Polarization of the endoplasmic reticulum by ER-septin tethering.

Polarization of the plasma membrane (PM) into domains is an important mechanism to compartmentalize cellular activities and to establish cell polarity. Polarization requires formation of diffusion barriers that prevent mixing of proteins between domains. Recent studies have uncovered that the endoplasmic reticulum (ER) of budding yeast and neurons is polarized by diffusion barriers, which in neurons controls glutamate signaling in dendritic spines. The molecular identity of these barriers is currently unknown. Here, we show that a direct interaction between the ER protein Scs2 and the septin Shs1 creates the ER diffusion barrier in yeast. Barrier formation requires Epo1, a novel ER-associated subunit of the polarisome that interacts with Scs2 and Shs1. ER-septin tethering polarizes the ER into separate mother and bud domains, one function of which is to position the spindle in the mother until M phase by confining the spindle capture protein Num1 to the mother ER.

Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions of OCA6, a tyrosine phosphatase gene, along with PMT1 or PMT2, two genes encoding ER membrane protein-O-mannosyltransferases involved in ER quality control, and SKI3, which encode for a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperone gene SCJ1 led to almost no secreted laccase activity. When we expressed SCJ1 from a low-copy plasmid, laccase secretion was restored. However, overexpression of SCJ1 had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.

Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility.

Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations.

A role for the budding yeast separase, Esp1, in Ty1 element retrotransposition.

Separase/Esp1 is a protease required at the onset of anaphase to cleave cohesin and thereby enable sister chromatid separation. Esp1 also promotes release of the Cdc14 phosphatase from the nucleolus to enable mitotic exit. To uncover other potential roles for separase, we performed two complementary genome-wide genetic interaction screens with a strain carrying the budding yeast esp1-1 separase mutation. We identified 161 genes that when mutated aggravate esp1-1 growth and 44 genes that upon increased dosage are detrimental to esp1-1 viability. In addition to the expected cell cycle and sister chromatid segregation genes that were identified, 24% of the genes identified in the esp1-1 genetic screens have a role in Ty1 element retrotransposition. Retrotransposons, like retroviruses, replicate through reverse transcription of an mRNA intermediate and the resultant cDNA product is integrated into the genome by a conserved transposon or retrovirus encoded integrase protein. We purified Esp1 from yeast and identified an interaction between Esp1 and Ty1 integrase using mass spectrometry that was subsequently confirmed by co-immunoprecipitation analysis. Ty1 transposon mobility and insertion upstream of the SUF16 tRNA gene are both reduced in an esp1-1 strain but increased in cohesin mutant strains. Securin/Pds1, which is required for efficient localization of Esp1 to the nucleus, is also required for efficient Ty1 transposition. We propose that Esp1 serves two roles to mediate Ty1 transposition - one to remove cohesin and the second to target Ty1-IN to chromatin.

A conserved endoplasmic reticulum membrane protein complex (EMC) facilitates phospholipid transfer from the ER to mitochondria.

Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER-mitochondria tethering complex called ERMES (the ER-mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER-mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.

Improving spine surgical access, appropriateness and efficiency in metropolitan, urban and rural settings.

BACKGROUND: The Inter-professional Spine Assessment and Education Clinics (ISAEC) were developed to improve primary care assessment, education and management of patients with persistent or recurrent low back pain-related symptoms. This study aims to determine the effect of ISAEC on access for surgical assessment, referral appropriateness and efficiency for patients meeting a priori referral criteria in rural, urban and metropolitan settings. METHODS: We conducted a retrospective review of prospective data from networked ISAEC clinics in Thunder Bay, Hamilton and Toronto, Ontario. For patients meeting surgical referral criteria, wait times for surgical assessment, surgical referral-related magnetic resonance imaging (MRI) scans and appropriateness of referral were recorded. RESULTS: Overall 422 patients, representing 10% of all ISAEC patients in the study period, were referred for surgical assessment. The average wait times for surgical assessment were 5.4, 4.3 and 2.2 weeks at the metropolitan, urban and rural centres, respectively. Referral MRI usage for the group decreased by 31%. Of the patients referred for formal surgical assessment, 80% had leg-dominant pain and 96% were deemed appropriate surgical referrals. CONCLUSION: Contrary to geographic concentration of health care resources in metropolitan settings, the greatest decrease in wait times was achieved in the rural setting. A networked, shared-cared model of care for patients with low back pain-related symptoms significantly improved access for surgical assessment despite varying geographic practice settings and barriers. The greatest reductions were noted in the rural setting. In addition, significant improvements in referral appropriateness and efficiency were achieved compared with historical reports across all sites.

