Yeast Nucleus Research Articles

Transcription factor condensates, 3D clustering, and gene expression enhancement of the MET regulon.

Some transcription factors (TFs) can form liquid-liquid phase separated (LLPS) condensates. However, the functions of these TF condensates in 3-Dimentional (3D) genome organization and gene regulation remain elusive. In response to methionine (met) starvation, budding yeast TF Met4 and a few co-activators, including Met32, induce a set of genes involved in met biosynthesis. Here, we show that the endogenous Met4 and Met32 form co-localized puncta-like structures in yeast nuclei upon met depletion. Recombinant Met4 and Met32 form mixed droplets with LLPS properties in vitro. In relation to chromatin, Met4 puncta co-localize with target genes, and at least a subset of these target genes is clustered in 3D in a Met4-dependent manner. A MET3pr-GFP reporter inserted near several native Met4-binding sites becomes co-localized with Met4 puncta and displays enhanced transcriptional activity. A Met4 variant with a partial truncation of an intrinsically disordered region (IDR) shows less puncta formation, and this mutant selectively reduces the reporter activity near Met4-binding sites to the basal level. Overall, these results support a model where Met4 and co-activators form condensates to bring multiple target genes into a vicinity with higher local TF concentrations, which facilitates a strong response to methionine depletion.

Transcription factor condensates, 3D clustering, and gene expression enhancement of the MET regulon

Some transcription factors (TFs) can form liquid–liquid phase separated (LLPS) condensates. However, the functions of these TF condensates in 3-Dimentional (3D) genome organization and gene regulation remain elusive. In response to methionine (met) starvation, budding yeast TF Met4 and a few co-activators, including Met32, induce a set of genes involved in met biosynthesis. Here, we show that the endogenous Met4 and Met32 form co-localized puncta-like structures in yeast nuclei upon met depletion. Recombinant Met4 and Met32 form mixed droplets with LLPS properties in vitro. In relation to chromatin, Met4 puncta co-localize with target genes, and at least a subset of these target genes is clustered in 3D in a Met4-dependent manner. A MET3pr-GFP reporter inserted near several native Met4-binding sites becomes co-localized with Met4 puncta and displays enhanced transcriptional activity. A Met4 variant with a partial truncation of an intrinsically disordered region (IDR) shows less puncta formation, and this mutant selectively reduces the reporter activity near Met4-binding sites to the basal level. Overall, these results support a model where Met4 and co-activators form condensates to bring multiple target genes into a vicinity with higher local TF concentrations, which facilitates a strong response to methionine depletion.

Nuclear Type I Myosins are Essential for Life and Genome Organization.

The active transport of large biomolecules within a cell is critical for homeostasis. While the cytoplasmic process is well-studied, how the spacing of nucleoplasmic cargo is coordinated is poorly understood. We investigated the impact of myosin motors in the nucleus of budding yeast. We found that life requires a nuclear type I myosin whereas the essential type II or V myosins were not requisite in the nucleus. Nuclear depletion of type I myosins triggered 3D genome disorganization, nucleolar disruption, broad gene expression changes, and nuclear membrane morphology collapse. Genome disorganization occurred first supporting a model where type I myosins actively maintain genome architecture that scaffolds nuclear membrane and nucleolar morphologies. Overall, nuclear myosin is critical for the form and function of the nucleus.

Transcription Factor Condensates Mediate Clustering of MET Regulon and Enhancement in Gene Expression.

Some transcription factors (TFs) can form liquid-liquid phase separated (LLPS) condensates. However, the functions of these TF condensates in 3D genome organization and gene regulation remain elusive. In response to methionine (met) starvation, budding yeast TF Met4 and a few co-activators, including Met32, induce a set of genes involved in met biosynthesis. Here, we show that the endogenous Met4 and Met32 form co-localized puncta-like structures in yeast nuclei upon met depletion. Recombinant Met4 and Met32 form mixed droplets with LLPS properties in vitro. In relation to chromatin, Met4 puncta co-localize with target genes, and at least a subset of these target genes is clustered in 3D in a Met4-dependent manner. A MET3pr-GFP reporter inserted near several native Met4 binding sites becomes co-localized with Met4 puncta and displays enhanced transcriptional activity. A Met4 variant with a partial truncation of an intrinsically disordered region (IDR) shows less puncta formation, and this mutant selectively reduces the reporter activity near Met4 binding sites to the basal level. Overall, these results support a model where Met4 and co-activators form condensates to bring multiple target genes into a vicinity with higher local TF concentrations, which facilitates a strong response to methionine depletion.

