ABSTRACT
PURPOSE: ABP 710 has been developed as a biosimilar to infliximab reference product (RP). The objective of this study was to assess analytical similarity (structural and functional) between ABP 710 and infliximab RP licensed by the United States Food and Drug Administration (infliximab [US]) and the European Union (infliximab [EU]), using sensitive, state-of-the-art analytical methods capable of detecting minor differences in product quality attributes. METHODS: Comprehensive analytical characterization utilizing orthogonal techniques was performed with 14 to 28 unique lots of ABP 710 or infliximab RP, depending on the assay. Comparisons were used to investigate the primary structure related to amino acid sequence; post-translational modifications (PTMs) including glycans; higher order structure; particles and aggregates; primary biological properties mediated by target and receptor binding; product-related substances and impurities; and general properties. RESULTS: ABP 710 had the same amino acid sequence, primary structure, higher order structure, PTM profiles and biological activities as infliximab RP. The finished drug product had the same strength (protein content and concentration) as infliximab RP. CONCLUSIONS: Based on the comprehensive analytical similarity assessment, ABP 710 was found to be highly analytically similar to infliximab RP for all biological activities relevant for clinical efficacy and safety.
Subject(s)
Antibodies, Monoclonal/analysis , Biosimilar Pharmaceuticals/analysis , Infliximab/analysis , Amino Acid Sequence , Biosimilar Pharmaceuticals/chemistry , Circular Dichroism , Humans , Infliximab/chemistry , Protein Processing, Post-Translational , Spectroscopy, Fourier Transform InfraredABSTRACT
Dynamic control of peroxisome proliferation is integral to the peroxisome's many functions. The endoplasmic reticulum (ER) serves as a source of preperoxisomal vesicles (PPVs) that mature into peroxisomes during de novo peroxisome biogenesis and support growth and division of existing peroxisomes. However, the mechanism of PPV formation and release from the ER remains poorly understood. In this study, we show that endosomal sorting complexes required for transport (ESCRT)-III are required to release PPVs budding from the ER into the cytosol. Absence of ESCRT-III proteins impedes de novo peroxisome formation and results in an aberrant peroxisome population in vivo. Using a cell-free PPV budding assay, we show that ESCRT-III proteins Vps20 and Snf7 are necessary to release PPVs from the ER. ESCRT-III is therefore a positive effector of membrane scission for vesicles budding both away from and toward the cytosol. These findings have important implications for the evolutionary timing of emergence of peroxisomes and the rest of the internal membrane architecture of the eukaryotic cell.
Subject(s)
Endoplasmic Reticulum/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Peroxisomes/metabolism , Saccharomyces cerevisiae/metabolism , Cytoplasmic Vesicles/drug effects , Cytoplasmic Vesicles/metabolism , Endoplasmic Reticulum/drug effects , Genetic Testing , Oleic Acid/pharmacology , Organelle Biogenesis , Peroxisomes/drug effects , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Peroxisome proliferation occurs by at least two routes, division of existing peroxisomes and de novo biogenesis from the endoplasmic reticulum (ER). The proteins and molecular mechanisms governing peroxisome emergence from the ER are poorly characterized. In this study, we report that two integral membrane peroxins (proteins required for peroxisome biogenesis) in Saccharomyces cerevisiae, Pex29 and Pex30, reside in distinct regions of the ER and associate with Rtn1 and Yop1, reticulon family members that contribute to ER morphology, to govern peroxisome emergence from the ER. In vivo and in vitro analyses reveal that peroxisome proliferation is therefore not restricted to the peroxisome but begins at the level of the ER.
