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1.  PhosphoChain: a novel algorithm to predict kinase and phosphatase networks from high-throughput expression data 
Bioinformatics  2013;29(19):2435-2444.
Motivation: Protein phosphorylation is critical for regulating cellular activities by controlling protein activities, localization and turnover, and by transmitting information within cells through signaling networks. However, predictions of protein phosphorylation and signaling networks remain a significant challenge, lagging behind predictions of transcriptional regulatory networks into which they often feed.
Results: We developed PhosphoChain to predict kinases, phosphatases and chains of phosphorylation events in signaling networks by combining mRNA expression levels of regulators and targets with a motif detection algorithm and optional prior information. PhosphoChain correctly reconstructed ∼78% of the yeast mitogen-activated protein kinase pathway from publicly available data. When tested on yeast phosphoproteomic data from large-scale mass spectrometry experiments, PhosphoChain correctly identified ∼27% more phosphorylation sites than existing motif detection tools (NetPhosYeast and GPS2.0), and predictions of kinase–phosphatase interactions overlapped with ∼59% of known interactions present in yeast databases. PhosphoChain provides a valuable framework for predicting condition-specific phosphorylation events from high-throughput data.
Availability: PhosphoChain is implemented in Java and available at http://virgo.csie.ncku.edu.tw/PhosphoChain/ or http://aitchisonlab.com/PhosphoChain
Contact: john.aitchison@systemsbiology.org or jchiang@mail.ncku.edu.tw
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btt387
PMCID: PMC3777105  PMID: 23832245
2.  Nuclear pore complex evolution: a trypanosome Mlp analogue functions in chromosomal segregation but lacks transcriptional barrier activity 
Molecular Biology of the Cell  2014;25(9):1421-1436.
The nuclear face of the nuclear pore complex (NPC) interfaces with chromatin, transcription, and transport intermediates. A novel architecture for the nuclear face of the trypanosome NPC provides insights into NPC function and evolution.
The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organization. In many eukaryotes the coiled-coil Mlp/Tpr proteins of the NPC nuclear basket have specific functions in interactions with chromatin and defining specialized regions of active transcription, whereas Mlp2 associates with the mitotic spindle/NPC in a cell cycle–dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and lowered fidelity of telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site but apparently has minimal roles in control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analogue of Mlp/Tpr proteins, and, together with the lamina analogue NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina.
doi:10.1091/mbc.E13-12-0750
PMCID: PMC4004592  PMID: 24600046
3.  Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity 
Nature  2013;505(7485):691-695.
The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors1–3. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses4–13. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.
doi:10.1038/nature12862
PMCID: PMC4077721  PMID: 24284630
4.  Peroxisomes take shape 
Peroxisomes carry out various oxidative reactions that are tightly regulated to adapt to the changing needs of the cell and varying external environments. Accordingly, they are remarkably fluid and can change dramatically in abundance, size, shape and content in response to numerous cues. These dynamics are controlled by multiple aspects of peroxisome biogenesis that are coordinately regulated with each other and with other cellular processes. Ongoing studies are deciphering the diverse molecular mechanisms that underlie biogenesis and how they cooperate to dynamically control peroxisome utility. These important challenges should lead to an understanding of peroxisome dynamics that can be capitalized upon for bioengineering and the development of therapies to improve human health.
doi:10.1038/nrm3700
PMCID: PMC4060825  PMID: 24263361
5.  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
6.  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
7.  Multifunctional Double-negative T Cells in Sooty Mangabeys Mediate T-helper Functions Irrespective of SIV Infection 
PLoS Pathogens  2013;9(6):e1003441.
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.
Author Summary
SIV infection of sooty mangabeys is generally characterized by maintained CD4 T cell levels and a lack of disease progression despite active virus replication. We have previously shown however, that dramatic loss of CD4 T cells can occur during SIV infection of mangabeys. This study investigates the potential for double negative (DN) T cells (which lack CD4 and CD8, and are refractory to SIV/HIV infection) to perform helper T cell functions. In our study, sooty mangabey DN T cells exhibited a memory phenotype and a diverse repertoire in their T cell receptors. Once stimulated, the DN T cells expressed multiple cytokines, indicating that they have the potential to function as helper T cells (a function normally undertaken by CD4+ T cells). In SIV infected mangabeys, DN T cells continue to function, proliferate in vivo, and maintain expression of homeostatic cytokine receptors on their surface. It is therefore likely that DN T cells have the potential to compensate for the loss of CD4 T cells during SIV infection. These studies indicate that increasing DN T cell levels and/or function during pathogenic HIV infection may provide one tangible component of a functional cure, and inhibit progression to clinical disease and AIDS
doi:10.1371/journal.ppat.1003441
PMCID: PMC3694849  PMID: 23825945
8.  A FOXO3/IRF7 gene regulatory circuit limits inflammatory sequelae of antiviral responses 
Nature  2012;490(7420):421-425.
