting in impaired cell growth. In vitro, H2A T120 phosphorylation and H2A K119 ubiquitylation are mutually inhibitory, suggesting that histone phosphorylation indirectly activates chromatin. Furthermore, expression of a phosphomimetic H2A T120D increased H3 K4 methylation. Finally, both VRK1 and the H2A T120D mutant histone transformed NIH/3T3 cells. These results suggest that histone H2A T120 phosphorylation by hVRK1 causes inappropriate gene expression, including upregulated cyclin D1, which promotes oncogenic transformation.

Effects of Charged Lipids on Giant Unilamellar Vesicle Fusion and Inner Content Mixing via Freeze-Thawing.

Fusion between giant unilamellar vesicles (GUVs) can incorporate and mix components of biochemical reactions. Recently, GUV fusion induced by freeze-thawing (F/T) was employed to construct artificial cells that can easily and repeatedly fuse GUVs with efficient content mixing. However, GUVs were ruptured during F/T, and the inner contents leaked. Herein, we investigated the effects of charged lipids on GUV fusion via F/T. The presence of 10 %-50 % (w/w%) negatively charged lipids in GUV membranes, mainly composed of the neutral charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), improved resistance to GUV rupture and decreased inner content leakage. Furthermore, we found that the presence of positively charged lipids in GUV membranes elevated GUV rupture compared with F/T between GUVs containing POPC alone. Modified GUVs may better incorporate nutrients and lipid membranes with less damage following GUV fusion via F/T, providing an improved artificial model.

Effects of Sugars on Giant Unilamellar Vesicle Preparation, Fusion, PCR in Liposomes, and Pore Formation.

The water-in-oil emulsion transfer method was developed for preparing giant unilamellar vesicles (GUVs) and is useful for studying cellular functions under conditions that mimic cellular environments. A shortcoming of this method for encapsulating biochemical reactions is that it requires high sugar concentrations to enable the density effect to transverse the oil-water interface. In this study, we investigated the effects of sugars on GUV preparation and several biochemical reactions. We found that changing the sugar in the inner solution from sucrose to maltose or trehalose improved GUV formation. The fusion ratio of the freeze-thaw method was better in the traditional glucose-sucrose condition compared with the other examined conditions. For the inner biochemical reaction, we performed PCR in liposomes. The presence of maltose in the inner solution improved the stability of GUVs against damage caused by thermal cycles. Finally, fructose in the outer solution reduced leakage of the inner solution via pores on the membranes of GUVs. Our findings provide new insight for optimizing sugar conditions for preparing GUVs and inner GUV reactions. This could increase the utilization of GUVs as artificial cell compartment models.

Exchange of Proteins in Liposomes through Streptolysin O Pores.

Liposomes, which are vesicles surrounded by lipid membranes, can be used as biochemical reactors by encapsulating various reactions. Accordingly, they are useful for studying cellular functions under controlled conditions that mimic the environment within a cell. However, one of the shortcomings of liposomes as biochemical reactors is the difficulty of introducing or removing proteins due to the impermeability of the membrane. In this study, we established a method for exchanging proteins in liposomes by forming reversible pores in the membrane. We used the toxic protein streptolysin O (SLO); this forms pores in membranes made of phospholipids containing cholesterol that can be closed by the addition of calcium ions. After optimizing the experimental procedure and lipid composition, we observed the exchange of fluorescent proteins (transferrin Alexa Fluor 488 and 647) in 9.9 % of liposomes. We also introduced T7 RNA polymerase, a 98-kDa enzyme, and observed RNA synthesis in ∼8 % of liposomes. Our findings establish a new method for controlling the internal protein composition of liposomes, thereby increasing their utility as bioreactors.

Altered metabolic regulation owing to gsp1 mutations encoding the nuclear small G protein in Saccharomyces cerevisiae.

Nutrient metabolism is regulated for adaptation to, for example, environmental alterations, cellular stress, cell cycle, and cellular ageing. This regulatory network consists of cross-talk between cytoplasmic organelles and the nucleus. The ras-like nuclear small G protein, Ran, functions in nuclear-cytosolic transport and regulatory signal transmission. In yeast, some genes involved in the Ran system in yeast are required for growth on glycerol medium. Growth deficiency, due to mutations in the GSP1 gene, which encodes Ran, is allele specific. Specifically in this study, the gsp1-1894 cells lost mitochondria, and could not grow on media containing glycerol, galactose or maltose. However, the gsp1-1894 cells grew better on a high salt medium (1 M NaCl) and had increased expression levels of GPD1-lacZ. Furthermore, disruption of the HOG1 gene suppressed their growth deficiency on glycerol medium. These findings suggest that altered activation of Hog1 in the gsp1-1894 cells resulted in the loss of mitochondria and inhibition of glycerol metabolism. Growth deficiency of the gsp1-1894 cells on galactose medium was further suppressed by high dosage of the SIP2 DNA, which encodes the cytosolic ß subunit of AMPK. This suggests that higher cytosolic activity of AMPK is required for the utilization of an alternative carbon source in gsp1-1894 cells.