Subject(s)
Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Membrane Transport Proteins/metabolism , Peroxisomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Endoplasmic Reticulum/genetics , Membrane Proteins/genetics , Membrane Transport Proteins/genetics , Peroxisomes/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The search for specific productivity (qP) determinants in Chinese hamster ovary (CHO) cells has been the focus of the biopharmaceutical cell line engineering efforts aimed at creating "super-producer" cell lines. In this study, we evaluated the impact of small-molecule inducers and temperature shift on recombinant protein production, and used transcriptomic analysis to define gene-phenotype correlations for qP in our biological system. Next-generation RNA Sequencing (RNA-Seq) analysis revealed that each individual inducer (caffeine, hexamethylene bisacetamide (HMBA) and sodium butyrate (NaBu)) or a combination treatment had a distinct impact on the gene expression program of the RANK-Fc cell line. Temperature shift to 31 °C impacted inducer action with respect to transcriptional changes and phenotypic cell line parameters. We showed that inducer treatment was able to increase expression level of the Fc- fusion mRNA and the selectable marker mRNA from 16% up to 45% of total mRNA in the cell. We further demonstrated that qP exhibited a strong positive linear correlation to transcript levels of both the RANK-Fc fusion protein and the dihydrofolate reductase (DHFR) selectable marker. In fact, these were 2 out of 7 transcripts with significant positive correlation to qP at both temperatures. Many more transcripts were anti- correlated to qP, and gene set enrichment analysis (GSEA) revealed that those were involved in cell cycle progression, transcription, mRNA processing, translation and protein folding. Therefore, we postulate that the transcript level of the recombinant protein is a major qP determinant in our biological system, while downregulation of routine activity within the cell is necessary to divert cellular resources towards recombinant protein production.
Subject(s)
Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Acetamides/pharmacology , Animals , Butyric Acid/pharmacology , CHO Cells , Caffeine/pharmacology , Cell Survival/drug effects , Cricetinae , Cricetulus , Gene Expression/drug effects , Gene Expression Profiling , Proteomics , RNA, Messenger/metabolism , Sequence Analysis, RNA , TemperatureABSTRACT
Non-enzymatic glycation is a challenging post-translational modification to characterize due to the structural heterogeneity it generates in proteins. Glycation has become increasingly recognized as an important product quality attribute to monitor, particularly for the biotechnology sector, which produces recombinant proteins under conditions that are amenable to protein glycation. The elucidation of sites of glycation can be problematic using conventional collision-induced dissociation (CID)-based mass spectrometry because of the predominance of neutral loss ions. A method to characterize glycation using an IgG1 monoclonal antibody (mAb) as a model is reported here. The sugars present on this mAb were derivatized using sodium borohydride chemistry to stabilize the linkage and identified using CID-based MS(2) mass spectrometry and spectral search engines. Quantification of specific glycation sites was then done using a targeted MS(1) based approach, which allowed the identification of a glycation hot spot in the heavy chain complementarity-determining region 3 of the mAb. This targeted approach provided a path forward to developing a structural understanding of the propensity of sites to become glycated on mAbs. Through structural analysis we propose a model in which the number and 3-dimensional distances of carboxylic acid amino acyl residues create a favorable environment for glycation to occur.
Subject(s)
Antibodies, Monoclonal, Murine-Derived/chemistry , Immunoglobulin G/chemistry , Protein Processing, Post-Translational , Antibodies, Monoclonal, Murine-Derived/metabolism , Glycosylation , Immunoglobulin G/metabolism , Mass SpectrometryABSTRACT
The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures.
Subject(s)
Cell Culture Techniques/methods , Cell Cycle Checkpoints/drug effects , Cyclin-Dependent Kinases/antagonists & inhibitors , Recombinant Proteins/metabolism , Animals , Bioreactors , CHO Cells , Cricetinae , Cricetulus , Enzyme Inhibitors/pharmacology , Recombinant Proteins/analysisABSTRACT
Systems scale models provide the foundation for an effective iterative cycle between hypothesis generation, experiment and model refinement. Such models also enable predictions facilitating the understanding of biological complexity and the control of biological systems. Here, we demonstrate the reconstruction of a globally predictive gene regulatory model from public data: a model that can drive rational experiment design and reveal new regulatory mechanisms underlying responses to novel environments. Specifically, using â¼ 1500 publically available genome-wide transcriptome data sets from Saccharomyces cerevisiae, we have reconstructed an environment and gene regulatory influence network that accurately predicts regulatory mechanisms and gene expression changes on exposure of cells to completely novel environments. Focusing on transcriptional networks that induce peroxisomes biogenesis, the model-guided experiments allow us to expand a core regulatory network to include novel transcriptional influences and linkage across signaling and transcription. Thus, the approach and model provides a multi-scalar picture of gene dynamics and are powerful resources for exploiting extant data to rationally guide experimentation. The techniques outlined here are generally applicable to any biological system, which is especially important when experimental systems are challenging and samples are difficult and expensive to obtain-a common problem in laboratory animal and human studies.
