Few crucial links assure checkpoint efficiency in the yeast cell-cycle network

Motivation: The ability of cells to complete mitosis with high fidelity relies on elaborate checkpoint mechanisms. We study S- and M-phase checkpoint responses in silico in the budding yeast with a stochastic dynamical model for the cell-cycle. We aim to provide an unbiased functional classification of network interactions that reflect the contribution of each link to checkpoint efficiency in the presence of cellular fluctuations. Results: We developed an algorithm BNetDyn to compute stochastic dynamical trajectories for an input gene network and its structural perturbations. User specified output measures like the mutual information between trigger and output nodes are then evaluated on the stationary state of the Markov process. Systematic perturbations of the yeast cell-cycle model by Li et al. classify each link according to its effect on checkpoint efficiencies and stabilities of the main cell-cycle phases. This points to the crosstalk in the cascades downstream of the SBF/MBF transcription activator complexes as determinant for checkpoint optimality; a finding that consistently reflects recent experiments. Finally our stochastic analysis emphasizes how dynamical stability in the yeast cell-cycle network crucially relies on backward inhibitory circuits next to forward induction. Availability: C++ source code and network models can be downloaded at Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

Few crucial links assure checkpoint efficiency in the yeast cell-cycle network

MOTIVATION: The ability of cells to complete mitosis with high fidelity relies on elaborate checkpoint mechanisms. We study S- and M-phase checkpoint responses in silico in the budding yeast with a stochastic dynamical model for the cell-cycle. We aim to provide an unbiased functional classification of network interactions that reflect the contribution of each link to checkpoint efficiency in the presence of cellular fluctuations. RESULTS: We developed an algorithm BNetDyn to compute stochastic dynamical trajectories for an input gene network and its structural perturbations. User specified output measures like the mutual information between trigger and output nodes are then evaluated on the stationary state of the Markov process. Systematic perturbations of the yeast cell-cycle model by Li et al. classify each link according to its effect on checkpoint efficiencies and stabilities of the main cell-cycle phases. This points to the crosstalk in the cascades downstream of the SBF/MBF transcription activator complexes as determinant for checkpoint optimality; a finding that consistently reflects recent experiments. Finally our stochastic analysis emphasizes how dynamical stability in the yeast cell-cycle network crucially relies on backward inhibitory circuits next to forward induction. AVAILABILITY: C++ source code and network models can be downloaded at http://www.vital-it.ch/Software

A discrete inhomogeneous model for the yeast cell cycle

We study the robustness and stability of the yeast cell regulatory network by using a general inhomogeneous discrete model. We find that inhomogeneity, on average, enhances the stability of the biggest attractor of the dynamics and that the large size of the basin of attraction is robust against changes in the parameters of inhomogeneity. We find that the most frequent orbit, which represents the cell-cycle pathway, has a better biological meaning than the one exhibited by the homogeneous model.Comment: 5 pages, 1 figur

Functional analysis of the MMS19 gene in Carica papaya

All organisms face DNA-damaging radiation in the form of UV light and must have mechanisms to repair or undo damage. Plants being stationary organisms must cope with damaging UV rays in a molecular fashion and so have developed many forms of resistance. MMS19 is implicated in DNA repair indirectly as a nuclear encoded gene that binds onto protein assembly complexes in the cytosol. A mutant within the SunUp variety of Carica papaya has a large deletion for MMS19 and presents a diminutive phenotype. Deficient DNA repair mechanisms will shunt cells out of the natural cell cycle and reduce cell proliferation and differentiation. In this study the diminutive phenotype was characterized using electron microscopy, fluorescent microscopy, and RT-PCR. Mutant cells within leaf tissues showed increased autolytic and apoptotic activity, reduced expression of downstream genes indicating that MMS19 is necessary for maintaining normal growth and development of papaya

Beyond hairballs: depicting complexity of a kinase-phosphatase network in the budding yeast

