Paike Jayadeva Bhat

Integration of Global Signaling Pathways, cAMP-PKA, MAPK and TOR in the Regulation of FLO11

The budding yeast, Saccharomyces cerevisiae, responds to various environmental cues by invoking specific adaptive mechanisms for their survival. Under nitrogen limitation, S. cerevisiae undergoes a dimorphic filamentous transition called pseudohyphae, which helps the cell to forage for nutrients and reach an environment conducive for growth. This transition is governed by a complex network of signaling pathways, namely cAMP-PKA, MAPK and TOR, which controls the transcriptional activation of FLO11, a flocculin gene that encodes a cell wall protein. However, little is known about how these pathways co-ordinate to govern the conversion of nutritional availability into gene expression. Here, we have analyzed an integrative network comprised of cAMP-PKA, MAPK and TOR pathways with respect to the availability of nitrogen source using experimental and steady state modeling approach. Our experiments demonstrate that the steady state expression of FLO11 was bistable over a range of inducing ammonium sulphate concentration based on the preculturing condition. We also show that yeast switched from FLO11 expression to accumulation of trehalose, a STRE response controlled by a transcriptional activator Msn2/4, with decrease in the inducing concentration to complete starvation. Steady state analysis of the integrative network revealed the relationship between the environment, signaling cascades and the expression of FLO11. We demonstrate that the double negative feedback loop in TOR pathway can elicit a bistable response, to differentiate between vegetative growth, filamentous growth and STRE response. Negative feedback on TOR pathway function to restrict the expression of FLO11 under nitrogen starved condition and also with re-addition of nitrogen to starved cells. In general, we show that these global signaling pathways respond with specific sensitivity to regulate the expression of FLO11 under nitrogen limitation. The holistic steady state modeling approach of the integrative network revealed how the global signaling pathways could differentiate between multiple phenotypes.

Autoregulation of regulatory proteins is key for dynamic operation of GAL switch in Saccharomyces cerevisiae

Autoregulation and nucleocytoplasmic shuttling play important roles in the operation of the GAL regulatory system. However, the significance of these mechanisms in the overall operation of the switch is unclear. In this work, we develop a dynamic model for the GAL system and further validate the same using steady‐state and dynamic experimental expression data. Next, the model is used to delineate the relevance of shuttling and autoregulation in response to inducing, repressing, and non‐inducing–non‐repressing media. The analysis indicates that autoregulation of the repressor, Gal80p, is key in obtaining three distinct steady states in response to the three media. In particular, the analysis rationalizes the intuitively paradoxical observation that the concentration of repressor, Gal80p, actually increases in response to an increase in the inducer concentration. On the other hand, although nucleocytoplasmic shuttling does not affect the dynamics of the system, it plays a dominant role in obtaining a sensitive response to galactose. The dynamic model was also used to obtain insights on the preculturing effect on the system behavior.

Expression of GAL genes in a mutant strain of Saccharomyces cerevisiae lacking GAL80: quantitative model and experimental verification.

The regulatory network of GAL genes is a model system for the production of foreign proteins. A mathematical model based on steady state was developed for the expression of GAL (galactosidase) genes in a mutant strain of Saccharomyces cerevisiae lacking GAL80. The transcriptional and translational responses of the GAL switch were predicted at various steady‐state glucose concentrations. The model predicted ultrasensitive transcriptional response with a Hill coefficient (h) of 1.9 and 3.2 for genes with one and two binding sites respectively. Further, a lesser degree of ultrasensitivity was predicted for translational response with an h value of 1.3 for genes with one binding site and 2.1 for genes with two binding sites. The ultrasensitivity was due to dimerization of regulatory protein Gal4p and co‐operative binding of Gal4p to DNA. The steady‐state predictions were experimentally verified through measurements of α‐galactosidase (for one binding site) and β‐galactosidase (for two binding sites). The steady state model was further extended to represent the dynamic expression profile and the same was verified experimentally. The growth phase and the synthesis of foreign protein could be distinctly separated using a mutant strain of Saccharomyces cerevisiae (bakers yeast).