CONTEXTE: Les cliniques interprofessionnelles d'évaluation de la colonne vertébrale et d'éducation (Inter-professional Spine Assessment and Education Clinics [ISAEC]) ont été mises sur pied pour améliorer les soins primaires d'évaluation, d'éducation et de prise en charge des patients atteints de symptômes persistants ou récurrents de lombalgie. Cette étude a pour but d'évaluer l'effet des ISAEC sur l'accès à une évaluation chirurgicale et sur la pertinence et l'efficacité de la référence des patients en milieux ruraux, urbains et métropolitains répondant a priori aux critères de référence. MÉTHODES: Nous avons mené une étude rétrospective de données prospectives issues de cliniques du réseau des ISAEC situées à Thunder Bay, à Hamilton et à Toronto, en Ontario. Nous avons retenu pour l'étude les patients répondant aux critères de référence en chirurgie; pour chacun de ces patients, nous avons consigné le temps d'attente pour obtenir une évaluation chirurgicale, les images obtenues par résonance magnétique (IRM) aux fins de référence et la pertinence de la référence. RÉSULTATS: Au total, 422 patients, soit 10 % des patients des ISAEC au cours de la période étudiée, ont été dirigés en évaluation chirurgicale. Les temps d'attente moyens pour obtenir une évaluation chirurgicale étaient de 5,4 semaines, de 4,3 semaines et de 2,2 semaines dans les centres métropolitains, urbains et ruraux, respectivement. Le recours à l'IRM aux fins de référence a diminué de 31 % par rapport à la situation initiale. Parmi les patients référés en évaluation chirurgicale formelle, 80 % présentaient une douleur principalement localisée dans les jambes. La référence de 96 % des patients a été jugée adéquate. CONCLUSION: Même si les ressources en soins de santé sont concentrées en milieu métropolitain, c'est le milieu rural qui a connu la plus grande baisse du temps d'attente. La mise sur pied d'un modèle de soins partagés en réseau pour les patients aux prises avec des symptômes de lombalgie a amélioré l'accès aux évaluations chirurgicales de façon significative, malgré la variété géographique des milieux de pratique et les divers obstacles rencontrés. Les baisses les plus importantes ont été observées en milieu rural. De plus, des améliorations significatives de la pertinence et de l'efficacité des références ont été observées lors de la comparaison avec les rapports antérieurs, pour tous les sites de l'étude.

Plasma membrane--endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis.

Synthesis of phospholipids, sterols and sphingolipids is thought to occur at contact sites between the endoplasmic reticulum (ER) and other organelles because many lipid-synthesizing enzymes are enriched in these contacts. In only a few cases have the enzymes been localized to contacts in vivo and in no instances have the contacts been demonstrated to be required for enzyme function. Here, we show that plasma membrane (PM)--ER contact sites in yeast are required for phosphatidylcholine synthesis and regulate the activity of the phosphatidylethanolamine N-methyltransferase enzyme, Opi3. Opi3 activity requires Osh3, which localizes to PM-ER contacts where it might facilitate in trans catalysis by Opi3. Thus, membrane contact sites provide a structural mechanism to regulate lipid synthesis.

ER-shaping proteins facilitate lipid exchange between the ER and mitochondria in S. cerevisiae.

The endoplasmic reticulum (ER) forms a network of sheets and tubules that extends throughout the cell. Proteins required to maintain this complex structure include the reticulons, reticulon-like proteins, and dynamin-like GTPases called atlastins in mammals and Sey1p in Saccharomyces cerevisiae. Yeast cells missing these proteins have abnormal ER structure, particularly defects in the formation of ER tubules, but grow about as well as wild-type cells. We screened for mutations that cause cells that have defects in maintaining ER tubules to grow poorly. Among the genes we found were members of the ER mitochondria encounter structure (ERMES) complex that tethers the ER and mitochondria. Close contacts between the ER and mitochondria are thought to be sites where lipids are moved from the ER to mitochondria, a process that is required for mitochondrial membrane biogenesis. We show that ER to mitochondria phospholipid transfer slows significantly in cells missing both ER-shaping proteins and the ERMES complex. These cells also have altered steady-state levels of phospholipids. We found that the defect in ER to mitochondria phospholipid transfer in a strain missing ER-shaping proteins and a component of the ERMES complex was corrected by expression of a protein that artificially tethers the ER and mitochondria. Our findings indicate that ER-shaping proteins play a role in maintaining functional contacts between the ER and mitochondria and suggest that the shape of the ER at ER-mitochondria contact sites affects lipid exchange between these organelles.