Antiviral responses must be tightly regulated to rapidly defend against infection while minimizing inflammatory damage. Type 1 interferons (IFN-I) are crucial mediators of antiviral responses1 and their transcription is regulated by a variety of transcription factors2; principal amongst these is the family of interferon regulatory factors (IRFs)3. The IRF gene regulatory networks are complex and contain multiple feedback loops. The tools of systems biology are well suited to elucidate the complex interactions that give rise to precise coordination of the interferon response. Here we have used an unbiased systems approach to predict that a member of the forkhead family of transcription factors, FOXO3, is a negative regulator of a subset of antiviral genes. This prediction was validated using macrophages isolated from Foxo3-null mice. Genome-wide location analysis combined with gene deletion studies identified the Irf7 gene as a critical target of FOXO3. FOXO3 was identified as a negative regulator of Irf7 transcription and we have further demonstrated that FOXO3, IRF7 and IFN-I form a coherent feed-forward regulatory circuit. Our data suggest that the FOXO3-IRF7 regulatory circuit represents a novel mechanism for establishing the requisite set points in the interferon pathway that balances the beneficial effects and deleterious sequelae of the antiviral response.
doi:10.1038/nature11428
PMCID: PMC3556990  PMID: 22982991
9.  QTIPS: A novel method of unsupervised determination of isotopic amino acid distribution in SILAC experiments 
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.
doi:10.1016/j.jasms.2010.04.002
PMCID: PMC2914207  PMID: 20451407
QTIPS; SILAC; auxotrophy; yeast
10.  Metallochaperones Regulate Intracellular Copper Levels 
PLoS Computational Biology  2013;9(1):e1002880.
Copper (Cu) is an important enzyme co-factor that is also extremely toxic at high intracellular concentrations, making active efflux mechanisms essential for preventing Cu accumulation. Here, we have investigated the mechanistic role of metallochaperones in regulating Cu efflux. We have constructed a computational model of Cu trafficking and efflux based on systems analysis of the Cu stress response of Halobacterium salinarum. We have validated several model predictions via assays of transcriptional dynamics and intracellular Cu levels, discovering a completely novel function for metallochaperones. We demonstrate that in addition to trafficking Cu ions, metallochaperones also function as buffers to modulate the transcriptional responsiveness and efficacy of Cu efflux. This buffering function of metallochaperones ultimately sets the upper limit for intracellular Cu levels and provides a mechanistic explanation for previously observed Cu metallochaperone mutation phenotypes.
Author Summary
Copper (Cu) toxicity is a problem of medical, agricultural, and environmental significance. Cu toxicity severely inhibits growth of plant roots significantly affecting their morphology; Cu overload also accounts for some of the most common metal-metabolism abnormalities and neuropsychiatric problems including Wilson's and Menkes diseases. There is a large body of literature on how Cu enters and exits the cell; the kinetic and structural details of Cu translocation between trafficking, sensing, metabolic, and pumping proteins; and phenotypes associated with defects in metalloregulatory and efflux functions. Although the role of metallochaperones in Cu-cytotoxicity has been poorly studied, it has been observed that in animals deletion of metallochaperones results in elevated intracellular Cu levels along with overexpression of the P1-type ATPase efflux pump, ultimately causing malformation with high mortality. These observations are mechanistically explained by a predictive model of the Cu circuit in Halobacterium salinarum, which serves as an excellent model system for Cu trafficking and regulation in organisms with multiple chaperones. Constructed through iterative modeling and experimentation, this model accurately recapitulates known dynamical properties of the Cu circuit and predicts that intracellular Cu-buffering emerges as a consequence of the interplay of paralogous metallochaperones that traffic and allocate Cu to distinct targets.
doi:10.1371/journal.pcbi.1002880
PMCID: PMC3551603  PMID: 23349626
11.  Systems cell biology of the mitotic spindle 
The Journal of Cell Biology  2010;188(1):7-9.
