Dissecting the fission yeast regulatory network reveals phase-specific control elements of its cell cycle

Background  

Fission yeast Schizosaccharomyces pombe and budding yeast Saccharomyces cerevisiae are among the original model organisms in the study of the cell-division cycle. Unlike budding yeast, no large-scale regulatory network has been constructed for fission yeast. It has only been partially characterized. As a result, important regulatory cascades in budding yeast have no known or complete counterpart in fission yeast.     

Boolean network model predicts cell cycle sequence of fission yeast

A Boolean network model of the cell-cycle regulatory network of fission yeast (Schizosaccharomyces Pombe) is constructed solely on the basis of the known biochemical interaction topology. Simulating the model in the computer faithfully reproduces the known activity sequence of regulatory proteins along the cell cycle of the living cell. Contrary to existing differential equation models, no parameters enter the model except the structure of the regulatory circuitry. The dynamical properties of the model indicate that the biological dynamical sequence is robustly implemented in the regulatory network, with the biological stationary state G1 corresponding to the dominant attractor in state space, and with the biological regulatory sequence being a strongly attractive trajectory. Comparing the fission yeast cell-cycle model to a similar model of the corresponding network in S. cerevisiae, a remarkable difference in circuitry, as well as dynamics is observed. While the latter operates in a strongly damped mode, driven by external excitation, the S. pombe network represents an auto-excited system with external damping.     

Modelling the fission yeast cell cycle.

The molecular networks regulating basic physiological processes in a cell can be converted into mathematical equations (eg differential equations) and solved by a computer. The division cycle of eukaryotic cells is an important example of such a control system, and fission yeast is an excellent test organism for the computational modelling approach. The mathematical model is tested by simulating wild-type cells and many known cell cycle mutants. This paper describes an example where this approach is useful in understanding multiple rounds of DNA synthesis (endoreplication) in fission yeast cells that lack the main (B-type) mitotic cyclin, Cdc13. It is proposed that the key physiological variable driving progression through the cell cycle during balanced growth and division is the mass/DNA ratio, rather than the mass/nucleus ratio.     

Preservation of dynamic properties in qualitative modeling frameworks for gene regulatory networks

Mathematical modeling often helps to provide a systems perspective on gene regulatory networks. In particular, qualitative approaches are useful when detailed kinetic information is lacking. Multiple methods have been developed that implement qualitative information in different ways, e.g., in purely discrete or hybrid discrete/continuous models. In this paper, we compare the discrete asynchronous logical modeling formalism for gene regulatory networks due to R. Thomas with piecewise affine differential equation models. We provide a local characterization of the qualitative dynamics of a piecewise affine differential equation model using the discrete dynamics of a corresponding Thomas model. Based on this result, we investigate the consistency of higher-level dynamical properties such as attractor characteristics and reachability. We show that although the two approaches are based on equivalent information, the resulting qualitative dynamics are different. In particular, the dynamics of the piecewise affine differential equation model is not a simple refinement of the dynamics of the Thomas model     

Neutral space analysis for a Boolean network model of the fission yeast cell cycle network

Background

Interactions between genes and their products give rise to complex circuits known as gene regulatory networks (GRN) that enable cells to process information and respond to external stimuli. Several important processes for life, depend of an accurate and context-specific regulation of gene expression, such as the cell cycle, which can be analyzed through its GRN, where deregulation can lead to cancer in animals or a directed regulation could be applied for biotechnological processes using yeast. An approach to study the robustness of GRN is through the neutral space. In this paper, we explore the neutral space of a Schizosaccharomyces pombe (fission yeast) cell cycle network through an evolution strategy to generate a neutral graph, composed of Boolean regulatory networks that share the same state sequences of the fission yeast cell cycle.

Results

Through simulations it was found that in the generated neutral graph, the functional networks that are not in the wildtype connected component have in general a Hamming distance more than 3 with the wildtype, and more than 10 between the other disconnected functional networks. Significant differences were found between the functional networks in the connected component of the wildtype network and the rest of the network, not only at a topological level, but also at the state space level, where significant differences in the distribution of the basin of attraction for the G₁ fixed point was found for deterministic updating schemes.

Conclusions

In general, functional networks in the wildtype network connected component, can mutate up to no more than 3 times, then they reach a point of no return where the networks leave the connected component of the wildtype. The proposed method to construct a neutral graph is general and can be used to explore the neutral space of other biologically interesting networks, and also formulate new biological hypotheses studying the functional networks in the wildtype network connected component.     