Subject(s)
Gene Regulatory Networks , Systems Biology/methods , Gene Expression Profiling , Gene Expression Regulation, Fungal , Saccharomyces cerevisiae/geneticsABSTRACT
Studying SIV infection of natural host monkey species, such as sooty mangabeys, has provided insights into the immune changes associated with these nonprogressive infections. Mangabeys maintain immune health despite high viremia or the dramatic CD4 T cell depletion that can occur following multitropic SIV infection. Here we evaluate double-negative (DN)(CD3+CD4-CD8-) T cells that are resistant to SIV infection due to a lack of CD4 surface expression, for their potential to fulfill a role as helper T cells. We first determined that DN T cells are polyclonal and predominantly exhibit an effector memory phenotype (CD95+CD62L-). Microarray analysis of TCR (anti-CD3/CD28) stimulated DN T cells indicated that these cells are multifunctional and upregulate genes with marked similarity to CD4 T cells, such as immune genes associated with Th1 (IFNγ), Th2 (IL4, IL5, IL13, CD40L), Th17 (IL17, IL22) and TFH (IL21, ICOS, IL6) function, chemokines such as CXCL9 and CXCL10 and transcription factors known to be actively regulated in CD4 T cells. Multifunctional T-helper cell responses were maintained in DN T cells from uninfected and SIV infected mangabeys and persisted in mangabeys exhibiting SIV mediated CD4 loss. Interestingly, TCR stimulation of DN T cells from SIV infected mangabeys results in a decreased upregulation of IFNγ and increased IL5 and IL13 expression compared to uninfected mangabeys. Evaluation of proliferative capacity of DN T cells in vivo (BrDU labeling) indicated that these cells maintain their ability to proliferate despite SIV infection, and express the homeostatic cytokine receptors CD25 (IL2 receptor) and CD127 (IL7 receptor). This study identifies the potential for a CD4-negative T cell subset that is refractory to SIV infection to perform T-helper functions in mangabeys and suggests that immune therapeutics designed to increase DN T cell function during HIV infection may have beneficial effects for the host immune system.
Subject(s)
Cytokines/immunology , Receptors, Antigen, T-Cell/immunology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Cercocebus atys , Cytokines/blood , Cytokines/genetics , Receptors, Antigen, T-Cell/blood , Receptors, Antigen, T-Cell/genetics , Simian Acquired Immunodeficiency Syndrome/blood , Simian Acquired Immunodeficiency Syndrome/genetics , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/metabolism , T-Lymphocytes, Regulatory/metabolismABSTRACT
Cellular control of protein activities by modulation of their abundance or compartmentalization is not easily measured on a large scale. We developed and applied a method to globally interrogate these processes that is widely useful for systems-level analyses of dynamic cellular responses in many cell types. The approach involves subcellular fractionation followed by comprehensive proteomic analysis of the fractions, which is enabled by a data-independent acquisition mass spectrometry approach that samples every available mass to charge channel systematically to maximize sensitivity. Next, various fraction-enrichment ratios are measured for all detected proteins across different environmental conditions and used to group proteins into clusters reflecting changes in compartmentalization and relative conditional abundance. Application of the approach to characterize the response of yeast proteins to fatty acid exposure revealed dynamics of peroxisomes and novel dynamics of MCC/eisosomes, specialized plasma membrane domains comprised of membrane compartment occupied by Can1 (MCC) and eisosome subdomains. It also led to the identification of Fat3, a fatty acid transport protein of the plasma membrane, previously annotated as Ykl187.