Les kinases et les phosphatases (KP) représentent la plus grande famille des enzymes dans la cellule. Elles régulent les unes les autres ainsi que 60 % du protéome, formant des réseaux complexes kinase-phosphatase (KP-Net) jouant un rôle essentiel dans la signalisation cellulaire. Ces réseaux caractérisés d’une organisation de type commandes-exécutions possèdent généralement une structure hiérarchique. Malgré les nombreuse études effectuées sur le réseau KP-Net chez la levure, la structure hiérarchique ainsi que les principes fonctionnels sont toujours peux connu pour ce réseau. Dans ce contexte, le but de cette thèse consistait à effectuer une analyse d’intégration des données provenant de différentes sources avec la structure hiérarchique d’un réseau KP-Net de haute qualité chez la levure, S. cerevisiae, afin de générer des hypothèses concernant les principes fonctionnels de chaque couche de la hiérarchie du réseau KP-Net. En se basant sur une curation de données d’interactions effectuée dans la présente et dans d’autres études, le plus grand et authentique réseau KP-Net reconnu jusqu’à ce jour chez la levure a été assemblé dans cette étude. En évaluant le niveau hiérarchique du KP-Net en utilisant la métrique de la centralisation globale et en élucidant sa structure hiérarchique en utilisant l'algorithme vertex-sort (VS), nous avons trouvé que le réseau KP-Net possède une structure hiérarchique ayant la forme d’un sablier, formée de trois niveaux disjoints (supérieur, central et inférieur). En effet, le niveau supérieur du réseau, contenant un nombre élevé de KPs, était enrichi par des KPs associées à la régulation des signaux cellulaire; le niveau central, formé d’un nombre limité de KPs fortement connectées les unes aux autres, était enrichi en KPs impliquées dans la régulation du cycle cellulaire; et le niveau inférieur, composé d’un nombre important de KPs, était enrichi en KPs impliquées dans des processus cellulaires diversifiés. En superposant une grande multitude de propriétés biologiques des KPs sur le réseau KP-Net, le niveau supérieur était enrichi en phosphatases alors que le niveau inférieur en était appauvri, suggérant que les phosphatases seraient moins régulées par phosphorylation et déphosphorylation que les kinases. De plus, le niveau central était enrichi en KPs représentant des « bottlenecks », participant à plus d’une voie de signalisation, codées par des gènes essentiels et en KPs qui étaient les plus strictement régulées dans l’espace et dans le temps. Ceci implique que les KPs qui jouent un rôle essentiel dans le réseau KP-Net devraient être étroitement contrôlées. En outre, cette étude a montré que les protéines des KPs classées au niveau supérieur du réseau sont exprimées à des niveaux d’abondance plus élevés et à un niveau de bruit moins élevé que celles classées au niveau inférieur du réseau, suggérant que l’expression des enzymes à des abondances élevées invariables au niveau supérieur du réseau KP-Net pourrait être importante pour assurer un système robuste de signalisation. L’étude de l’algorithme VS a montré que le degré des nœuds affecte leur classement dans les différents niveaux d’un réseau hiérarchique sans biaiser les résultats biologiques du réseau étudié. En outre, une analyse de robustesse du réseau KP-Net a montré que les niveaus du réseau KP-Net sont modérément stable dans des réseaux bruités générés par ajout d’arrêtes au réseau KP-Net. Cependant, les niveaux de ces réseaux bruités et de ceux du réseau KP-Net se superposent significativement. De plus, les propriétés topologiques et biologiques du réseau KP-Net étaient retenues dans les réseaux bruités à différents niveaux. Ces résultats indiquant que bien qu’une robustesse partielle de nos résultats ait été observée, ces derniers représentent l’état actuel de nos connaissances des réseaux KP-Nets. Finalement, l’amélioration des techniques dédiées à l’identification des substrats des KPs aideront davantage à comprendre comment les réseaux KP-Nets fonctionnent. À titre d’exemple, je décris, dans cette thèse, une stratégie que nous avons conçu et qui permet à déterminer les interactions KP-substrats et les sous-unités régulatrices sur lesquelles ces interactions dépendent. Cette stratégie est basée sur la complémentation des fragments de protéines basée sur la cytosine désaminase chez la levure (OyCD PCA). L’OyCD PCA représente un essai in vivo à haut débit qui promet une description plus précise des réseaux KP-Nets complexes. En l’appliquant pour déterminer les