Two distinct ubiquitin-proteolysis pathways in the fission yeast cell cycle

The SCF complex (Skp1-Cullin-1-F-box) and the APC/cyclosome (anaphase-promoting complex) are two ubiquitin ligases that play a crucial role in eukaryotic cell cycle control. In fission yeast F-box/WD-repeat proteins Pop1 and Pop2, components of SCF are required for cell-cycle-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor Rum1 and the S-phase regulator Cdc18. Accumulation of these proteins in pop1 and pop2 mutants leads to re-replication and defects in sexual differentiation. Despite structural and functional similarities, Pop1 and Pop2 are not redundant homologues. Instead, these two proteins form heterodimers as well as homodimers, such that three distinct complexes, namely SCFPop1/Pop1, SCFPop1/Pop2 and SCFPop2/Pop2, appear to exist in the cell. The APC/cyclosome is responsible for inactivation of CDK/cyclins through the degradation of B-type cyclins. We have identified two novel components or regulators of this complex, called Apc10 and Ste9, which are evolutionarily highly conserved. Apc10 (and Ste9), together with Rum1, are required for the establishment of and progression through the G1 phase in fission yeast. We propose that dual downregulation of CDK, one via the APC/cyclosome and the other via the CDK inhibitor, is a universal mechanism that is used to arrest the cell cycle at G1.     

Controlling cell cycle progress in the fission yeast Schizosaccharomyces pombe.

The suitability of fission yeast as a model for understanding the eukaryotic cell cycle has been validated in five years of exciting developments. We review recent advances in understanding the nature of the controls that regulate progression through the cell cycle and the coordination of DNA replication and mitosis.     

The regulatory network of cell cycle progression is fundamentally different in plants versus yeast or metazoans

Plant growth and proliferation control is coming into a global focus due to recent ecological and economical developments. Plants represent not only the largest food supply for mankind but also may serve as a global source of renewable energies. However, plant breeding has to accomplish a tremendous boost in yield to match the growing demand of a still rapidly increasing human population. Moreover, breeding has to adjust to changing environmental conditions, in particular increased drought. Regulation of cell cycle control is a major determinant of plant growth and therefore an obvious target for plant breeding. Furthermore, cell cycle control is also crucial for the DNA damage response, for instance upon irradiation. Thus, an in-depth understanding of plant cell cycle regulation is of importance beyond a scientific point of view. The mere presence of many conserved core cell cycle regulators, e.g., CDKs, cyclins or CDK inhibitors, has formed the idea that the cell cycle in plants is exactly or at least very similarly controlled as in yeast or human cells. Here together with a recent publication we demonstrate that this dogma is not true and show that the control of entry into mitosis is fundamentally different in plants versus yeast or metazoans. Our findings build an important base for the understanding and ultimate modulation of plant growth not only during unperturbed but also under harsh environmental conditions.Key words: cell cycle, phosphorylation, checkpoint, DNA damage, cyclin-dependent kinase, CDK, WEE1, CDC25, ArabidopsisProgression through the cell cycle is not only a decisive event for a single cell but also of key importance for organ growth in multicellular organisms such as plants.^1,2 Moreover, coupled to and overlapping in space and time with proliferation, cell differentiation takes place and thus, a tight control of the cell cycle is one of the foundations of development.³ Thus, not very surprisingly, an elaborated machinery controlling cell cycle regulation has evolved and overall, many proteins appear to be conserved between humans and plants.^4,5 However, there are also clear differences in the repertoire of cell cycle regulators in plants and functional studies have often not yet been conducted to elucidate the specific role of many regulators.In metazoans, a switch-like activation of the central cyclin-dependent kinase, Cdk1 (or its homologous proteins, e.g., Cdc2⁺ or CDC28p) plays one of the most important roles in cell cycle control.⁶ Wee1-type kinases, e.g., Wee1 or Myt1, phosphorylate Cdk1-type kinases at Thr14 and Tyr15 (or the homologous positions) and inhibit their activity (Fig. 1A).⁷ The function of these kinases is opposed by Cdc25 that acts as dual specificity phosphatase and removes these phosphate groups leading to the rapid activation of Cdk1-type kinases. This inhibition of Cdk1 activity by Wee1 and its release by Cdc25 fulfill a fundamental function during metazoan cell cycle control ensures the unidirectionality of the cell cycle.^8,9 The underlying molecular mechanism for this is a wiring of Cdk1 with Cdc25 or Wee1 by positive and antagonistic (double-negative) feedback loops, i.e., Cdk1 activates its activator Cdc25 and inactivates its inhibitor Wee1 (Fig. 1C). Thus, there are only two stable steady states, inactive or active; this bistability generates a biological switch. The transition from one state to the other is thought to be brought about by rising and falling levels of cyclins as activating subunits of CDKs. Moreover, due to the positive feedback wiring, the two steady states are buffered against small changes in cyclin levels, i.e., it takes a much higher concentration of cyclins to switch from G₂-phase into mitosis than to stay in mitosis. This property of feed-back wiring, called hysteresis, greatly reinforces the unidirectionality of the cell cycle (Fig. 1A and C).^10,11Open in a separate windowFigure 1Computational analysis of the switch-like activation of Cdk1-like kinases. (A and B) show steady-state activity of CDKs as a function of cyclin levels. (A) CDK/cyclin activity regulated via inhibitory Tyr15-phosphorylation of the CDK catalytic subunit of the complex. (B) CDK/cyclin activity control is achieved by stoichiometrically acting CDK inhibitors (CKIs). Both switches allow building up inactivated kinase and once a cyclin level has reached a threshold, high levels of kinase activity are rapidly available that can forcefully promote the entry into the next cell cycle phase. Importantly, a small drop in cyclin levels is not sufficient to change the activity state, thus the system is buffered and once the decision is taken to enter the next cell cycle phase, this cannot easily be reverted. (C) Double-negative and positive feedback loops targeting the status of inhibitory CDK phosphorylation. CDK activity is governed via inhibitory phosphorylation by WEE1/MYT1 kinases and activatory dephosphorylation by CDC25 phosphatases. CDK can phosphorylate WEE1/MYT1 to inactivate its own inactivator and CDK activates its own activator CDC25 by phosphorylation.¹¹ (D) Double-negative feedback loop of the CDK-CKI module.⁴³ CKIs inhibit CDKs and, in turn, CDKs promote CKI degradation.⁴²Cdc25 and the feedback loops sketched above are also major targets of a checkpoint response and interruption of these can effectively arrest the cell cycle. For instance, in animals, DNA damage is sensed by ATM and ATR kinases that in turn activate Chk1 and Chk2 kinases which then will phosphorylate and inactivate Cdc25 allowing the cell to repair its damage.^12,13 In parallel, Chk1/2 activate Wee1 by phosphorylation and reinforce the checkpoint.Previously, candidate genes for Cdc25 and Wee1 homologs have been identified in Arabidopsis as well as in other plants.^14–18 Along with the finding that plants contain Cdk1-like kinases with a PSTAIRE cyclin binding signature, designated CDKAs, which can rescue yeast cdc2/cdc28 mutants,^19–22 this has given rise to the notion that the wiring of the regulatory triangle CDKA-CDC25-WEE1 is conserved in plants.Here and in an accompanying publication by Dissmeyer et al.²³ we have probed this notion by a detailed structure-function analysis. Our data demonstrate that the regulatory connection between these three components is not conserved and that plants must have evolved different mechanisms to stably progress through a mitotic cycle and arrest the cell cycle upon DNA damage.     