Balony: a software package for analysis of data generated by synthetic genetic array experiments.

BACKGROUND: Synthetic Genetic Array (SGA) analysis is a procedure which has been developed to allow the systematic examination of large numbers of double mutants in the yeast Saccharomyces cerevisiae. The aim of these experiments is to identify genetic interactions between pairs of genes. These experiments generate a number of images of ordered arrays of yeast colonies which must be analyzed in order to quantify the extent of the genetic interactions. We have designed software that is able to analyze virtually any image of regularly arrayed colonies and allows the user significant flexibility over the analysis procedure. RESULTS: "Balony" is freely available software which enables the extraction of quantitative data from array-based genetic screens. The program follows a multi-step process, beginning with the optional preparation of plate images from single or composite images. Next, the colonies are identified on a plate and the pixel area of each is measured. This is followed by a scoring module which normalizes data and pairs control and experimental data files. The final step is analysis of the scored data, where the strength and reproducibility of genetic interactions can be visualized and cross-referenced with information on each gene to provide biological insights into the results of the screen. CONCLUSIONS: Analysis of SGA screens with Balony can be either automated or highly interactive, enabling the user to customize the process to their specific needs. Quantitative data can be extracted at each stage for external analysis if required. Beyond SGA, this software can be used for analyzing many types of plate-based high-throughput screens.

Synthesis and crystal structure of bis-(2-phthal-imido-eth-yl)ammonium chloride dihydrate.

The title compound {systematic name: bis-[2-(1,3-dioxoisoindol-2-yl)eth-yl]aza-nium chloride dihydrate}, C20H18N3O4 +·Cl-·2H2O, is a phthalimide-protected polyamine that was synthesized by a previous method. It was characterized by ESI-MS, 1H NMR, and FT-IR. Crystals were grown from a solution of H2O and 0.1 M HCl. The central nitro-gen atom is protonated and forms hydrogen bonds with the chloride ion and a water mol-ecule. The two phthalimide units make a dihedral angle of 22.07 (3)°. The crystal packing features a hydrogen-bond network, two-coordinated chloride, and off-set π-π stacking.

Interobserver reliability in the histopathological diagnosis of cartilaginous tumors in patients with multiple osteochondromas.

The distinction between benign and malignant cartilaginous tumors located peripherally in the bone may be a challenging task in surgical pathology. The aim of this study was to investigate interobserver reliability in histological diagnosis of cartilaginous tumors in the setting of multiple osteochondromas and to evaluate possible histological parameters that could differentiate among osteochondroma, low- and high-grade secondary peripheral chondrosarcoma. Interobserver reliability was assessed by 12 specialized bone-tumor pathologists in a set of 38 cases. Substantial agreement on diagnosis among all the reviewers was observed (intraclass correlation coefficient=0.78). Our study confirmed that mitotic figures and nuclear pleomorphism are hallmarks of high-grade secondary peripheral chondrosarcoma. However, despite the substantial agreement, we demonstrated that histology alone cannot distinguish osteochondroma from low-grade secondary peripheral chondrosarcoma in the setting of multiple osteochondromas, as nodularity, the presence of binucleated cells, irregular calcification, cystic/mucoid changes and necrosis were not helpful to indicate malignant transformation of an osteochondroma. On the other hand, among the concordant cases, the cartilage cap in osteochondroma was significantly less thicker than in low- and high-grade secondary peripheral chondrosarcoma. Therefore, our study showed that a multidisciplinary approach integrating clinical and radiographical features and the size of the cartilaginous cap in combination with a histological assessment are crucial to the diagnosis of cartilaginous tumors.

Inheritance of cortical ER in yeast is required for normal septin organization.

How cells monitor the distribution of organelles is largely unknown. In budding yeast, the largest subdomain of the endoplasmic reticulum (ER) is a network of cortical ER (cER) that adheres to the plasma membrane. Delivery of cER from mother cells to buds, which is termed cER inheritance, occurs as an orderly process early in budding. We find that cER inheritance is defective in cells lacking Scs2, a yeast homologue of the integral ER membrane protein VAP (vesicle-associated membrane protein-associated protein) conserved in all eukaryotes. Scs2 and human VAP both target yeast bud tips, suggesting a conserved action of VAP in attaching ER to sites of polarized growth. In addition, the loss of either Scs2 or Ice2 (another protein involved in cER inheritance) perturbs septin assembly at the bud neck. This perturbation leads to a delay in the transition through G2, activating the Saccharomyces wee1 kinase (Swe1) and the morphogenesis checkpoint. Thus, we identify a mechanism involved in sensing the distribution of ER.

