PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-15 (15)
 

Clipboard (0)
None

Select a Filter Below

Journals
more »
Year of Publication
Document Types
author:("Wan, yakut")
1.  Streptavidin-biotin-based directional double Nanobody sandwich ELISA for clinical rapid and sensitive detection of influenza H5N1 
Background
Influenza H5N1 is one subtype of the influenza A virus which can infect human bodies and lead to death. Timely diagnosis before its breakout is vital to the human health. The current clinical biochemical diagnosis for influenza virus are still flawed, and the diagnostic kits of H5N1 are mainly based on traditional monoclonal antibodies that hardly meet the requirements for clinical applications. Nanobody is a promising tool for diagnostics and treatment due to its smallest size, high specificity and stability. In this study, a novel Nanobody-based bioassay was developed for rapid, low-cost and sensitive detection of the influenza H5N1 virus.
Methods
Nanobodies specific to H5N1 virus were selected from a VHH library by phage display technology. In this system, the biotinylated Nanobody was directionally captured by streptavidin coated on ELISA plate, which can specifically capture the H5N1 virus. Another Nanobody conjugated with HRP was used as a detector. A novel directional enzyme-linked immunosorbent assay for H5N1 using specific Nanobodies was established and compared to the conventional undirected ELISA assay.
Results
We have successfully constructed a high quality phage display Nanobody library and isolated two Nanobodies against H5N1 with high affinity and specificity. These two Nanobodies were further used to prepare the biosensor detection system. This streptavidin-biotin-based directional double Nanobodies sandwich ELISA for H5N1 detection showed superiority over the commonly undirectional ELISA protocol. The linear range of detection for standards in this immunoassay was approximately 50–1000 ng/mL and the detection limit was 14.1 ng/mL. The average recoveries of H5N1 virus from human serum samples were in the range from 94.58% to 114.51%, with a coefficient of variation less than 6.5%.
Conclusion
Collectively, these results demonstrated that the proposed detection system is an alternative diagnostic tool that enables a rapid, inexpensive, sensitive and specific detection of the influenza virus.
doi:10.1186/s12967-014-0352-5
PMCID: PMC4274719  PMID: 25526777
Nanobody; Influenza H5N1 virus; Directional; Biotinylation; Streptavidin
2.  Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications 
Background
Nanobodies (Nbs) have proved their great value as therapeutic molecules and clinical diagnostic tools. Although the routine procedure to obtain Nbs is to immunize camels with antigens, it is unavailable to immunize a camel when the antigens are highly toxic, pathogenic or nonimmunogenic. A synthetic phage display library is an alternative to generate Nbs against such targets, besides all the other ones.
Methods
We constructed a large and diverse synthetic phage display Nanobody (Nb) library based on the conserved camel single-domain antibody fragment (VHH) framework of cAbBCII10. Diversity was introduced in the complementarity-determining region 3 (CDR3) by means of randomization of synthetic oligonucleotides. Then human prealbumin (PA) and neutrophil gelatinase-associated lipocalin (NGAL) were used to select specific Nbs from this library. Furthermore, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect PA based on horseradish peroxidase (HRP)-conjugated anti-PA Nb isolated from this study and another biotinylated anti-PA Nb obtained from an immune library, in our previous study.
Results
A large and diverse synthetic phage display Nb library with CDR3 regions randomized by trinucleotide cassettes was constructed. The library size was 1.65 × 109 CFU/mL and the correct insertion ratio was nearly 100%. A Nb against human PA and against NGAL was successfully isolated from the synthetic library. The obtained anti-PA Nb was effectively used to develop a sandwich ELISA for PA detection and it demonstrated a working range from 50 to 1000 ng/mL, with a limit of detection (LOD) of 27.1 ng/mL.
Conclusion
This proposed novel synthetic library was a good source for obtaining some antigen-specific Nbs. This approach could provide crucial support to an immune library and a naïve library in the acquisition of specific Nbs, potentially functioning as a great resource for medical diagnostic applications. In addition, we have successfully developed a novel sandwich ELISA to detect PA, which could provide great assistance for clinical PA detection.
doi:10.1186/s12967-014-0343-6
PMCID: PMC4269866  PMID: 25496223
Nanobody; Trinucleotide cassettes; Human prealbumin; Phage display; Diagnostic application
3.  Uniform Orientation of Biotinylated Nanobody as an Affinity Binder for Detection of Bacillus thuringiensis (Bt) Cry1Ac Toxin 
Toxins  2014;6(12):3208-3222.
Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies) sandwich-ELISA (DAS-ELISA) assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac) were selected as capture antibody (Nb61) and detection antibody (Nb44). The capture antibody (Nb61) was biotinylated in vivo for directional immobilization on wells coated with streptavidin matrix. Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 μg·mL−1 and a working range 0.010–1.0 μg·mL−1. The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system.
doi:10.3390/toxins6123208
PMCID: PMC4280530  PMID: 25474492
nanobody; Cry1Ac toxin; streptavidin; DAS-ELISA
4.  Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2 
Nanoscale Research Letters  2014;9(1):528.
Our objective is to develop a rapid and sensitive assay based on magnetic beads to detect the concentration of influenza H3N2. The possibility of using variable domain heavy-chain antibodies (nanobody) as diagnostic tools for influenza H3N2 was investigated. A healthy camel was immunized with inactivated influenza H3N2. A nanobody library of 8 × 108 clones was constructed and phage displayed. After three successive biopanning steps, H3N2-specific nanobodies were successfully isolated, expressed in Escherichia coli, and purified. Sequence analysis of the nanobodies revealed that we possessed four classes of nanobodies against H3N2. Two nanobodies were further used to prepare our rapid diagnostic kit. Biotinylated nanobody was effectively immobilized onto the surface of streptavidin magnetic beads. The modified magnetic beads with nanobody capture specifically influenza H3N2 and can still be recognized by nanobodies conjugated to horseradish peroxidase (HRP) conjugates. Under optimized conditions, the present immunoassay exhibited a relatively high sensitive detection with a limit of 50 ng/mL. In conclusion, by combining magnetic beads with specific nanobodies, this assay provides a promising influenza detection assay to develop a potential rapid, sensitive, and low-cost diagnostic tool to screen for influenza infections.
doi:10.1186/1556-276X-9-528
PMCID: PMC4199786  PMID: 25328501
Influenza A grade 2 (H3N2); Nanobody; Magnetic nanoparticles; Biotinylation
5.  Distinct roles for histone chaperones in the deposition of Htz1 in chromatin 
Bioscience Reports  2014;34(5):e00139.
Histone variant Htz1 substitution for H2A plays important roles in diverse DNA transactions. Histone chaperones Chz1 and Nap1 (nucleosome assembly protein 1) are important for the deposition Htz1 into nucleosomes. In literatures, it was suggested that Chz1 is a Htz1–H2B-specific chaperone, and it is relatively unstructured in solution but it becomes structured in complex with the Htz1–H2B histone dimer. Nap1 (nucleosome assembly protein 1) can bind (H3–H4)2 tetramers, H2A–H2B dimers and Htz1–H2B dimers. Nap1 can bind H2A–H2B dimer in the cytoplasm and shuttles the dimer into the nucleus. Moreover, Nap1 functions in nucleosome assembly by competitively interacting with non-nucleosomal histone–DNA. However, the exact roles of these chaperones in assembling Htz1-containing nucleosome remain largely unknown. In this paper, we revealed that Chz1 does not show a physical interaction with chromatin. In contrast, Nap1 binds exactly at the genomic DNA that contains Htz1. Nap1 and Htz1 show a preferential interaction with AG-rich DNA sequences. Deletion of chz1 results in a significantly decreased binding of Htz1 in chromatin, whereas deletion of nap1 dramatically increases the association of Htz1 with chromatin. Furthermore, genome-wide nucleosome-mapping analysis revealed that nucleosome occupancy for Htz1p-bound genes decreases upon deleting htz1 or chz1, suggesting that Htz1 is required for nucleosome structure at the specific genome loci. All together, these results define the distinct roles for histone chaperones Chz1 and Nap1 to regulate Htz1 incorporation into chromatin.
doi:10.1042/BSR20140092
PMCID: PMC4168335  PMID: 25338502
histone chaperone; Htz1 deposition; nucleosome structure; ChIP-chip, chromatin immunoprecipitation and tiling microarray chips; GO, gene ontology; HA, haemagglutinin; HAT, histone acetyltransferase; Nap1, nucleosome assembly protein 1; NFR or NDR, nucleosome-free (depleted) region; PCC, Pearson correlation coefficient; Pol II, polymerase II; TSS, transcription start sites; TTS, transcription termination site
6.  Molecular mechanisms of system responses to novel stimuli are predictable from public data 
Nucleic Acids Research  2013;42(3):1442-1460.