Four domains of Ada1 form a heterochromatin boundary through different mechanisms.

In eukaryotic cells, there are two chromatin states, silenced and active, and the formation of a so-called boundary plays a critical role in demarcating these regions; however, the mechanisms underlying boundary formation are not well understood. In this study, we focused on S. cerevisiae ADA1, a gene previously shown to encode a protein with a robust boundary function. Ada1 is a component of the histone modification complex Spt-Ada-Gcn5-acetyltransferase (SAGA) and the SAGA-like (SLIK) complex, and it helps to maintain the integrity of these complexes. Domain analysis showed that four relatively small regions of Ada1 (Region I; 66-75 aa, II; 232-282 aa, III; 416-436 aa and IV; 476-488 aa) have a boundary function. Among these, Region II could form an intact SAGA complex, whereas the other regions could not. Investigation of cellular factors that interact with these small regions identified a number of proteasome-associated proteins. Interestingly, the boundary functions of Region II and Region III were affected by depletion of Ump1, a maturation and assembly factor of the 20S proteasome. These results suggest that the boundary function of Ada1 is functionally linked to proteasome processes and that the four relatively small regions in ADA1 form a boundary via different mechanisms.

Single cell visualization of yeast gene expression shows correlation of epigenetic switching between multiple heterochromatic regions through multiple generations.

Differences in gene expression between individual cells can be mediated by epigenetic regulation; thus, methods that enable detailed analyses of single cells are crucial to understanding this phenomenon. In this study, genomic silencing regions of Saccharomyces cerevisiae that are subject to epigenetic regulation, including the HMR, HML, and telomere regions, were investigated using a newly developed single cell analysis method. This method uses fluorescently labeled proteins to track changes in gene expression over multiple generations of a single cell. Epigenetic control of gene expression differed depending on the specific silencing region at which the reporter gene was inserted. Correlations between gene expression at the HMR-left and HMR-right regions, as well as the HMR-right and HML-right regions, were observed in the single-cell level; however, no such correlations involving the telomere region were observed. Deletion of the histone acetyltransferase GCN5 gene from a yeast strain carrying a fluorescent reporter gene at the HMR-left region reduced the frequency of changes in gene expression over a generation. The results presented here suggest that epigenetic control within an individual cell is reversible and can be achieved via regulation of histone acetyltransferase activity.

C-terminus of the Sgf73 subunit of SAGA and SLIK is important for retention in the larger complex and for heterochromatin boundary function.

The budding yeast Saccharomyces cerevisiae contains active and inactive chromatin separated by boundary domains. Previously, we used genome-wide screening to identify 55 boundary-related genes. Here, we focus on Sgf73, a boundary protein that is a component of the Spt-Ada-Gcn5 acetyltransferase (SAGA) and SLIK (SAGA-like) complexes. These complexes have histone acetyltransferase (HAT) and histone deubiquitinase activity, and Sgf73 is one of the factors necessary to anchor the deubiquitination module. Domain analysis of Sgf73 was carried out, and the minimum region (373-402 aa) essential for boundary function was identified. This minimum region does not include the domain involved in anchoring the deubiquitination module, suggesting that the histone deubiquitinase activity of Sgf73 is not important for its boundary function. Next, Sgf73-mediated boundary function was analyzed in disruption strains in which different protein subunits of the SAGA/SLIK/ADA complexes were deleted. Deletion of ada2, ada3 or gcn5 (a HAT module component) caused complete loss of the boundary function of Sgf73. The importance of SAGA or SLIK complex binding to the boundary function of Sgf73 was also analyzed. Western blot analysis detected both the full-length and truncated forms of Spt7, suggesting that SAGA and SLIK complex formation is important for the boundary function of Sgf73.

IMD2, located near the boundary of heterochromatin regions, is regulated by multiple HAT-related factors.