substrats de la kinase cycline-dépendante de type 1 (Cdk1, appelée aussi Cdc28) chez la levure et l’implication des cyclines dans la phosphorylation de ces substrats par Cdk1, l’essai OyCD PCA a montré un comportement compensatoire collectif des cyclines pour la majorité des substrats. De plus, cet essai a montré que la tubuline- γ est phosphorylée spécifiquement par Clb3-Cdk1, établissant ainsi le moment pendant lequel cet événement contrôle l'assemblage du fuseau mitotique.Kinases and phosphatases (KP) form the largest family of enzymes in living cells. They regulate each other and 60 % of the proteome forming complex kinase-phosphatase networks (KP-Net) essential for cell signaling. Such networks having the command-execution aspect tend to have a hierarchical structure. Despite the extensive study of the KP-Net in the budding yeast, the hierarchical structure as well as the functional principles of this network are still not known. In this context, this thesis aims to perform an integrative analysis of multi-omics data with the hierarchical structure of a bona fide KP-Net in the budding yeast Saccharomyces cerevisiae, in order to generate hypotheses about the functional principles of each layer in the KP-Net hierarchy. Based on a literature curation effort accomplished in this and in other studies, the largest bona fide KP-Net of the S. cerevisiae known to date was assembled in this thesis. By assessing the hierarchical level of the KP-Net using the global reaching centrality and by elucidating the its hierarchical structure using the vertex-sort (VS) algorithm, we found that the KP-Net has a moderate hierarchical structure made of three disjoint layers (top, core and bottom) resembling a bow tie shape. The top layer having a large size was found enriched for signaling regulation; the core layer made of few strongly connected KPs was found enriched mostly for cell cycle regulation; and the bottom layer having a large size was found enriched for diverse biological processes. On overlaying a wide range of KP biological properties on top of the KP-Net hierarchical structure, the top layer was found enriched for and the bottom layer was found depleted for phosphatases, suggesting that phosphatases are less regulated by phosphorylation and dephosphoryation interactions (PDI) than kinases. Moreover, the core layer was found enriched for KPs representing bottlenecks, pathway-shared components, essential genes and for the most tightly regulated KPs in time and space, implying that KPs playing an essential role in the KP-Net should be firmly controlled. Interestingly, KP proteins in the top layer were found more abundant and less noisy than those of the bottom layer, suggesting that availability of enzymes at invariable protein expression level at the top of the network might be important to ensure a robust signaling. Analysis of the VS algorithm showed that node degrees affect their classification in the different layers of a network hierarchical structure without biasing biological results of the sorted network. Robustness analysis of the KP-Net showed that KP-Net layers are moderately stable in noisy networks generated by adding edges to the KP-Net. However, layers of these noisy overlap significantly with those of the KP-Net. Moreover, topological and biological properties of the KP-Net were retained in the noisy networks to different levels. These findings indicate that despite the observed partial robustness of our results, they mostly represent our current knowledge about KP-Nets. Finally, enhancement of techniques dedicated to identify KPs substrates will enhance our understanding about how KP-Nets function. As an example, I describe here a strategy that we devised to help in determining KP-substrate interactions and the regulatory subunits on which these interactions depend. The strategy is based on a protein-fragment complementation assay based on the optimized yeast cytosine deaminase (OyCD PCA). The OyCD PCA represents a large scale in vivo screen that promises a substantial improvement in delineating the complex KP-Nets. We applied the strategy to determine substrates of the cyclin-dependent kinase 1 (Cdk1; also called Cdc28) and cyclins implicated in phosphorylation of these substrates by Cdk1 in S. cerevisiae. The OyCD PCA showed a wide compensatory behavior of cyclins for most of the substrates and the phosphorylation of γ-tubulin specifically by Clb3-Cdk1, thus establishing the timing of the latter event in controlling assembly of the mitotic spindle