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 ∼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.
doi:10.1093/nar/gkt938
PMCID: PMC3919619  PMID: 24185701
7.  A role for the nucleoporin Nup170p in chromatin structure and gene silencing 
Cell  2013;152(5):969-983.
Embedded in the nuclear envelope, nuclear pore complexes (NPCs) not only regulate nuclear transport, but also interface with transcriptionally active euchromatin, largely silenced heterochromatin, as well as the boundaries between these regions. It is unclear what functional role NPCs play in establishing or maintaining these distinct chromatin domains. We report that the yeast NPC protein Nup170p interacts with regions of the genome containing ribosomal protein and subtelomeric genes. Here, it functions in nucleosome positioning and as a repressor of transcription. We show that the role of Nup170p in subtelomeric gene silencing is linked to its association with the RSC chromatin-remodeling complex and the silencing factor Sir4p, and that the binding of Nup170p and Sir4p to subtelomeric chromatin is cooperative and necessary for the association of telomeres with the nuclear envelope. Our results establish the NPC as an active participant in silencing and the formation of peripheral heterochromatin.
doi:10.1016/j.cell.2013.01.049
PMCID: PMC3690833  PMID: 23452847
nuclear pore complex; chromatin-remodeling; telomere; heterochromatin; ribosomal protein genes; RSC; Sth1p; Sir4p; Rap1p; Nup170p
8.  NUP-1 Is a Large Coiled-Coil Nucleoskeletal Protein in Trypanosomes with Lamin-Like Functions 
PLoS Biology  2012;10(3):e1001287.
NUP1, the first example of a nuclear lamin analog in nonmetazoans, performs roles similar to those of lamins in maintaining the structure and organization of the nucleus in Trypanosoma brucei.
A unifying feature of eukaryotic nuclear organization is genome segregation into transcriptionally active euchromatin and transcriptionally repressed heterochromatin. In metazoa, lamin proteins preserve nuclear integrity and higher order heterochromatin organization at the nuclear periphery, but no non-metazoan lamin orthologues have been identified, despite the likely presence of nucleoskeletal elements in many lineages. This suggests a metazoan-specific origin for lamins, and therefore that distinct protein elements must compose the nucleoskeleton in other lineages. The trypanosomatids are highly divergent organisms and possess well-documented but remarkably distinct mechanisms for control of gene expression, including polycistronic transcription and trans-splicing. NUP-1 is a large protein localizing to the nuclear periphery of Trypanosoma brucei and a candidate nucleoskeletal component. We sought to determine if NUP-1 mediates heterochromatin organization and gene regulation at the nuclear periphery by examining the influence of NUP-1 knockdown on morphology, chromatin positioning, and transcription. We demonstrate that NUP-1 is essential and part of a stable network at the inner face of the trypanosome nuclear envelope, since knockdown cells have abnormally shaped nuclei with compromised structural integrity. NUP-1 knockdown also disrupts organization of nuclear pore complexes and chromosomes. Most significantly, we find that NUP-1 is required to maintain the silenced state of developmentally regulated genes at the nuclear periphery; NUP-1 knockdown results in highly specific mis-regulation of telomere-proximal silenced variant surface glycoprotein (VSG) expression sites and procyclin loci, indicating a disruption to normal chromatin organization essential to life-cycle progression. Further, NUP-1 depletion leads to increased VSG switching and therefore appears to have a role in control of antigenic variation. Thus, analogous to vertebrate lamins, NUP-1 is a major component of the nucleoskeleton with key roles in organization of the nuclear periphery, heterochromatin, and epigenetic control of developmentally regulated loci.
Author Summary
Eukaryotes—fungi, plants, animals, and many unicellular organisms—are defined by the presence of a cell nucleus that contains the chromosomes and is enveloped by a lipid membrane lined on the inner face with a protein network called the lamina. Among other functions, the lamina serves as an anchorage site for the ends of chromosomes. In multicellular animals (metazoa), the lamina comprises a few related proteins called lamins, which are very important for many functions related to the nucleus; abnormal lamins result in multiple nuclear defects and diseases, including inappropriate gene expression and premature aging. Until now, however, lamins had been found only in metazoa; no protein of equivalent function had been identified in plants, fungi, or unicellular organisms. Here, we describe a protein from African trypanosomes—the single-cell parasites that cause sleeping sickness—that fulfils many lamin-like roles, including maintaining nuclear structure and organizing the chromosomes of this organism. We show that this protein, which we call NUP-1 for nuclear periphery protein-1, is vital for the antigenic variation mechanisms that allow the parasite to escape the host immune response. We propose that NUP-1 is a lamin analogue that performs similar functions in trypanosomes to those of authentic lamins in metazoa. These findings, we believe, have important implications for understanding the evolution of the nucleus.