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.
doi:10.1083/jcb.200912028
PMCID: PMC2812858  PMID: 20065087
12.  Asymmetric positive feedback loops reliably control biological responses 
A common regulatory motif, where a heterodimeric transcriptional regulator positively autoregulates only one of its components, is found to have particular properties that enable precise and robust control of cellular responses to environmental stimuli, providing an explanation for the prevalence of this motif in evolved regulatory networks.
Many important biological systems rely on regulation by dimers of proteins which upregulate the transcription of numerous targets, including one, and only one, of the dimer pair. This is termed asymmetric self-upregulation.ASymmetric Self-UpREgulated (ASSURE) networks confer rapid induction of their targets and their network behaviors are robust to parameter variation—both features appear to have contributed to the prevalence of the network across widely different biological systems.Likely evolutionary precursors to ASSURE networks are symmetrically self-upregulated network mediated by homodimers. In silico and experimental studies demonstrate that the ASSURE network confers a competitive advantage over its symmetrical counterpart.
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.
doi:10.1038/msb.2012.10
PMCID: PMC3361002  PMID: 22531117
heterodimer; kinetic model; positive feedback; regulatory network motif; robustness
13.  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
14.  PAcIFIC goes faster, quantitative and accurate 
Analytical chemistry  2011;83(6):2250-2257.
Data-dependent precursor ion selection is widely used in shotgun proteomics to profile the protein components of complex samples. Although very popular, this bottom-up method presents major drawbacks in terms of detectable dynamic range. Recently, we demonstrated the superior performance of a data-independent method we termed Peptide Acquisition Independent From Ion Count (PAcIFIC). Here, we report a faster, accurate, multiplexed and quantitative PAcIFIC method. Our results show that the time needed to perform such analysis can be decreased by 33% to 66% using modern ion trap instruments and that high mass accuracy can be applied to such a strategy. Quantification capability is demonstrated on protein standards and a whole bacterial cell lysate using isobaric tagging. Finally, we confirm in yeast the dynamic range capabilities of such a method where proteins down to less than 50 copies per cell can be monitored without sample pre-fractionation.
doi:10.1021/ac103079q
PMCID: PMC3217585  PMID: 21341720
15.  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
16.  The Yeast Nuclear Pore Complex and Transport Through It 
Genetics  2012;190(3):855-883.
Exchange of macromolecules between the nucleus and cytoplasm is a key regulatory event in the expression of a cell’s genome. This exchange requires a dedicated transport system: (1) nuclear pore complexes (NPCs), embedded in the nuclear envelope and composed of proteins termed nucleoporins (or “Nups”), and (2) nuclear transport factors that recognize the cargoes to be transported and ferry them across the NPCs. This transport is regulated at multiple levels, and the NPC itself also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes. Here we summarize how the yeast Saccharomyces has been used extensively as a model system to understand the fundamental and highly conserved features of this transport system, revealing the structure and function of the NPC; the NPC’s role in the regulation of gene expression; and the interactions of transport factors with their cargoes, regulatory factors, and specific nucleoporins.
doi:10.1534/genetics.111.127803
PMCID: PMC3296253  PMID: 22419078
17.  Genome-wide analysis of signaling networks regulating fatty acid–induced gene expression and organelle biogenesis 
The Journal of Cell Biology  2008;181(2):281-292.
Reversible phosphorylation is the most common posttranslational modification used in the regulation of cellular processes. This study of phosphatases and kinases required for peroxisome biogenesis is the first genome-wide analysis of phosphorylation events controlling organelle biogenesis. We evaluate signaling molecule deletion strains of the yeast Saccharomyces cerevisiae for presence of a green fluorescent protein chimera of peroxisomal thiolase, formation of peroxisomes, and peroxisome functionality. We find that distinct signaling networks involving glucose-mediated gene repression, derepression, oleate-mediated induction, and peroxisome formation promote stages of the biogenesis pathway. Additionally, separate classes of signaling proteins are responsible for the regulation of peroxisome number and size. These signaling networks specify the requirements of early and late events of peroxisome biogenesis. Among the numerous signaling proteins involved, Pho85p is exceptional, with functional involvements in both gene expression and peroxisome formation. Our study represents the first global study of signaling networks regulating the biogenesis of an organelle.
doi:10.1083/jcb.200710009
PMCID: PMC2315675  PMID: 18426976
18.  DETERMINISTIC AND STOCHASTIC MODELS OF GENETIC REGULATORY NETWORKS 
Methods in enzymology  2009;467:335-356.