In Saccharomyces cerevisiae, boundaries formed by DNA sequence-dependent or -independent histone modifications stop the spread of the heterochromatin region formed via the Sir complex. However, it is unclear whether the histone modifiers that control DNA sequence-independent boundaries function in a chromosome-specific or -nonspecific manner. In this study, we evaluated the effects of the SAGA complex, a histone acetyltransferase (HAT) complex, and its relationship with other histone-modifying enzymes to clarify the mechanism underlying boundary regulation of the IMD2 gene on the right subtelomere of chromosome VIII. We found that Spt8, a component of the SAGA complex, is important for boundary formation in this region and that the inclusion of Spt8 in the SAGA complex is more important than its interaction with TATA-binding protein and TFIIS. In addition to SAGA, various HAT-related factors, such as NuA4 and Rtt109, also functioned in this region. In particular, the SAGA complex induced weak IMD2 expression throughout the cell, whereas NuA4 induced strong expression. These results indicate that multiple HATs contribute to the regulation of boundary formation and IMD2 expression on the right subtelomere of chromosome VIII and that IMD2 expression is determined by the balance between these factors.

RNA synthesis in liposomes with negatively charged lipids after fusion via freezing-thawing.

The freezing-thawing (F/T) method for fusing giant unilamellar vesicles (GUVs) can provide substrates, enzymes and membrane material simultaneously and repetitively, and is useful for constructing a recursive model of an artificial cell. However, enzymatic efficiency after F/T is reduced due to rupture of the GUVs and leakage of the inner solution during F/T. Previously, liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and a negatively charged lipid, such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), showed lower rupture and leakage rates during F/T. In this study, we investigated the effect of POPG on the supply of components required for T7 RNA polymerase reactions via F/T by flow cytometry analysis. We found that the addition of POPG to liposome preparations reduced the efficiency of RNA synthesis. In addition, DNA was concentrated during F/T and RNA synthesis occurred after F/T in liposomes composed of POPC and POPG. Our results provide new insights for more efficient supply of substrates and enzymes by the F/T method, thereby increasing the utility of the F/T method for the construction of recursive bioreactors.

A novel tracking and analysis system for time-lapse cellular imaging of Schizosaccharomyces pombe.

The importance of the parent-progeny relationship tracking technique in single-cell analysis has grown with the passage of time. In this study, fundamental image-processing techniques were combined to develop software capable of inferring cell cycle alterations in fission yeast cells, which exhibit equipartition during division. These methods, exclusively relying on bright-field images as input, could track parent-progeny relationships after cell division by assessing the temporal morphological transformation of these cells. In the application of this technique, the software was employed for calculating intracellular fluorescent dots during every stage of the cell cycle, using a yeast strain expressing EGFP-fused Swi6, which binds to chromatin. The results obtained with this software were consistent with those of previous studies. This software facilitated single-cell-level tracking of parent-progeny relationships in cells exhibiting equipartition during division and enabled the monitoring of spatial fluctuations in a cell cycle-dependent protein. This method, expediting the analysis of extensive datasets, may also empower large-scale screening experiments that cannot be conducted manually.

HIF-1 inhibition reverses opacity in a rat model of galactose-induced cataract.

Cataract is an eye disease, in which the lens becomes opaque, causing vision loss and blindness. The detailed mechanism of cataract development has not been characterized, and effective drug therapies remain unavailable. Here, we investigated the effects of Hypoxia-inducible factor 1 (HIF-1) inhibitors using an ex vivo model, in which rat lenses were cultured in galactose-containing medium to induce opacity formation. We found that treatment with the HIF-1 inhibitors 2-Methoxyestradiol (2ME2), YC-1, and Bavachinin decreased lens opacity. Microarray analysis on 2ME2-treated samples, in which opacity was decreased, identified genes upregulated by galactose and downregulated by inhibitor treatment. Subsequent STRING analysis on genes that showed expression change by RT-qPCR identified two clusters. First cluster related to the cytoskeleton and epithelial-mesenchymal transition (EMT). Second cluster related to the oxidative stress, and apoptosis. ACTA2, a known marker for EMT, and TXNIP, a suppressor of cell proliferation and activator of apoptosis, were present in each cluster. Thus, suppression of EMT and apoptosis, as well as activation of cell proliferation, appear to underlie the decrease in lens opacity.

Glutamate is effective in decreasing opacity formed in galactose-induced cataract model.

Although cataract is the leading cause of blindness worldwide, the detailed pathogenesis of cataract remains unclear, and clinically useful drug treatments are still lacking. In this study, we examined the effects of glutamate using an ex vivo model in which rat lens is cultured in a galactose-containing medium to induce opacity formation. After inducing lens opacity formation in galactose medium, glutamate was added, and the opacity decreased when the culture was continued. Next, microarray analysis was performed using samples in which the opacity was reduced by glutamate, and genes whose expression increased with galactose culture and decreased with the addition of glutamate were extracted. Subsequently, STRING analysis was performed on a group of genes that showed variation as a result of quantitative measurement of gene expression by RT-qPCR. The results suggest that apoptosis, oxidative stress, endoplasmic reticulum (ER) stress, cell proliferation, epithelial-mesenchymal transition (EMT), cytoskeleton, and histones are involved in the formation and reduction of opacity. Therefore, glutamate may reduce opacity by inhibiting oxidative stress and its downstream functions, and by regulating the cytoskeleton and cell proliferation.