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.

Zymolyase Treatment of Saccharomyces cerevisiae Affects Cellular Proteins and Degrades Tyrosyl-DNA Phosphodiesterase I.

Saccharomyces cerevisiae is a genetically tractable, affordable, and extensively documented eukaryotic single-cell model organism. This budding yeast is amenable for the development of genetic and biochemical experiments and is frequently used to investigate the function, activity, and mechanism of mammalian proteins. However, yeast contains a cell wall that hinders select assays including organelle isolation. Lytic enzymes, with Zymolyase as the most effective and frequently used tool, are utilized to weaken the yeast cell wall resulting in yeast spheroplasts. Spheroplasts are easily lysed by, for example, osmotic-shock conditions to isolate yeast nuclei or mitochondria. However, during our studies of the DNA repair enzyme tyrosyl-DNA phosphodiesterase I (Tdp1), we encountered a negative effect of Zymolyase. We observed that Zymolyase treatment affected the steady-state protein levels of Tdp1. This was revealed by inconsistencies in technical and biological replicate lysates of plasmid-born galactose-induced expression of Tdp1. This off-target effect of Zymolyase is rarely discussed in articles and affects a select number of intracellular proteins, including transcription factors and assays such as chromatin immunoprecipitations. Following extensive troubleshooting, we concluded that the culprit is the Ser-protease, Zymolyase B, component of the Zymolyase enzyme mixture that causes the degradation of Tdp1. In this study, we report the protocols we have used, and our final protocol with an easy, affordable adaptation to any assay/protocol involving Zymolyase.

Examining chromatin heterogeneity through PacBio long-read sequencing of M.EcoGII methylated genomes: an m6A detection efficiency and calling bias correcting pipeline.

Recent studies have combined DNA methyltransferase footprinting of genomic DNA in nuclei with long-read sequencing, resulting in detailed chromatin maps for multi-kilobase stretches of genomic DNA from one cell. Theoretically, nucleosome footprints and nucleosome-depleted regions can be identified using M.EcoGII, which methylates adenines in any sequence context, providing a high-resolution map of accessible regions in each DNA molecule. Here, we report PacBio long-read sequence data for budding yeast nuclei treated with M.EcoGII and a bioinformatic pipeline which corrects for three key challenges undermining this promising method. First, detection of m6A in individual DNA molecules by the PacBio software is inefficient, resulting in false footprints predicted by random gaps of seemingly unmethylated adenines. Second, there is a strong bias against m6A base calling as AT content increases. Third, occasional methylation occurs within nucleosomes, breaking up their footprints. After correcting for these issues, our pipeline calculates a correlation coefficient-based score indicating the extent of chromatin heterogeneity within the cell population for every gene. Although the population average is consistent with that derived using other techniques, we observe a wide range of heterogeneity in nucleosome positions at the single-molecule level, probably reflecting cellular chromatin dynamics.

Dynamics of DNA damage-induced nuclear inclusions are regulated by SUMOylation of Btn2

Spatial compartmentalization is a key facet of protein quality control that serves to store disassembled or non-native proteins until triage to the refolding or degradation machinery can occur in a regulated manner. Yeast cells sequester nuclear proteins at intranuclear quality control bodies (INQ) in response to various stresses, although the regulation of this process remains poorly understood. Here we reveal the SUMO modification of the small heat shock protein Btn2 under DNA damage and place Btn2 SUMOylation in a pathway promoting protein clearance from INQ structures. Along with other chaperones, and degradation machinery, Btn2-SUMO promotes INQ clearance from cells recovering from genotoxic stress. These data link small heat shock protein post-translational modification to the regulation of protein sequestration in the yeast nucleus.