Systems biology of GAL regulon in Saccharomyces cerevisiae

Evolutionary success of an organism depends on its ability to express or adapt to constantly changing environmental conditions. Saccharomyces cerevisiae has evolved an elaborate genetic circuit to regulate the expression of galactose‐metabolizing enzymes in the presence of galactose but in the absence of glucose. The circuit possesses molecular mechanisms such as multiple binding sites, cooperativity, autoregulation, nucleocytoplasmic shuttling, and substrate sensing mechanism. Furthermore, the GAL system consists of two positive (activating) feedback and one negative (repressing) feedback loops. These individual mechanisms, elucidated through experimental approach, can be integrated to obtain a system‐wide behavior. Mathematical models in conjunction with guided experiments have demonstrated system‐level properties such as ultrasensitivity, memory, noise attenuation, rapid response, and sensitive response arising out of the molecular interactions. These system‐level properties allow S. cerevisiae to adapt and grow in a galactose medium under noisy and changing environments. This review focuses on system‐level models and properties of the GAL regulon Copyright

Epigenetics of the yeast galactose genetic switch

The transcriptional activation of enzymes involved in galactose utilization (GAL genes) in Saccharomyces cerevisiae is regulated by a complex interplay between three regulatory proteins encoded by GAL4 (transcriptional activator), GAL3 (signal transducer) and GAL80 (repressor). The relative concentrations of the signal transducer and the repressor are maintained by autoregulation. Cells disabled for autoregulation exhibit phenotypes distinctly different from that of the wild type cells, enabling us to explore the biological significance of autoregulation. The redundancy in signal transduction due to the presence of GAL1 (alternate signal transducer) also makes it a suitable model to understand the phenomenon of epigenetics. In this article we review some of the recent attempts made to understand the importance of epigenetics in the establishment of cellular and transcriptional memory.

Stochastic galactokinase expression underlies GAL gene induction in a GAL3 mutant of Saccharomyces cerevisiae

GAL1 and GAL3 are paralogous signal transducers that functionally inactivate Gal80p to activate the Gal4p‐dependent transcriptional activation of GAL genes in Saccharomyces cerevisiae in response to galactose. Unlike a wild‐type strain, the gal3∆ strain shows delayed growth kinetics as a result of the signaling function of GAL1. The mechanism ensuring that GAL1 is eventually expressed to turn on the GAL switch in the gal3∆ strain remains a paradox. Using galactose and histidine growth complementation assays, we demonstrate that 0.3% of the gal3∆ cell population responds to galactose. This is corroborated by flow cytometry and microscopic analysis. The galactose responders and nonresponders isolated from the galactose‐adapted population attain the original bimodal state and this phenotype is found to be as hard wired as a genetic trait. Computational analysis suggests that the log‐normal distribution in GAL4 synthesis can lead to bimodal expression of GAL80, resulting in the bimodal expression of GAL genes. Heterozygosity at the GAL80 but not at the GAL1, GAL2 or GAL4 locus alters the extent of bimodality of the gal3∆ cell population. We suggest that the asymmetric expression pattern between GAL1 and GAL3 results in the ability of S. cerevisiae to activate the GAL pathway by conferring nongenetic heterogeneity.

Can metabolic plasticity be a cause for cancer? Warburg–Waddington legacy revisited

Fermentation of glucose to lactate in the presence of sufficient oxygen, known as aerobic glycolysis or Warburg effect, is a universal phenotype of cancer cells. Understanding its origin and role in cellular immortalization and transformation has attracted considerable attention in the recent past. Intriguingly, while we now know that Warburg effect is essential for tumor growth and development, it is thought to arise because of genetic and/or epigenetic changes. In contrast to the above, we propose that Warburg effect can also arise due to normal biochemical fluctuations, independent of genetic and epigenetic changes. Cells that have acquired Warburg effect proliferate rapidly to give rise to a population of heterogeneous progenitors of cancer cells. Such cells also generate more lactate and alter the fitness landscape. This dynamic fitness landscape facilitates evolution of cancer cells from its progenitors, in a fashion analogous to Darwinian evolution. Thus, sporadic cancer can also occur first by the acquisition of Warburg effect, then followed by mutation and selection. The idea proposed here circumvents the inherent difficulties associated with the current understanding of tumorigenesis, and is also consistent with many experimental and epidemiological observations. We discuss this model in the context of epigenetics as originally enunciated by Waddington.