Subject(s)
Proteome/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cell Fractionation , Culture Media , Glucose/metabolism , Lipid Metabolism , Microscopy, Fluorescence , Molecular Sequence Annotation , Oleic Acid/metabolism , Organelles/chemistry , Organelles/metabolism , Protein Transport , Proteome/chemistry , Proteomics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/chemistry , Subcellular Fractions/chemistryABSTRACT
Positive feedback is a common mechanism enabling biological systems to respond to stimuli in a switch-like manner. Such systems are often characterized by the requisite formation of a heterodimer where only one of the pair is subject to feedback. This ASymmetric Self-UpREgulation (ASSURE) motif is central to many biological systems, including cholesterol homeostasis (LXRα/RXRα), adipocyte differentiation (PPARγ/RXRα), development and differentiation (RAR/RXR), myogenesis (MyoD/E12) and cellular antiviral defense (IRF3/IRF7). To understand why this motif is so prevalent, we examined its properties in an evolutionarily conserved transcriptional regulatory network in yeast (Oaf1p/Pip2p). We demonstrate that the asymmetry in positive feedback confers a competitive advantage and allows the system to robustly increase its responsiveness while precisely tuning the response to a consistent level in the presence of varying stimuli. This study reveals evolutionary advantages for the ASSURE motif, and mechanisms for control, that are relevant to pharmacologic intervention and synthetic biology applications.
Subject(s)
Feedback, Physiological , Gene Expression Regulation, Fungal , Models, Molecular , Saccharomyces cerevisiae/genetics , Systems Biology/methods , Flow Cytometry , Gene Regulatory Networks , Saccharomyces cerevisiae/growth & development , Signal Transduction , Up-RegulationABSTRACT
High-field asymmetric waveform ion mobility spectrometry (FAIMS) is an atmospheric pressure ion mobility technique that can be used to reduce sample complexity and increase dynamic range in tandem mass spectrometry experiments. FAIMS fractionates ions in the gas-phase according to characteristic differences in mobilities in electric fields of different strengths. Undesired ion species such as solvated clusters and singly charged chemical background ions can be prevented from reaching the mass analyzer, thus decreasing chemical noise. To date, there has been limited success using the commercially available Thermo Fisher FAIMS device with both standard ESI and nanoLC-MS. We have modified a Thermo Fisher electrospray source to accommodate a fused silica pulled tip capillary column for nanospray ionization, which will enable standard laboratories access to FAIMS technology. Our modified source allows easily obtainable stable spray at flow rates of 300 nL/min when coupled with FAIMS. The modified electrospray source allows the use of sheath gas, which provides a fivefold increase in signal obtained when nanoLC is coupled to FAIMS. In this work, nanoLC-FAIMS-MS and nanoLC-MS were compared by analyzing a tryptic digest of a 1:1 mixture of SILAC-labeled haploid and diploid yeast to demonstrate the performance of nanoLC-FAIMS-MS, at different compensation voltages, for post-column fractionation of complex protein digests. The effective dynamic range more than doubled when FAIMS was used. In total, 10,377 unique stripped peptides and 1649 unique proteins with SILAC ratios were identified from the combined nanoLC-FAIMS-MS experiments, compared with 6908 unique stripped peptides and 1003 unique proteins with SILAC ratios identified from the combined nanoLC-MS experiments. This work demonstrates how a commercially available FAIMS device can be combined with nanoLC to improve proteome coverage in shotgun and targeted type proteomics experiments.
Subject(s)
Proteomics/methods , Saccharomyces cerevisiae Proteins/analysis , Spectrometry, Mass, Electrospray Ionization/methods , Chromatography, Liquid/methods , Nanotechnology/methods , Proteome , Saccharomyces cerevisiae , Tandem Mass Spectrometry/methodsABSTRACT
Here, we present a detailed method for generating a dynamic transcriptional regulatory network from large-scale chromatin immunoprecipitation data, and functional analysis of participating factors through the identification and characterization of significantly overrepresented multi-input motifs in the network. This is done by visualizing interactive data using a network analysis tool, such as Cytoscape, clustering DNA targets of the transcription factors based on their network topologies, and statistically analyzing each cluster based on its size and properties of its members. These analyses yield testable predictions about the conditional and cooperative functions of the factors. This is a versatile approach that allows the visualization of network architecture on a genome-wide level and is applicable to understanding combinatorial control mechanisms of DNA-binding regulators that conditionally cooperate in a wide variety of biological models.