Traditionally molecular biology research has tended to reduce biological pathways to composite units studied as isolated parts of the cellular system. With the advent of high throughput methodologies that can capture thousands of data points, and powerful computational approaches, the reality of studying cellular processes at a systems level is upon us. As these approaches yield massive datasets, systems level analyses have drawn upon other fields such as engineering and mathematics, adapting computational and statistical approaches to decipher relationships between molecules. Guided by high quality datasets and analyses, one can begin the process of predictive modeling. The findings from such approaches are often surprising and beyond normal intuition. We discuss four classes of dynamical systems used to model genetic regulatory networks. The discussion is divided into continuous and discrete models, as well as deterministic and stochastic model classes. For each combination of these categories, a model is presented and discussed in the context of the yeast cell cycle, illustrating how different types of questions can be addressed by different model classes.
doi:10.1016/S0076-6879(09)67013-0
PMCID: PMC3230268  PMID: 19897099
19.  The role of karyopherins in the regulated sumoylation of septins 
The Journal of Cell Biology  2007;177(1):39-49.
In the yeast Saccharomyces cerevisiae, several components of the septin ring are sumoylated during anaphase and then abruptly desumoylated at cytokinesis. We show that septin sumoylation is controlled by the interactions of two enzymes of the sumoylation pathway, Siz1p and Ulp1p, with the nuclear transport machinery. The E3 ligase Siz1p is imported into the nucleus by the karyopherin Kap95p during interphase. In M phase, Siz1p is exported from the nucleus by the karyopherin Kap142p/Msn5p and subsequently targeted to the septin ring, where it participates in septin sumoylation. We also show that the accumulation of sumoylated septins during mitosis is dependent on the interactions of the SUMO isopeptidase Ulp1p with Kap121p and Kap95p–Kap60p and the nuclear pore complex (NPC). In addition to sequestering Ulp1 at the NPC, Kap121p is required for targeting Ulp1p to the septin ring during mitosis. We present a model in which Ulp1p is maintained at the NPC during interphase and transiently interacts with the septin ring during mitosis.
doi:10.1083/jcb.200608066
PMCID: PMC2064105  PMID: 17403926
20.  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
21.  The peroxin Pex34p functions with the Pex11 family of peroxisomal divisional proteins to regulate the peroxisome population in yeast 
Molecular Biology of the Cell  2011;22(10):1727-1738.
Pex34p is a novel peroxisomal protein involved in controlling peroxisome abundance in Saccharomyces cerevisiae. Pex34p acts to control peroxisome numbers both alone and in cooperation with the Pex11 protein family of peroxisome divisional proteins.
Peroxisomes are ubiquitous organelles involved in diverse metabolic processes, most notably the metabolism of lipids and the detoxification of reactive oxygen species. Peroxisomes are highly dynamic and change in size and number in response to both intra- and extracellular cues. In the yeast Saccharomyces cerevisiae, peroxisome growth and division are controlled by both the differential import of soluble matrix proteins and a specialized divisional machinery that includes peroxisome-specific factors, such as members of the Pex11 protein family, and general organelle divisional factors, such as the dynamin-related protein Vps1p. Global yeast two-hybrid analyses have demonstrated interactions between the product of the S. cerevisiae gene of unknown function, YCL056c, and Pex proteins involved in peroxisome biogenesis. Here we show that the protein encoded by YCL056c, renamed Pex34p, is a peroxisomal integral membrane protein that acts independently and also in concert with the Pex11 protein family members Pex11p, Pex25p, and Pex27p to control the peroxisome populations of cells under conditions of both peroxisome proliferation and constitutive peroxisome division. Yeast two-hybrid analysis showed that Pex34p interacts physically with itself and with Pex11p, Pex25p, and Pex27p but not with Vps1p. Pex34p can act as a positive effector of peroxisome division as its overexpression leads to increased numbers of peroxisomes in wild type and pex34Δ cells. Pex34p requires the Pex11 family proteins to promote peroxisome division. Our discovery of Pex34p as a protein involved in the already complex control of peroxisome populations emphasizes the necessity of cells to strictly regulate their peroxisome populations to be able to respond appropriately to changing environmental conditions.
doi:10.1091/mbc.E11-01-0084
PMCID: PMC3093324  PMID: 21441307
22.  Trade-off between Responsiveness and Noise Suppression in Biomolecular System Responses to Environmental Cues 
PLoS Computational Biology  2011;7(6):e1002091.