Perturbation of the interaction between Gal4p and Gal80p of the Saccharomyces cerevisiae GAL switch results in altered responses to galactose and glucose

In S. cerevisiae, following the Whole Genome Duplication (WGD), GAL1‐encoded galactokinase retained its signal transduction function but lost basal expression. On the other hand, its paralogue GAL3, lost kinase activity but retained its signalling function and basal expression, thus making it indispensable for the rapid induction of the S. cerevisiae GAL switch. However, a gal3Δ strain exhibits delayed growth kinetics due to the redundant signalling function of GAL1. The subfunctionalization between the paralogues GAL1 and GAL3 is due to expression divergence and is proposed to be due to the alteration in the Upstream Activating Sequences (UASG). We demonstrate that the GAL switch becomes independent of GAL3 by altering the interaction between Gal4p and Gal80p without altering the configuration of UASG. In addition to the above, the altered switch of S. cerevisiae loses ultrasensitivity and stringent glucose repression. These changes caused an increase in fitness in the disaccharide melibiose at the expense of a decrease in fitness in galactose. The above altered features of the ScGAL switch are similar to the features of the GAL switch of K. lactis that diverged from S. cerevisiae before the WGD.

Fermentative metabolism impedes p53-dependent apoptosis in a Crabtree-positive but not in Crabtree-negative yeast

Tumour cells distinguish from normal cells by fermenting glucose to lactate in presence of sufficient oxygen and functional mitochondria (Warburg effect). Crabtree effect was invoked to explain the biochemical basis of Warburg effect by suggesting that excess glucose suppresses mitochondrial respiration. It is known that the Warburg effect and Crabtree effect are displayed by Saccharomyces cerevisiae, during growth on abundant glucose. Beyond this similarity, it was also demonstrated that expression of human pro-apoptotic proteins in S. cerevisiae such as Bax and p53 caused apoptosis. Here, we demonstrate that p53 expression in S. cerevisiae (Crabtree-positive yeast) causes increase in ROS levels and apoptosis when cells are growing on non-fermentable carbon sources but not on fermentable carbon sources, a feature similar to tumour cells. In contrast, in Kluyveromyces lactis (Crabtree-negative yeast) p53 causes increase in ROS levels and apoptosis regardless of the carbon source. Interestingly, the increased ROS levels and apoptosis are correlated to increased oxygen uptake in both S. cerevisiae and K. lactis. Based on these results, we suggest that at least in yeast, fermentation per se does not prevent the escape from apoptosis. Rather, the Crabtree effect plays a crucial role in determining whether the cells should undergo apoptosis or not.

KRH1 and KRH2 are functionally non-redundant in signaling for pseudohyphal differentiation in Saccharomyces cerevisiae

Diploid cells of Saccharomyces cerevisiae undergo pseudohyphal differentiation in response to nutrient depletion. Although this dimorphic transition occurs due to signals originating from carbon and nitrogen limitation, how these signals are coordinated and integrated is not understood. Results of this study indicate that the pseudohyphal defect of the mep2∆ mutant is overcome upon disruption of KRH2/GPB1 but not KRH1/GPB2. Further, the agar invasion defect observed in a mep2 mutant strain is suppressed only by deleting KRH2 and not KRH1. Thus, the results presented indicate that MEP2 functions by inhibiting KRH2 to trigger filamentation response when glucose becomes limiting. Biochemical data and phenotypic response to glucose replenishment reveal that KRH1 and KRH2 are differentially regulated by glucose and ammonium to induce pseudohyphae formation via the cAMP-PKA pathway. In contrast to the current view, this study clearly demonstrates that, KRH1 and KRH2 are not functionally redundant.