Subject(s)
Gene Regulatory Networks/genetics , Transcription Factors/genetics , Chromatin Immunoprecipitation , Cluster Analysis , Data Interpretation, Statistical , Databases, Genetic , Humans , Polymerase Chain ReactionABSTRACT
The histone chaperones play an important role in chromatin assembly and disassembly during replication and transcription. We have assessed the global roles of histone chaperones in Saccharomyces cerevisiae. Microarray transcriptional analyzes indicate that histone chaperones have their own specific target genes, and various histone chaperones have partially overlapping functions during transcriptional regulation. The histone deacetylase inhibitor TSA and histone chaperones Asf1, Vps75 and Rtt106 can function in parallel pathways to regulate transcription. Moreover, TSA can specifically antagonize histone chaperone Chz1-mediated telomere anti-silencing. This study demonstrates that a mutual cross-talk mechanism exists between histone chaperones and histone deacetylation in transcriptional regulation.
Subject(s)
Gene Expression Profiling , Histone Chaperones/antagonists & inhibitors , Hydroxamic Acids/pharmacology , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Telomere/genetics , Gene Expression Regulation, Fungal/drug effects , Gene Silencing/drug effects , Histone Deacetylases , Transcription, GeneticABSTRACT
Peroxisomes are intracellular organelles that house a number of diverse metabolic processes, notably those required for beta-oxidation of fatty acids. Peroxisomes biogenesis can be induced by the presence of peroxisome proliferators, including fatty acids, which activate complex cellular programs that underlie the induction process. Here, we used multi-parameter quantitative phenotype analyses of an arrayed mutant collection of yeast cells induced to proliferate peroxisomes, to establish a comprehensive inventory of genes required for peroxisome induction and function. The assays employed include growth in the presence of fatty acids, and confocal imaging and flow cytometry through the induction process. In addition to the classical phenotypes associated with loss of peroxisomal functions, these studies identified 169 genes required for robust signaling, transcription, normal peroxisomal development and morphologies, and transmission of peroxisomes to daughter cells. These gene products are localized throughout the cell, and many have indirect connections to peroxisome function. By integration with extant data sets, we present a total of 211 genes linked to peroxisome biogenesis and highlight the complex networks through which information flows during peroxisome biogenesis and function.
Subject(s)
Genomics , Peroxisomes/metabolism , Computational Biology , Databases, Factual , Flow Cytometry , Fungal Proteins/metabolism , Genes, Fungal/genetics , Protein Transport , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
Stable incorporation of labeled amino acids in cell culture is a simple approach to label proteins in vivo for mass spectrometric quantification. Full incorporation of isotopically heavy amino acids facilitates accurate quantification of proteins from different cultures, yet analysis methods for determination of incorporation are cumbersome and time-consuming. We present QTIPS, Quantification by Total Identified Peptides for SILAC, a straightforward, accurate method to determine the level of heavy amino acid incorporation throughout a population of peptides detected by mass spectrometry. Using QTIPS, we show that the incorporation of heavy amino acids in baker's yeast is unaffected by the use of prototrophic strains, indicating that auxotrophy is not a requirement for SILAC experiments in this organism. This method has general utility for multiple applications where isotopic labeling is used for quantification in mass spectrometry.
Subject(s)
Amino Acids/chemistry , Isotope Labeling/methods , Isotopes/chemistry , Peptide Fragments/chemistry , Amino Acid Sequence , Amino Acids/metabolism , Mass Spectrometry , Metabolic Networks and Pathways , Molecular Sequence Data , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolismABSTRACT
Phosphorylation of proteins is a key posttranslational modification in cellular signaling, regulating many aspects of cellular responses. We used a quantitative, integrated, phosphoproteomics approach to characterize the cellular responses of the yeast Saccharomyces cerevisiae to the fatty acid oleic acid, a molecule with broad human health implications and a potent inducer of peroxisomes. A combination of cryolysis and urea solubilization was used to minimize the opportunity for reorientation of the phosphoproteome, and hydrophilic interaction liquid chromatography and IMAC chemistries were used to fractionate and enrich for phosphopeptides. Using these approaches, numerous phosphorylated peptides specific to oleate-induced and glucose-repressed conditions were identified and mapped to known signaling pathways. These include several transcription factors, two of which, Pip2p and Cst6p, must be phosphorylated for the normal transcriptional response of fatty acid-responsive loci encoding peroxisomal proteins. The phosphoproteome data were integrated with results from genome-wide assays studying the effects of signaling molecule deletions and known protein-protein interactions to generate a putative fatty acid-responsive signaling network. In this network, the most highly connected nodes are those with the largest effects on cellular responses to oleic acid. These properties are consistent with a scale-free topology, demonstrating that scale-free properties are conserved in condition-specific networks.