Spt3 and Spt8 Are Involved in the Formation of a Silencing Boundary by Interacting with TATA-Binding Protein.

In Saccharomyces cerevisiae, a heterochromatin-like chromatin structure called the silencing region is present at the telomere as a complex of Sir2, Sir3, and Sir4. Although spreading of the silencing region is blocked by histone acetylase-mediated boundary formation, the details of the factors and mechanisms involved in the spread and formation of the boundary at each telomere are unknown. Here, we show that Spt3 and Spt8 block the spread of the silencing regions. Spt3 and Spt8 are members of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, which has histone acetyltransferase activity. We performed microarray analysis of the transcriptome of spt3Δ and spt8Δ strains and RT-qPCR analysis of the transcript levels of genes from the subtelomeric region in mutants in which the interaction of Spt3 with TATA-binding protein (TBP) is altered. The results not only indicated that both Spt3 and Spt8 are involved in TBP-mediated boundary formation on the right arm of chromosome III, but also that boundary formation in this region is DNA sequence independent. Although both Spt3 and Spt8 interact with TBP, Spt3 had a greater effect on genome-wide transcription. Mutant analysis showed that the interaction between Spt3 and TBP plays an important role in the boundary formation.

Gene profiles and mutations in the development of cataracts in the ICR rat model of hereditary cataracts.

Cataracts are opacifications of the lens that cause loss of visual acuity and ultimately of eyesight. Age-related cataract develops in most elderly people, but the mechanisms of cataract onset are incompletely understood. The Ihara Cataract Rat (ICR) is an animal model of hereditary cataracts showing cortical opacity that commonly develops prematurely. We identified putative mechanisms of cataract onset in the ICR rat model by measuring gene expression changes before and after cortical cataract development and conducting point mutation analysis. Genes differentially expressed between 4-week-old animals without cortical cataracts and 8-10-week-old animals with cortical cataracts were selected from microarray analysis. Three connections were identified by STRING analysis: (i) Epithelial-Mesenchymal Transition (EMT), including Col1a2, and Pik3r1. (ii) Lens homeostasis, including Aqp5, and Cpm. (iii) Lipid metabolism, including Scd1, Srebf1, and Pnpla3. Subsequently, mutation points were selected by comparing ICR rats with 12 different rats that do not develop cataracts. The apolipoprotein Apoc3 was mutated in ICR rats. Analyses of gene expression changes and point and mutations suggested that abnormalities in EMT or lipid metabolism could contribute to cataract development in ICR rats.

Chronic Antioxidant Capacity Loss in Anterior Chamber Environment After Iridectomy.

Purpose: To compare the ascorbic acid concentration and total antioxidant capacity in the aqueous humor of pigmented Rex rabbits after sham operation (control), iridectomy, and trabeculectomy. Methods: Pigmented Rex rabbits were divided into control, iridectomy, and trabeculectomy groups and followed up for 12 months after surgery. Ascorbic acid concentration and total antioxidant capacity in the aqueous humor, intraocular pressure, and the occurrence of cataracts were examined in each group. Results: The ascorbic acid concentration and total antioxidant capacity after iridectomy and trabeculectomy were significantly lower at one week and at one, six, and 12 months after operation than those in the control group (P ≤ 0.03). Ascorbic acid concentration was positively and significantly correlated with total antioxidant capacity in the aqueous humor (P < 0.01). Compared to the control and the iridectomy groups, intraocular pressure in the trabeculectomy group was significantly lower at one week and at one and six months after surgery (one week: P < 0.01 and P < 0.01, respectively; one month: P < 0.01 and P = 0.03, respectively; six months: P = 0.03). Histological findings in the iridectomy and trabeculectomy groups included the appearance of vacuoles in the lens at six and 12 months after surgery. Conclusions: Iridectomy causes a sustained decrease in ascorbic acid concentration, followed by a long-term decrease in the total antioxidant capacity within the aqueous humor. Translational Relevance: The animal model possibly predicts the vulnerability focusing on the antioxidant level in the anterior chamber environment after trabeculectomy and iridectomy per se in clinical settings.