doi:10.1371/journal.pbio.1001287
PMCID: PMC3313915  PMID: 22479148
9.  Role of the nuclear envelope on genome organization and gene expression 
doi:10.1002/wsbm.101
PMCID: PMC3050641  PMID: 21305702
Nuclear pore; genome organization; gene expression; boundary activity
10.  A regression model approach to enable cell morphology correction in high-throughput flow cytometry 
Large variations in cell size and shape can undermine traditional gating methods for analyzing flow cytometry data. Correcting for these effects enables analysis of high-throughput data sets, including >5000 yeast samples with diverse cell morphologies.
The regression model approach corrects for the effects of cell morphology on fluorescence, as well as an extremely small and restrictive gate, but without removing any of the cells.In contrast to traditional gating, this approach enables the quantitative analysis of high-throughput flow cytometry experiments, since the regression model can compare between biological samples that show no or little overlap in terms of the morphology of the cells.The analysis of a high-throughput yeast flow cytometry data set consisting of >5000 biological samples identified key proteins that affect the time and intensity of the bifurcation event that happens after the carbon source transition from glucose to fatty acids. Here, some yeast cells undergo major structural changes, while others do not.
Flow cytometry is a widely used technique that enables the measurement of different optical properties of individual cells within large populations of cells in a fast and automated manner. For example, by targeting cell-specific markers with fluorescent probes, flow cytometry is used to identify (and isolate) cell types within complex mixtures of cells. In addition, fluorescence reporters can be used in conjunction with flow cytometry to measure protein, RNA or DNA concentration within single cells of a population.
One of the biggest advantages of this technique is that it provides information of how each cell behaves instead of just measuring the population average. This can be essential when analyzing complex samples that consist of diverse cell types or when measuring cellular responses to stimuli. For example, there is an important difference between a 50% expression increase of all cells in a population after stimulation and a 100% increase in only half of the cells, while the other half remains unresponsive. Another important advantage of flow cytometry is automation, which enables high-throughput studies with thousands of samples and conditions. However, current methods are confounded by populations of cells that are non-uniform in terms of size and granularity. Such variability affects the emitted fluorescence of the cell and adds undesired variability when estimating population fluorescence. This effect also frustrates a sensible comparison between conditions, where not only fluorescence but also cell size and granularity may be affected.
Traditionally, this problem has been addressed by using ‘gates' that restrict the analysis to cells with similar morphological properties (i.e. cell size and cell granularity). Because cells inside the gate are morphologically similar to one another, they will show a smaller variability in their response within the population. Moreover, applying the same gate in all samples assures that observed differences between these samples are not due to differential cell morphologies.
Gating, however, comes with costs. First, since only a subgroup of cells is selected, the final number of cells analyzed can be significantly reduced. This means that in order to have sufficient statistical power, more cells have to be acquired, which, if even possible in the first place, increases the time and cost of the experiment. Second, finding a good gate for all samples and conditions can be challenging if not impossible, especially in cases where cellular morphology changes dramatically between conditions. Finally, gating is a very user-dependent process, where both the size and shape of the gate are determined by the researcher and will affect the outcome, introducing subjectivity in the analysis that complicates reproducibility.
In this paper, we present an alternative method to gating that addresses the issues stated above. The method is based on a regression model containing linear and non-linear terms that estimates and corrects for the effect of cell size and granularity on the observed fluorescence of each cell in a sample. The corrected fluorescence thus becomes ‘free' of the morphological effects.
Because the model uses all cells in the sample, it assures that the corrected fluorescence is an accurate representation of the sample. In addition, the regression model can predict the expected fluorescence of a sample in areas where there are no cells. This makes it possible to compare between samples that have little overlap with good confidence. Furthermore, because the regression model is automated, it is fully reproducible between labs and conditions. Finally, it allows for a rapid analysis of big data sets containing thousands of samples.