When living systems detect changes in their external environment their response must be measured to balance the need to react appropriately with the need to remain stable, ignoring insignificant signals. Because this is a fundamental challenge of all biological systems that execute programs in response to stimuli, we developed a generalized time-frequency analysis (TFA) framework to systematically explore the dynamical properties of biomolecular networks. Using TFA, we focused on two well-characterized yeast gene regulatory networks responsive to carbon-source shifts and a mammalian innate immune regulatory network responsive to lipopolysaccharides (LPS). The networks are comprised of two different basic architectures. Dual positive and negative feedback loops make up the yeast galactose network; whereas overlapping positive and negative feed-forward loops are common to the yeast fatty-acid response network and the LPS-induced network of macrophages. TFA revealed remarkably distinct network behaviors in terms of trade-offs in responsiveness and noise suppression that are appropriately tuned to each biological response. The wild type galactose network was found to be highly responsive while the oleate network has greater noise suppression ability. The LPS network appeared more balanced, exhibiting less bias toward noise suppression or responsiveness. Exploration of the network parameter space exposed dramatic differences in system behaviors for each network. These studies highlight fundamental structural and dynamical principles that underlie each network, reveal constrained parameters of positive and negative feedback and feed-forward strengths that tune the networks appropriately for their respective biological roles, and demonstrate the general utility of the TFA approach for systems and synthetic biology.
Author Summary
Biological systems constantly balance noise suppression with responsiveness. In a fluctuating environment, some changes are insignificant to living cells while others represent cues to which they must respond. These stimuli are interpreted by molecular circuits that enable the cell to strike an appropriate balance between responsiveness and noise suppression. This trade-off is governed by the structure and kinetic parameters of molecular networks, which have been tuned by evolutionary selection for different stimuli and responses. We consider three regulatory circuits (two from yeast and one from mammalian cells), which respond to different environments and involve very different physiological processes. To investigate the responses to a time varying signal, we developed a generalized time-frequency analysis framework for studying such trade-offs using mathematical models of regulatory circuits and explore how the structure and parameters of the circuit affect the trade-offs between noise suppression and responsiveness. The generalized TFA approach represents an effective tool for exploring and analyzing different systems-level dynamical properties. Making use of such properties can facilitate prediction and network control for systems- and synthetic biology applications.
doi:10.1371/journal.pcbi.1002091
PMCID: PMC3127798  PMID: 21738459
23.  Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane 
The Journal of Cell Biology  2004;167(6):1099-1112.
We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.
doi:10.1083/jcb.200404119
PMCID: PMC2172632  PMID: 15596542
24.  Characterization of Karyopherin Cargoes Reveals Unique Mechanisms of Kap121p-Mediated Nuclear Import 
Molecular and Cellular Biology  2004;24(19):8487-8503.
In yeast there are at least 14 members of the β-karyopherin protein family that govern the movement of a diverse set of cargoes between the nucleus and cytoplasm. Knowledge of the cargoes carried by each karyopherin and insight into the mechanisms of transport are fundamental to understanding constitutive and regulated transport and elucidating how they impact normal cellular functions. Here, we have focused on the identification of nuclear import cargoes for the essential yeast β-karyopherin, Kap121p. Using an overlay blot assay and coimmunopurification studies, we have identified 30 putative Kap121p cargoes. Among these were Nop1p and Sof1p, two essential trans-acting protein factors required at the early stages of ribosome biogenesis. Characterization of the Kap121p-Nop1p and Kap121p-Sof1p interactions demonstrated that, in addition to lysine-rich nuclear localization signals (NLSs), Kap121p recognizes a unique class of signals distinguished by the abundance of arginine and glycine residues and consequently termed rg-NLSs. Kap104p is also known to recognize rg-NLSs, and here we show that it compensates for the loss of Kap121p function. Sof1p is also transported by Kap121p; however, its import can be mediated by a piggyback mechanism with Nop1p bridging the interaction between Sof1p and Kap121p. Together, our data elucidate additional levels of complexity in these nuclear transport pathways.
doi:10.1128/MCB.24.19.8487-8503.2004
PMCID: PMC516728  PMID: 15367670
25.  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

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