Histone acetyltransferase inhibition reverses opacity in rat galactose-induced cataract.

Cataract, a disease that causes opacity of the lens, is the leading cause of blindness worldwide. Cataracts secondary to diabetes are common, even in young patients, so they are of significant clinical importance. Here, we used an ex vivo model of galactose-induced cataracts in the rat lens to investigate the therapeutic effects of histone acetyltransferase (HAT) inhibitors. Among the tested HAT inhibitors, TH1834 was the only one that could reverse most of the opacity once it had formed in the lens. Combination treatment with C646/CPTH2 and CBP30/CPTH2 also had therapeutic effects. In lens cross-sections, vacuoles were present in the tissue of the cortical equatorial region of untreated cataract samples. In treated cataract samples, lens tissue regenerated to fill the vacuoles. To identify the genes regulated by HAT inhibitors, qRT-PCR was performed on treated and untreated cataract samples to determine candidate genes. Expression of Acta1 and Stmn4, both of which are involved in the cytoskeleton, were altered significantly in C646+CPTH2 samples. Expression of Emd, a nuclear membrane protein, and Prtfdc1, which is involved in cancer cell proliferation, were altered significantly in CBP30+CPTH2 samples. Acta1, Acta2, Arrdc3, Hebp2, Hist2h2ab, Pmf1, Ppdpf, Rbm3, RGD1561694, Slc16a6, Slfn13, Tagln, Tgfb1i1, and Tuba1c in TH1834 samples were significantly altered. These genes were primarily related to regulation of cell proliferation, the cytoskeleton, and cell differentiation. Expression levels increased with the onset of cataracts and was suppressed in samples treated with HAT inhibitors.

Analysis of cataract-regulated genes using chemical DNA damage induction in a rat ex vivo model.

Although cataracts affect almost all people at advanced age and carry a risk of blindness, the mechanisms of cataract development remain incompletely understood. Oxidative stress, which is a causative factor in cataract, results in DNA breakage, which suggests that DNA damage could contribute to the formation of cataracts. We developed an ex vivo experimental system to study changes in gene expression during the formation of opacities in the lens by culturing explanted rat lenses with Methylmethanesulfonate (MMS) or Bleomycin, which induce DNA damage. Lenses cultured using this experimental system developed cortical opacity, which increased in a concentration- and time-dependent manner. In addition, we compared expression profiles at the whole gene level using microarray analysis of lenses subjected to MMS or Bleomycin stress. Microarray findings in MMS-induced opacity were validated and gene expression was measured from Days 1-4 using RT-qPCR. Altered genes were classified into four groups based on the days of peak gene expression: Group 1, in which expression peaked on Day 1; Group 2, in which expression peaked on Day 2; Group 3, in which expression progressively increased from Days 1-4 or were upregulated on Day 1 and sustained through Day 4; and Group 4, in which expression level oscillated from Days 1-4. Genes involved in lipid metabolism were restricted to Group 1. DNA repair- and cell cycle-related genes were restricted to Groups 1 and 2. Genes associated with oxidative stress and drug efflux were restricted to Group 2. These findings suggest that in temporal changes of MMS-induced opacity formation, the activated pathways could occur in the following order: lipid metabolism, DNA repair and cell cycle, and oxidative stress and drug efflux.

Atm inhibition decreases lens opacity in a rat model of galactose-induced cataract.

Cataract causes vision loss and blindness due to formation of opacities of the lens. The regulatory mechanisms of cataract formation and progression remain unclear, and no effective drug treatments are clinically available. In the present study, we tested the effect of ataxia telangiectasia mutated (Atm) inhibitors using an ex vivo model in which rat lenses were cultured in galactose-containing medium to induce opacity formation. After lens opacities were induced by galactose, the lenses were further incubated with the Atm inhibitors AZD0156 or KU55933, which decreased lens opacity. Subsequently, we used microarray analysis to investigate the underlying molecular mechanisms of action, and extracted genes that were upregulated by galactose-induced opacity, but not by inhibitor treatment. Quantitative measurement of mRNA levels and subsequent STRING analysis revealed that a functional network consisting primarily of actin family and actin-binding proteins was upregulated by galactose treatment and downregulated by both Atm inhibitors. In particular, Acta2 is a known marker of epithelial-mesenchymal transition (EMT) in epithelial cells, and other genes connected in this functional network (Actn1, Tagln, Thbs1, and Angptl4) also suggested involvement of EMT. Abnormal differentiation of lens epithelial cells via EMT could contribute to formation of opacities; therefore, suppression of these genes by Atm inhibition is a potential therapeutic target for reducing opacities and alleviating cataract-related visual impairment.