Yeast 26S proteasome nuclear import is coupled to nucleus-specific degradation of the karyopherin adaptor protein Sts1

In eukaryotes, the ubiquitin–proteasome system is an essential pathway for protein degradation and cellular homeostasis. 26S proteasomes concentrate in the nucleus of budding yeast Saccharomyces cerevisiae due to the essential import adaptor protein Sts1 and the karyopherin-α protein Srp1. Here, we show that Sts1 facilitates proteasome nuclear import by recruiting proteasomes to the karyopherin-α/β heterodimer. Following nuclear transport, the karyopherin proteins are likely separated from Sts1 through interaction with RanGTP in the nucleus. RanGTP-induced release of Sts1 from the karyopherin proteins initiates Sts1 proteasomal degradation in vitro. Sts1 undergoes karyopherin-mediated nuclear import in the absence of proteasome interaction, but Sts1 degradation in vivo is only observed when proteasomes successfully localize to the nucleus. Sts1 appears to function as a proteasome import factor during exponential growth only, as it is not found in proteasome storage granules (PSGs) during prolonged glucose starvation, nor does it appear to contribute to the rapid nuclear reimport of proteasomes following glucose refeeding and PSG dissipation. We propose that Sts1 acts as a single-turnover proteasome nuclear import factor by recruiting karyopherins for transport and undergoing subsequent RanGTP-initiated ubiquitin-independent proteasomal degradation in the nucleus.

The CRISPR/Cas9 system forms a condensate in the yeast nucleus.

CRISPR/Cas9 gene editing technology has revolutionized genetic engineering. However, the nuclear dynamics of Cas9 in eukaryotic cells, particularly in the model organism Saccharomyces cerevisiae , remains poorly understood. Here, we constructed yeast strains expressing fluorescently tagged Cas9 variants, revealing their accumulation in the nucleus over time. Notably, Cas9 was non-uniformly distributed in the nucleoplasm during the initial hours, suggesting the formation of a condensate. This condensate often co-localizes with the nucleolus and associates the target site to its periphery. Our findings provide insights into Cas9 nuclear dynamics in yeast, advancing our understanding of CRISPR/Cas9-based genetic manipulation.

The serine-arginine (SR) protein UmRrm75 from Ustilago maydis is a functional ortholog of yeast ScHrb1.

The Basidiomycete fungus Ustilago maydis is a biotrophic pathogen of maize. The U. maydis UmRrm75 gene encodes an RNA-binding protein (RBP). In a previous study, we reported that ΔUmRrm75 null mutant strains accumulate H2O2, exhibit slow growth, and have decreased virulence in maize. Herein, we describe UmRrm75 as an ortholog of the ScHrb1, a serine-arginine (SR) protein identified in the yeast Saccharomyces cerevisiae, which plays a role in nuclear quality control, specifically in mRNA splicing and export processes. The yeast ScHrb1 mutant (ΔScHrb1) exhibits an increased sensitivity to elevated levels of boron. We noticed that the ΔScHrb1 displayed sensitivity to H2O2, which is consistent with previous findings in the ΔUmRrm75 mutant. We reversed the sensitivity phenotypes of boron and H2O2 by introducing the UmRrm75 gene into the ΔScHrb1 mutant. Furthermore, we generated complementary strains of U. maydis by expressing UmRrm75-GFP under its native promoter in the ∆UmRrm75 mutants. The UmRrm75-GFP/∆UmRrm75 complementary strains successfully recovered their growth capability under stressors, H2O2 and boron, resembling the parental strains FB2 and AB33. The subcellular localization experiments conducted in U. maydis revealed that the UmRrm75 protein is localized within the nucleus of both yeast and hyphae. The nuclear localization of the UmRrm75 protein remains unaltered even under conditions of heat or oxidative stress. This suggests that UmRrm75 might perform its RBP activity in the nucleus, as previously reported for ScHrb1. Our data contribute to understanding the role of the nuclear RBP UmRrm75 from the corn smut fungus U. maydis.