Ist2 recruits the lipid transporters Osh6/7 to ER-PM contacts to maintain phospholipid metabolism.

ER-plasma membrane (PM) contacts are proposed to be held together by distinct families of tethering proteins, which in yeast include the VAP homologues Scs2/22, the extended-synaptotagmin homologues Tcb1/2/3, and the TMEM16 homologue Ist2. It is unclear whether these tethers act redundantly or whether individual tethers have specific functions at contacts. Here, we show that Ist2 directly recruits the phosphatidylserine (PS) transport proteins and ORP family members Osh6 and Osh7 to ER-PM contacts through a binding site located in Ist2's disordered C-terminal tethering region. This interaction is required for phosphatidylethanolamine (PE) production by the PS decarboxylase Psd2, whereby PS transported from the ER to the PM by Osh6/7 is endocytosed to the site of Psd2 in endosomes/Golgi/vacuoles. This role for Ist2 and Osh6/7 in nonvesicular PS transport is specific, as other tethers/transport proteins do not compensate. Thus, we identify a molecular link between the ORP and TMEM16 families and a role for endocytosis of PS in PE synthesis.

Sentinel interaction mapping - a generic approach for the functional analysis of human disease gene variants using yeast.

Advances in sequencing technology have led to an explosion in the number of known genetic variants of human genes. A major challenge is to now determine which of these variants contribute to diseases as a result of their effect on gene function. Here, we describe a generic approach using the yeast Saccharomyces cerevisiae to quickly develop gene-specific in vivo assays that can be used to quantify the level of function of a genetic variant. Using synthetic dosage lethality screening, 'sentinel' yeast strains are identified that are sensitive to overexpression of a human disease gene. Variants of the gene can then be functionalized in a high-throughput fashion through simple growth assays using solid or liquid media. Sentinel interaction mapping (SIM) has the potential to create functional assays for the large majority of human disease genes that do not have a yeast orthologue. Using the tumour suppressor gene PTEN as an example, we show that SIM assays can provide a fast and economical means to screen a large number of genetic variants.

Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction.

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PTEN hamartoma syndrome (PHTS), we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occur across the protein, resulting in partial-to-complete loss-of-function (LoF), which is well correlated across models. However, assays are selectively sensitive to variants located in substrate binding and catalytic domains, which exhibit complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.

Early microrecanalization of vas deferens following biodegradable graft implantation in bilaterally vasectomized rats.

We evaluated a biodegradable graft for reconstruction of rat vasa deferentia with long obstructed or missing segments. A total of 47 Sprague-Dawley rats underwent bilateral vasectomy and were divided into groups according to length of the vas deferens affected (0.5, 1, 1.5 cm). After 8 weeks, poly-(D,L-lactide) (PDLA) grafts were used to reconnect the vas deferens. Grafts and adjoining vasa deferentia were excised 8 and 12 weeks later and evaluated microscopically. At 8 weeks, microscopic changes included a robust inflammatory response around the grafts. All grafts were still intact but in the early stages of degradation. No microtubules, indicative of vas deferens recanalization, were identified. One specimen showed evidence of healing and neovascularization at the interface zone between the vas deferens and the graft. At 12 weeks, grafts were further degraded but still present. Microscopic evaluation showed decreased inflammation. Seven specimens showed neovascularization at the interface zone; two of these showed distinct epithelialized vas deferens microcanals at the graft edges. One specimen showed a microcanal spanning the entire 0.5-cm graft. A time period of 8 weeks is not ample enough for vas deferens regeneration in the setting of a biodegradable PDLA graft; however, early evidence of re-growth was seen at 12 weeks. A longer healing time should permit further biodegradation of the graft, as well as re-growth and possible eventual reconnection of the vas deferens, allowing passage of sperm. These findings suggest a potential role for biodegradable grafts in the reconstruction of vas deferens with long obstructed segments.

A nuclear role for the DEAD-box protein Dbp5 in tRNA export.