To probe the validity of the model, we performed several experiments. We show how the regression model is able to remove the morphological-associated variability as well as an extremely small and restrictive gate, but without the caveat of removing cells. We test the method in different organisms (yeast and human) and applications (protein level detection, separation of mixed subpopulations). We then apply this method to unveil new biological insights in the mechanistic processes involved in transcriptional noise.
Gene transcription is a process subjected to the randomness intrinsic to any molecular event. Although such randomness may seem to be undesirable for the cell, since it prevents consistent behavior, there are situations where some degree of randomness is beneficial (e.g. bet hedging). For this reason, each gene is tuned to exhibit different levels of randomness or noise depending on its functions. For core and essential genes, the cell has developed mechanisms to lower the level of noise, while for genes involved in the response to stress, the variability is greater.
This gene transcription tuning can be determined at many levels, from the architecture of the transcriptional network, to epigenetic regulation. In our study, we analyze the latter using the response of yeast to the presence of fatty acid in the environment. Fatty acid can be used as energy by yeast, but it requires major structural changes and commitments. We have observed that at the population level, there is a bifurcation event whereby some cells undergo these changes and others do not. We have analyzed this bifurcation event in mutants for all the non-essential epigenetic regulators in yeast and identified key proteins that affect the time and intensity of this bifurcation. Even though fatty acid triggers major morphological changes in the cell, the regression model still makes it possible to analyze the over 5000 flow cytometry samples in this data set in an automated manner, whereas a traditional gating approach would be impossible.
Cells exposed to stimuli exhibit a wide range of responses ensuring phenotypic variability across the population. Such single cell behavior is often examined by flow cytometry; however, gating procedures typically employed to select a small subpopulation of cells with similar morphological characteristics make it difficult, even impossible, to quantitatively compare cells across a large variety of experimental conditions because these conditions can lead to profound morphological variations. To overcome these limitations, we developed a regression approach to correct for variability in fluorescence intensity due to differences in cell size and granularity without discarding any of the cells, which gating ipso facto does. This approach enables quantitative studies of cellular heterogeneity and transcriptional noise in high-throughput experiments involving thousands of samples. We used this approach to analyze a library of yeast knockout strains and reveal genes required for the population to establish a bimodal response to oleic acid induction. We identify a group of epigenetic regulators and nucleoporins that, by maintaining an ‘unresponsive population,' may provide the population with the advantage of diversified bet hedging.
doi:10.1038/msb.2011.64
PMCID: PMC3202802  PMID: 21952134
flow cytometry; high-throughput experiments; statistical regression model; transcriptional noise
11.  Environment-responsive transcription factors bind subtelomeric elements and regulate gene silencing 
Chromosome position analysis of ChIP-chip data revealed that several carbon source and stress-responsive yeast transcription factors conditionally bind subtelomeric X elements.Integration of several microarray gene expression data sets showed that, in this context, the factors conditionally control the boundaries and strength of subtelomeric silencing.Regulation of silencing by a fatty acid-responsive factor was found to be dependent on Sir2p and independent of Hda1p.These findings provide a critical link for establishing the mechanisms by which telomere biology is coordinated with other cellular processes including responses to environmental stimuli, aging and adaptation.
It is well established that environmental conditions modulate gene expression through local binding of a variety of conditionally active transcription factors, each responsive to specific environmental cues. However, another prevalent mechanism of gene regulation in eukaryotic cells is the long-range control of groups of genes by chromatin modifications or other position-dependent mechanisms. One such phenomenon, gene silencing, is an important and evolutionarily conserved mode of regulation that controls expression of subtelomeric genes. These genes are enriched for stress response and metabolic genes and their regulation is controlled by the spreading of silencing molecules from chromosome ends (telomeres) into subtelomeric regions. Levels of subtelomeric silencing have been linked to cellular lifespan, and study of the regulation of silencing is fundamental to our understanding of human aging. The spread of silencing in subtelomeric regions is discontinuous, and is controlled by various genomic elements that can either relay and enhance silencing from telomeres (proto-silencing) or create boundaries that protect some genomic regions from silencing. In yeast, every subtelomeric region contains an X element that proto-silences centromere-proximal genes, and also insulates telomere-proximal genes from silencing.