Single-molecule study reveals Hmo1, not Hho1, promotes chromatin assembly in budding yeast.

Linker histone H1 plays a crucial role in various biological processes, including nucleosome stabilization, high-order chromatin structure organization, gene expression, and epigenetic regulation in eukaryotic cells. Unlike higher eukaryotes, little about the linker histone in Saccharomyces cerevisiae is known. Hho1 and Hmo1 are two long-standing controversial histone H1 candidates in budding yeast. In this study, we directly observed at the single-molecule level that Hmo1, but not Hho1, is involved in chromatin assembly in the yeast nucleoplasmic extracts (YNPE), which can replicate the physiological condition of the yeast nucleus. The presence of Hmo1 facilitates the assembly of nucleosomes on DNA in YNPE, as revealed by single-molecule force spectroscopy. Further single-molecule analysis showed that the lysine-rich C-terminal domain (CTD) of Hmo1 is essential for the function of chromatin compaction, while the second globular domain at the C-terminus of Hho1 impairs its ability. In addition, Hmo1, but not Hho1, forms condensates with double-stranded DNA via reversible phase separation. The phosphorylation fluctuation of Hmo1 coincides with metazoan H1 during the cell cycle. Our data suggest that Hmo1, but not Hho1, possesses some functionality similar to that of linker histone in Saccharomyces cerevisiae, even though some properties of Hmo1 differ from those of a canonical linker histone H1. Our study provides clues for the linker histone H1 in budding yeast and provides insights into the evolution and diversity of histone H1 across eukaryotes. IMPORTANCE There has been a long-standing debate regarding the identity of linker histone H1 in budding yeast. To address this issue, we utilized YNPE, which accurately replicate the physiological conditions in yeast nuclei, in combination with total internal reflection fluorescence microscopy and magnetic tweezers. Our findings demonstrated that Hmo1, rather than Hho1, is responsible for chromatin assembly in budding yeast. Additionally, we found that Hmo1 shares certain characteristics with histone H1, including phase separation and phosphorylation fluctuations throughout the cell cycle. Furthermore, we discovered that the lysine-rich domain of Hho1 is buried by its second globular domain at the C-terminus, resulting in the loss of function that is similar to histone H1. Our study provides compelling evidence to suggest that Hmo1 shares linker histone H1 function in budding yeast and contributes to our understanding of the evolution of linker histone H1 across eukaryotes.

Ultrastructure and fractal property of chromosomes in close-to-native yeast nuclei visualized using X-ray laser diffraction

Genome compaction and activity in the nucleus depend on spatiotemporal changes in the organization of chromatins in chromosomes. However, the direct imaging of the chromosome structures in the nuclei has been difficult and challenging. Herein, we directly visualized the structure of chromosomes in frozen-hydrated nuclei of budding yeast in the interphase using X-ray laser diffraction. The reconstructed projection electron density maps revealed inhomogeneous distributions of chromosomes, such as a 300 nm assembly and fibrous substructures in the elliptic-circular shaped nuclei of approximately 800 nm. In addition, from the diffraction patterns, we confirmed the absence of regular arrangements of chromosomes and chromatins with 400–20 nm spacing, and demonstrated that chromosomes were composed of self-similarly assembled substructural domains with an average radius of gyration of 58 nm and smooth surfaces. Based on these analyses, we constructed putative models to discuss the organization of 16 chromosomes, carrying DNA of 4.1 mm in 800 nm ellipsoid of the nucleus at the interphase. We anticipate the structural parameters on the fractal property of chromosomes and the experimental images to be a starting point for constructing more sophisticated 3D structural models of the nucleus.

Host- and virus-induced gene silencing of HOG1-MAPK cascade genes in Rhizophagus irregularis inhibit arbuscule development and reduce resistance of plants to drought stress.