A fission yeast B-type cyclin functioning early in the cell cycle.

We have cloned a fission yeast gene, cig1+, encoding a 48 kd product that is most similar to cyclin B proteins. The cig1+ protein has a "cyclin box" approximately 40% identical to B-type cyclins of other species, but lacks the "destruction box" required for proteolysis of mitotic cyclins. Deletion of cig1+ had no observable effect on cell viability or progression through G2 or M phase, but instead caused a marked lag in the progression from G1 to S phase. G1 constituted approximately 70% of the cell cycle in cig1 deletion strains, as compared with less than 10% in cig1+ strains. Constitutive cig1+ overexpression was lethal, causing cessation of growth and arrest in G1. Expression of cig1+ failed to rescue an S. cerevisiae strain lacking CLN Start cyclins. Thus, cig1+ identifies a new class of B-type cyclin acting in G1 or S phase that appears to be functionally distinct from all previously described cyclin proteins.     

Logical analysis of the budding yeast cell cycle

The budding yeast Saccharomyces cerevisiae is a model organism that is commonly used to investigate control of the eukaryotic cell cycle. Moreover, because of the extensive experimental data on wild type and mutant phenotypes, it is also particularly suitable for mathematical modelling and analysis. Here, I present a new Boolean model of the budding yeast cell cycle. This model is consistent with a wide range of wild type and mutant phenotypes and shows remarkable robustness against perturbations, both to reaction times and the states of component genes/proteins. Because of its simple logical nature, the model is suitable for sub-network analysis, which can be used to identify a four node core regulatory circuit underlying cell cycle regulation. Sub-network analysis can also be used to identify key sub-dynamics that are essential for viable cell cycle control, as well as identifying the sub-dynamics that are most variable between different mutants.     

Coordination of DNA damage tolerance mechanisms with cell cycle progression in fission yeast

DNA damage tolerance (DDT) mechanisms allow cells to synthesize a new DNA strand when the template is damaged. Many mutations resulting from DNA damage in eukaryotes are generated during DDT when cells use the mutagenic translesion polymerases, Rev1 and Polζ, rather than mechanisms with higher fidelity. The coordination among DDT mechanisms is not well understood. We used live-cell imaging to study the function of DDT mechanisms throughout the cell cycle of the fission yeast Schizosaccharomyces pombe. We report that checkpoint-dependent mitotic delay provides a cellular mechanism to ensure the completion of high fidelity DDT, largely by homology-directed repair (HDR). DDT by mutagenic polymerases is suppressed during the checkpoint delay by a mechanism dependent on Rad51 recombinase. When cells pass the G2/M checkpoint and can no longer delay mitosis, they completely lose the capacity for HDR and simultaneously exhibit a requirement for Rev1 and Polζ. Thus, DDT is coordinated with the checkpoint response so that the activity of mutagenic polymerases is confined to a vulnerable period of the cell cycle when checkpoint delay and HDR are not possible.