In this paper, we identify a regulatory mechanism to control X element-mediated proto-silencing and insulating activities in response to environmental cues. The mechanism was identified using chromosome position analysis of microarray-based chromatin immunoprecipitation (ChIP-chip) data for environment-responsive TFs and genome-wide gene expression data under the same conditions. The mechanism involves the conditional association of environment-responsive transcription factors to X elements. The binding at X elements results in regulation of proto-silencing of centromere-proximal genes, or insulation of telomere-proximal genes (depending on the factor) in response to environmental stimuli related to stress response and metabolism. One example is shown below (Figure 4B). Transcription factor, Oaf1p, conditionally binds X elements in the presence of fatty acids and enhances proto-silencing specifically under this condition. Oaf1p and several other factors implicated here are known to control adjacent genes at intrachromosomal positions, suggesting their dual functionality in both gene-specific transcriptional regulation, and long-range position-dependent mechanism. Investigation of this mechanism during the response to fatty acid exposure showed that conditional proto-silencing activity is dependent on Sir2p, a molecule known to be involved in subtelomeric silencing related to aging. This study reveals a path cells can use to coordinate subtelomeric silencing related to aging with cellular environment, and with the activities of other cellular processes.
Subtelomeric chromatin is subject to evolutionarily conserved complex epigenetic regulation and is implicated in numerous aspects of cellular function including formation of heterochromatin, regulation of stress response pathways and control of lifespan. Subtelomeric DNA is characterized by the presence of specific repeated segments that serve to propagate silencing or to protect chromosomal regions from spreading epigenetic control. In this study, analysis of genome-wide chromatin immunoprecipitation and expression data, suggests that several yeast transcription factors regulate subtelomeric silencing in response to various environmental stimuli through conditional association with proto-silencing regions called X elements. In this context, Oaf1p, Rox1p, Gzf1p and Phd1p control the propagation of silencing toward centromeres in response to stimuli affecting stress responses and metabolism, whereas others, including Adr1p, Yap5p and Msn4p, appear to influence boundaries of silencing, regulating telomere-proximal genes in Y′ elements. The factors implicated here are known to control adjacent genes at intrachromosomal positions, suggesting their dual functionality. This study reveals a path for the coordination of subtelomeric silencing with cellular environment, and with activities of other cellular processes.
doi:10.1038/msb.2010.110
PMCID: PMC3049408  PMID: 21206489
chromatin; proto-silencer; Sir2; subtelomeric silencing; X element
12.  Genome-Wide Analysis of Effectors of Peroxisome Biogenesis 
PLoS ONE  2010;5(8):e11953.
Peroxisomes are intracellular organelles that house a number of diverse metabolic processes, notably those required for β-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.
doi:10.1371/journal.pone.0011953
PMCID: PMC2915925  PMID: 20694151
13.  Histone chaperone Chz1p regulates H2B ubiquitination and subtelomeric anti-silencing 
Nucleic Acids Research  2009;38(5):1431-1440.
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 Δchz1. Taken together, the results demonstrate a novel role for Chz1p in epigenetic regulation, through H2B de-ubiquitination by Ubp10p.
doi:10.1093/nar/gkp1099
PMCID: PMC2836552  PMID: 20008511
14.  Role of the Histone Variant H2A.Z/Htz1p in TBP Recruitment, Chromatin Dynamics, and Regulated Expression of Oleate-Responsive Genes▿  
Molecular and Cellular Biology  2009;29(9):2346-2358.
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.
doi:10.1128/MCB.01233-08
PMCID: PMC2668375  PMID: 19273605
15.  Molecular architecture of the kinetochore-microtubule attachment site is conserved between point and regional centromeres 
The Journal of Cell Biology  2008;181(4):587-594.
Point and regional centromeres specify a unique site on each chromosome for kinetochore assembly. The point centromere in budding yeast is a unique 150-bp DNA sequence, which supports a kinetochore with only one microtubule attachment. In contrast, regional centromeres are complex in architecture, can be up to 5 Mb in length, and typically support many kinetochore-microtubule attachments. We used quantitative fluorescence microscopy to count the number of core structural kinetochore protein complexes at the regional centromeres in fission yeast and Candida albicans. We find that the number of CENP-A nucleosomes at these centromeres reflects the number of kinetochore-microtubule attachments instead of their length. The numbers of kinetochore protein complexes per microtubule attachment are nearly identical to the numbers in a budding yeast kinetochore. These findings reveal that kinetochores with multiple microtubule attachments are mainly built by repeating a conserved structural subunit that is equivalent to a single microtubule attachment site.
doi:10.1083/jcb.200803027
PMCID: PMC2386099  PMID: 18474626

Results 1-15 (15)