Arbuscular mycorrhizal (AM) fungi can form beneficial associations with the most terrestrial vascular plant species. AM fungi not only facilitate plant nutrient acquisition but also enhance plant tolerance to various environmental stresses such as drought stress. However, the molecular mechanisms by which AM fungal mitogen-activated protein kinase (MAPK) cascades mediate the host adaptation to drought stimulus remains to be investigated. Recently, many studies have shown that virus-induced gene silencing (VIGS) and host-induced gene silencing (HIGS) strategies are used for functional studies of AM fungi. Here, we identify the three HOG1 (High Osmolarity Glycerol 1)-MAPK cascade genes RiSte11, RiPbs2 and RiHog1 from Rhizophagus irregularis. The expression levels of the three HOG1-MAPK genes are significantly increased in mycorrhizal roots of the plant Astragalus sinicus under severe drought stress. RiHog1 protein was predominantly localized in the nucleus of yeast in response to 1M sorbitol treatment, and RiPbs2 interacts with RiSte11 or RiHog1 directly by pull-down assay. Importantly, VIGS or HIGS of RiSte11, RiPbs2 or RiHog1 hampers arbuscule development and decreases relative water content in plants during AM symbiosis. Moreover, silencing of HOG1-MAPK cascade genes led to the decreased expression of drought-resistant genes (RiAQPs, RiTPSs, RiNTH1 and Ri14-3-3) in the AM fungal symbiont in response to drought stress. Taken together, this study demonstrates that VIGS or HIGS of AM fungal HOG1-MAPK cascade inhibits arbuscule development and expression of AM fungal drought-resistant genes under drought stress.

Expression and functional analysis of various structural domains of tobacco topoisomerase II: To understand the mechanistic insights of plant type II topoisomerases

In contrast to bacterial, yeast and animal systems, topoisomerases (topo) from plants have not been well studied. In this report, we generated four truncated topoisomerase II (Topo II) cDNA fragments encoding different functional domains of Nicotiana tabacum topo II (NtTopoII). Each of these recombinant polypeptides was expressed alone or in combination in temperature-sensitive topoisomerase II yeast mutants. Recombinant NtTopoII with truncated polypeptides fails to target the yeast nuclei and does not rescue the temperature-sensitive phenotype. In contrast complementation was achieved with the full-length NtTopoII, which localized to the yeast nucleus. These observations suggested the presence of a potent nuclear localization signal (NLS) in the extreme C-terminal 314 amino acid residues of NtTopoII that functioned effectively in the heterologous yeast system. Biochemical characterization of purified recombinant full-length and the partial NtTopoII polypeptides revealed that the ATP-binding and hydrolysis region of NtTopoIIwas located at 413 amino acid N-terminal region and this ATPase domain is functional both when it is expressed as a separate polypeptide or as part of the holoenzyme. The present findings also revealed that all NtTopoII truncated polypeptides were detrimental for in vitro supercoiled DNA relaxation and/or DNA nicking and ligation activity. Further, we discuss the possible disruption of coordinated macromolecular interface movements and the dimer interactions in truncated NtTopoII that are required for functional topoisomerase activity.

DNA damage-induced nuclear import of HSP90α is promoted by Aha1.

The interplay between yHSP90α (Hsp82) and Rad51 has been implicated in the DNA double-strand break repair (DSB) pathway in yeast. Here we report that nuclear translocation of yHSP90α and its recruitment to the DSB end are essential for homologous recombination (HR)-mediated DNA repair in yeast. The HsHSP90α possesses an amino-terminal extension which is phosphorylated upon DNA damage. We find that the absence of the amino-terminal extension in yHSP90α does not compromise its nuclear import, and the nonphosphorylatable-mutant HsHSP90αT7A could be imported to the yeast nucleus upon DNA damage. Interestingly, the flexible charged-linker (CL) domains of both yHSP90α and HsHSP90α play a critical role during their nuclear translocation. The conformational restricted CL mutant yHSP90α∆(211-259), but not a shorter deletion version yHSP90α∆(211-242), fails to reach the nucleus. As the CL domain of yHSP90α is critical for its interaction with Aha1, we investigated whether Aha1 promotes the nuclear import of yHSP90α. We found that the nuclear import of yHSP90α is severely affected in ∆aha1 strain. Moreover, Aha1 is accumulated in the nucleus during DNA damage. Hence Aha1 may serve as a potential target for inhibiting nuclear function of yHSP90α. The increased sensitivity of ∆aha1 strain to genotoxic agents strengthens this notion.