Dbp5 is an essential DEAD-box protein that mediates nuclear mRNP export. Dbp5 also shuttles between nuclear and cytoplasmic compartments with reported roles in transcription, ribosomal subunit export, and translation; however, the mechanism(s) by which nucleocytoplasmic transport occurs and how Dbp5 specifically contributes to each of these processes remains unclear. Towards understanding the functions and transport of Dbp5 in Saccharomyces cerevisiae, alanine scanning mutagenesis was used to generate point mutants at all possible residues within a GFP-Dbp5 reporter. Characterization of the 456 viable mutants led to the identification of an N-terminal Xpo1-dependent nuclear export signal in Dbp5, in addition to other separation-of-function alleles, which together provide evidence that Dbp5 nuclear shuttling is not essential for mRNP export. Rather, disruptions in Dbp5 nucleocytoplasmic transport result in tRNA export defects, including changes in tRNA shuttling dynamics during recovery from nutrient stress.

Involvement of growth factors in the process of post-vasectomy micro-recanalization.

PURPOSE: We investigated the role of growth factors in the process of post-vasectomy micro-recanalization using real-time polymerase chain reaction, enzyme-linked immunosorbent assay and histopathological analyses of the vasectomy sites and controls at different time points in a rat model. MATERIALS AND METHODS: Unilateral vasectomies were performed in 18 rats with sham surgery on the contralateral side. Vasectomy sites and vas segments of similar length from the sham operated sides were taken for analysis at 2, 8 and 12 weeks. Real-time polymerase chain reaction was used to test the expression of mRNA of 7 common growth factors and select growth factor receptors. Enzyme-linked immunosorbent assay was performed for growth factors with strong positive polymerase chain reaction findings. Histopathological examination was performed by a staff pathologist (BRD) to detect micro-recanalization, defined as tubules visible on hematoxylin and eosin staining with an epithelial lining of cuboidal or columnar cells. RESULTS: Micro-canals were found in 2 of 18 rat specimens. Real-time polymerase chain reaction of all specimens demonstrated a 12-fold increase in platelet-derived growth factor-beta, a 6-fold increase in platelet-derived growth factor-beta receptor, an 11-fold increase in platelet-derived growth factor-alpha, a 7-fold increase in platelet-derived growth factor-alpha receptor and a 9-fold increase in transforming growth factor-beta compared to the sham operated side. All increases were sustained and statistically significant (p <0.05). Enzyme-linked immunosorbent assay revealed statistically significantly increased expression of platelet-derived growth factor-beta protein. CONCLUSIONS: The demonstrated micro-recanalization and sustained growth factor up-regulation at vasectomy sites suggest a possible mechanism for post-vasectomy ejaculate sperm identification. There is a need for further research on the potential role of select growth factors in reconstruction of the male reproductive tract.

Viral proteins as a potential driver of histone depletion in dinoflagellates.

Within canonical eukaryotic nuclei, DNA is packaged with highly conserved histone proteins into nucleosomes, which facilitate DNA condensation and contribute to genomic regulation. Yet the dinoflagellates, a group of unicellular algae, are a striking exception to this otherwise universal feature as they have largely abandoned histones and acquired apparently viral-derived substitutes termed DVNPs (dinoflagellate-viral-nucleoproteins). Despite the magnitude of this transition, its evolutionary drivers remain unknown. Here, using Saccharomyces cerevisiae as a model, we show that DVNP impairs growth and antagonizes chromatin by localizing to histone binding sites, displacing nucleosomes, and impairing transcription. Furthermore, DVNP toxicity can be relieved through histone depletion and cells diminish their histones in response to DVNP expression suggesting that histone reduction could have been an adaptive response to these viral proteins. These findings provide insights into eukaryotic chromatin evolution and highlight the potential for horizontal gene transfer to drive the divergence of cellular systems.

Histone Acetylation, Not Stoichiometry, Regulates Linker Histone Binding in Saccharomyces cerevisiae.

Linker histones play a fundamental role in shaping chromatin structure, but how their interaction with chromatin is regulated is not well understood. In this study, we used a combination of genetic and genomic approaches to explore the regulation of linker histone binding in the yeast, Saccharomyces cerevisiae We found that increased expression of Hho1, the yeast linker histone, resulted in a severe growth defect, despite only subtle changes in chromatin structure. Further, this growth defect was rescued by mutations that increase histone acetylation. Consistent with this, genome-wide analysis of linker histone occupancy revealed an inverse correlation with histone tail acetylation in both yeast and mouse embryonic stem cells. Collectively, these results suggest that histone acetylation negatively regulates linker histone binding in S. cerevisiae and other organisms and provide important insight into how chromatin structure is regulated and maintained to both facilitate and repress transcription.