Shedding light on the role of plant miRNAs in DNA damage response (DDR) and trans-kingdom transfer

One of the challenges that living organisms face is to respond promptly to genotoxic stress to avoid DNA damage. To this purpose, they developed complex DNA damage response (DDR) mechanisms. These mechanisms are highly conserved among organisms, including plants, and need to be finely regulated to take place properly. In this scenario, microRNAs are emerging as active players, thus attracting the attention of the research community. The involvement of miRNAs in DDR has been investigated prominently in human cells wherease studies on plants are still scarce. In addition, recently, miRNAs started to be envisioned as trans-kingdom molecules able to exert regulatory functions in evolutionary distant organisms. Particularly, attention is drawn to plant miRNAs ingested with the diet; evidence is accumulating on their ability to regulate genes in organisms other than the one in which they were synthesized, including humans and pathogens.In the present PhD thesis, different bioinformatics approaches have been developed aiming at identifying plant miRNAs along with their endogenous and cross-kingdom targets to pinpoint conserved pathways between evolutionary distant species. Alonside model organisms, the developed pipeline may find application on any species of interest to address species-specific cross-kingdom interactions or to performe large-scale investigations involving several plant/animal species. The emergence of DDR-related miRNAs in plants and humans constitutes fundamental informations obtained from these approaches.To experimentally investigate the involvement of plant miRNAs in the regulation of DDR-associated pathways, an ad hoc system was developed, using the model legume Medicago truncatula. Specific treatments with camptothecin (CPT) and/or NSC120686 (NSC) targeting compoments of DDR, namely topoisomerase I (Top1) and tyrosyl-DNA phosphodiesterase 1 (Tdp1), were used. These treatments, imposed to M. truncatula seeds for a 7-day time period, do not influence the germination process, but result in inhibition of seedling development, causing an increase in cell death and accumulation of DNA damage. To demonstrate that the imposed treatments had an effect on DDR, the expression of SOG1 (suppressor of gamma response 1) master-regulator was investigated by qRT-PCR. Importantly, a phylogenetic study demonstrated that M. truncatula possessed a small SOG1 gene family, composed by MtSOG1A and MtSOG1B genes. The expression of both genes was significantly enhanced in treatment-specific manner. Additionally, the espression of multiple genes playing important roles in different DNA repair pathways, cell cycle regulation and chromatin remodelling, were differentially expressed in a treatment-specific manner. Subsequently, specific miRNAs identifyed from the bioinformatics approach as targeting genes involved in DDR processes, were investigated along side their targets, thus providing a first step in their function validation.To investigate plant miRNAs trans-kingdom potential, additional studies were conducted using apple (M. domestica) since it can be eaten raw and hence, can be a better system for feeding trials. As a proof of concept, artificial miRNAs (amiRNAs) were delivered to human colorectal adenocarcinoma cells and the expression of these microRNAs and their in silico predicted targets were evaluated by qRT-PCR. Specifically, amiRNAs mimicking mdm-miR482a-3p and mdm-miR858 were transfected into HT-29 cell lines. After 72 h, amiRNAs were clearly detected inside the cells and the performed qRT-PCR analysis showed a significant downregulation of the IL4R (Interleukin 4 Receptor) gene, involved in promoting Th2 differentiation, suggesting the possibility of apple miRNAs to regulate the activity of human genes in vitro. Taken together, the results presented in the current PhD thesis demonstrate the involvement of plant miRNAs in DDR-associated processes as well as present evidence on the plant miRNAs trans-kingdom potential.One of the challenges that living organisms face is to respond promptly to genotoxic stress to avoid DNA damage. To this purpose, they developed complex DNA damage response (DDR) mechanisms. These mechanisms are highly conserved among organisms, including plants, and need to be finely regulated to take place properly. In this scenario, microRNAs are emerging as active players, thus attracting the attention of the research community. The involvement of miRNAs in DDR has been investigated prominently in human cells wherease studies on plants are still scarce. In addition, recently, miRNAs started to be envisioned as trans-kingdom molecules able to exert regulatory functions in evolutionary distant organisms. Particularly, attention is drawn to plant miRNAs ingested with the diet; evidence is accumulating on their ability to regulate genes in organisms other than the one in which they were synthesized, including humans and pathogens.In the present PhD thesis, different bioinformatics approaches have been developed aiming at identifying plant miRNAs along with their endogenous and cross-kingdom targets to pinpoint conserved pathways between evolutionary distant species. Alonside model organisms, the developed pipeline may find application on any species of interest to address species-specific cross-kingdom interactions or to performe large-scale investigations involving several plant/animal species. The emergence of DDR-related miRNAs in plants and humans constitutes fundamental informations obtained from these approaches.To experimentally investigate the involvement of plant miRNAs in the regulation of DDR-associated pathways, an ad hoc system was developed, using the model legume Medicago truncatula. Specific treatments with camptothecin (CPT) and/or NSC120686 (NSC) targeting compoments of DDR, namely topoisomerase I (Top1) and tyrosyl-DNA phosphodiesterase 1 (Tdp1), were used. These treatments, imposed to M. truncatula seeds for a 7-day time period, do not influence the germination process, but result in inhibition of seedling development, causing an increase in cell death and accumulation of DNA damage. To demonstrate that the imposed treatments had an effect on DDR, the expression of SOG1 (suppressor of gamma response 1) master-regulator was investigated by qRT-PCR. Importantly, a phylogenetic study demonstrated that M. truncatula possessed a small SOG1 gene family, composed by MtSOG1A and MtSOG1B genes. The expression of both genes was significantly enhanced in treatment-specific manner. Additionally, the espression of multiple genes playing important roles in different DNA repair pathways, cell cycle regulation and chromatin remodelling, were differentially expressed in a treatment-specific manner. Subsequently, specific miRNAs identifyed from the bioinformatics approach as targeting genes involved in DDR processes, were investigated along side their targets, thus providing a first step in their function validation.To investigate plant miRNAs trans-kingdom potential, additional studies were conducted using apple (M. domestica) since it can be eaten raw and hence, can be a better system for feeding trials. As a proof of concept, artificial miRNAs (amiRNAs) were delivered to human colorectal adenocarcinoma cells and the expression of these microRNAs and their in silico predicted targets were evaluated by qRT-PCR. Specifically, amiRNAs mimicking mdm-miR482a-3p and mdm-miR858 were transfected into HT-29 cell lines. After 72 h, amiRNAs were clearly detected inside the cells and the performed qRT-PCR analysis showed a significant downregulation of the IL4R (Interleukin 4 Receptor) gene, involved in promoting Th2 differentiation, suggesting the possibility of apple miRNAs to regulate the activity of human genes in vitro. Taken together, the results presented in the current PhD thesis demonstrate the involvement of plant miRNAs in DDR-associated processes as well as present evidence on the plant miRNAs trans-kingdom potential