Control of nuclear size by osmotic forces in Schizosaccharomyces pombe.

The size of the nucleus scales robustly with cell size so that the nuclear-to-cell volume ratio (N/C ratio) is maintained during cell growth in many cell types. The mechanism responsible for this scaling remains mysterious. Previous studies have established that the N/C ratio is not determined by DNA amount but is instead influenced by factors such as nuclear envelope mechanics and nuclear transport. Here, we developed a quantitative model for nuclear size control based upon colloid osmotic pressure and tested key predictions in the fission yeast Schizosaccharomyces pombe. This model posits that the N/C ratio is determined by the numbers of macromolecules in the nucleoplasm and cytoplasm. Osmotic shift experiments showed that the fission yeast nucleus behaves as an ideal osmometer whose volume is primarily dictated by osmotic forces. Inhibition of nuclear export caused accumulation of macromolecules in the nucleoplasm, leading to nuclear swelling. We further demonstrated that the N/C ratio is maintained by a homeostasis mechanism based upon synthesis of macromolecules during growth. These studies demonstrate the functions of colloid osmotic pressure in intracellular organization and size control.

Quantitative analysis of nuclear pore complex organization in Schizosaccharomyces pombe.

The number, distribution, and composition of nuclear pore complexes (NPCs) in the nuclear envelope varies between cell types and changes during cellular differentiation and in disease. To understand how NPC density and organization are controlled, we analyzed the NPC number and distribution in the fission yeast Schizosaccharomyces pombe using structured illumination microscopy. The small size of yeast nuclei, genetic features of fungi, and our robust image analysis pipeline allowed us to study NPCs in intact nuclei under multiple conditions. Our data revealed that NPC density is maintained across a wide range of nuclear sizes. Regions of reduced NPC density are observed over the nucleolus and surrounding the spindle pole body (SPB). Lem2-mediated tethering of the centromeres to the SPB is required to maintain NPC exclusion near SPBs. These findings provide a quantitative understanding of NPC number and distribution in S. pombe and show that interactions between the centromere and the nuclear envelope influences local NPC distribution.

How the fission yeast nucleus gets its shape: altering force balance via laser ablation during mitosis

The fission yeast S. pombe undergoes closed cell division, meaning that the mitotic spindle forms, elongates, and segregates chromosomes inside an intact nuclear envelope. As it does so, its mitotic spindle forms a single bundle of parallel and antiparallel microtubules. This process puts unique force constraints on both the spindle and the envelope. During spindle elongation, the nucleus simultaneously undergoes a series of shape changes: from a single spherical nucleus, through a peanut-shaped and then barbell-shaped nucleus, and finally becoming two new nuclei, one for each new daughter cell.

Proteomic Mapping by APEX2-Catalyzed Proximity Labeling in Saccharomyces cerevisiae Semipermeabilized Cells.

Enzyme-catalyzed proximity labeling (PL) has proven to be a valuable resource for proteomic mapping of subcellular compartments and protein networks in living cells. We have used engineered ascorbate peroxidase (APEX2) to develop a PL approach for budding yeast. It is based on semipermeabilized cells to overcome poor cellular permeability of the APEX2 substrate biotin-phenol and difficulties in its delivery into the cell. The use of semipermeabilized cells has several advantages, in particular the avoidance of generating fragile spheroplasts and the opportunity of employing cells from a glucose-containing medium for APEX2 tagging. In this protocol we describe how to perform a ratiometric three-state stable isotope labeling by amino acids in cell culture (SILAC) approach that allows to map an open cellular compartment like the yeast nucleus. In particular, we focus on the proteomic sample preparation and provide instructions to achieve high-resolution mapping of a subcellular yeast proteome.