Subject(s)
Peroxisomes , Phosphoproteins/metabolism , Proteomics , Saccharomyces cerevisiae/metabolism , Signal Transduction , Mass Spectrometry , Oligonucleotide Array Sequence AnalysisABSTRACT
Cell division depends critically on the temporally controlled assembly of mitotic spindles, which are responsible for the distribution of duplicated chromosomes to each of the two daughter cells. To gain insight into the process, Vizeacoumar et al., in this issue (Vizeacoumar et al. 2010. J. Cell Biol. doi:10.1083/jcb.200909013), have combined systems genetics with high-throughput and high-content imaging to comprehensively identify and classify novel components that contribute to the morphology and function of the mitotic spindle.
Subject(s)
Cytological Techniques/methods , Genetic Techniques , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/cytology , Spindle Apparatus/chemistry , Systems Biology/methods , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Spindle Apparatus/geneticsABSTRACT
Chz1p is a histone chaperone that interacts physically and functionally with the histone variant Htz1p, which has been implicated in establishing and maintaining boundaries between transcriptionally inactive heterochromatin and active euchromatin. To investigate the role of Chz1p in chromatin organization, we performed genome-wide expression arrays and chromatin immunoprecipitations of SIR complex components and modified histones in a CHZ1 deletion strain. Deletion of CHZ1 led to reduced ubiquitination of subtelomere-associated H2B, reduced subtelomeric H3K79 di-methylation, and increased binding of Sir3p, and Sir4p at telomere-distal euchromatin regions, correlating with decreased gene expression in subtelomeric regions. This anti-silencing defect appears to be mediated by enhanced association of de-ubiquitinase Ubp10p with subtelomeric DNA, as detected by chromatin immunoprecipitation analysis. In support of this, we show that deletion of UBP10 can antagonize the subtelomeric silencing phenotype of Deltachz1. Taken together, the results demonstrate a novel role for Chz1p in epigenetic regulation, through H2B de-ubiquitination by Ubp10p.
Subject(s)
Gene Expression Regulation, Fungal , Histone Chaperones/physiology , Histones/metabolism , Saccharomyces cerevisiae Proteins/physiology , Ubiquitination , Gene Deletion , Gene Silencing , Histone Chaperones/genetics , Histones/chemistry , Methylation , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Telomere/metabolism , Transcription, Genetic , Ubiquitin Thiolesterase/metabolismABSTRACT
This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis. Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics. Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.
Subject(s)
Oleic Acid/pharmacology , Phosphoproteins/metabolism , Proteomics/methods , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Phosphoproteins/analysis , Phosphoproteins/isolation & purification , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae Proteins/isolation & purificationABSTRACT
The histone variant H2A.Z (Htz1p) has been implicated in transcriptional regulation in numerous organisms, including Saccharomyces cerevisiae. Genome-wide transcriptome profiling and chromatin immunoprecipitation studies identified a role for Htz1p in the rapid and robust activation of many oleate-responsive genes encoding peroxisomal proteins, in particular POT1, POX1, FOX2, and CTA1. The Swr1p-, Gcn5p-, and Chz1p-dependent association of Htz1p with these promoters in their repressed states appears to establish an epigenetic marker for the rapid and strong expression of these highly inducible promoters. Isw2p also plays a role in establishing the nucleosome state of these promoters and associates stably in the absence of Htz1p. An analysis of the nucleosome dynamics and Htz1p association with these promoters suggests a complex mechanism in which Htz1p-containing nucleosomes at fatty acid-responsive promoters are disassembled upon initial exposure to oleic acid leading to the loss of Htz1p from the promoter. These nucleosomes reassemble at later stages of gene expression. While these new nucleosomes do not incorporate Htz1p, the initial presence of Htz1p appears to mark the promoter for sustained gene expression and the recruitment of TATA-binding protein.