Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.

In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and -1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these "fragile" nucleosomes play an important role in regulating RPG transcriptional output.

Sugar Influx Sensing by the Phosphotransferase System of Escherichia coli.

The phosphotransferase system (PTS) plays a pivotal role in the uptake of multiple sugars in Escherichia coli and many other bacteria. In the cell, individual sugar-specific PTS branches are interconnected through a series of phosphotransfer reactions, thus creating a global network that not only phosphorylates incoming sugars but also regulates a number of cellular processes. Despite the apparent importance of the PTS network in bacterial physiology, the holistic function of the network in the cell remains unclear. Here we used Förster resonance energy transfer (FRET) to investigate the PTS network in E. coli, including the dynamics of protein interactions and the processing of different stimuli and their transmission to the chemotaxis pathway. Our results demonstrate that despite the seeming complexity of the cellular PTS network, its core part operates in a strikingly simple way, sensing the overall influx of PTS sugars irrespective of the sugar identity and distributing this information equally through all studied branches of the network. Moreover, it also integrates several other specific metabolic inputs. The integrated output of the PTS network is then transmitted linearly to the chemotaxis pathway, in stark contrast to the amplification of conventional chemotactic stimuli. Finally, we observe that default uptake through the uninduced PTS network correlates well with the quality of the carbon source, apparently representing an optimal regulatory strategy.

Sharing of Phosphatases Promotes Response Plasticity in Phosphorylation Cascades.

Sharing of positive or negative regulators between multiple targets is frequently observed in cellular signaling cascades. For instance, phosphatase sharing between multiple kinases is ubiquitous within the MAPK pathway. Here we investigate how such phosphatase sharing could shape robustness and evolvability of the phosphorylation cascade. Through modeling and evolutionary simulations, we demonstrate that 1) phosphatase sharing dramatically increases robustness of a bistable MAPK response, and 2) phosphatase-sharing cascades evolve faster than nonsharing cascades. This faster evolution is particularly pronounced when evolving from a monostable toward a bistable phenotype, whereas the transition speed of a population from a bistable to monostable response is not affected by phosphatase sharing. This property may enable the phosphatase-sharing design to adapt better in a changing environment. Analysis of the respective mutational landscapes reveal that phosphatase sharing reduces the number of limiting mutations required for transition from monostable to bistable responses, hence facilitating a faster transition to such response types. Taken together, using MAPK cascade as an example, our study offers a general theoretical framework to explore robustness and evolutionary plasticity of signal transduction cascades.

Country-specific optimization strategy for testing through contact tracing can help maintain a low reproduction number ([Formula: see text]) during unlock.

In response to the COVID19 pandemic, many countries have implemented lockdowns in multiple phases to ensure social distancing and quarantining of the infected subjects. Subsequent unlocks to reopen the economies started next waves of infection and imposed an extra burden on quarantine to keep the reproduction number ([Formula: see text]) < 1. However, most countries could not effectively contain the infection spread, suggesting identification of the potential sources weakening the effect of lockdowns could help design better informed lockdown-unlock cycles in the future. Here, through building quantitative epidemic models and analyzing the metadata of 50 countries from across the continents we first found that the estimated value of [Formula: see text], adjusted w.r.t the distribution of medical facilities and virus clades correlates strongly with the testing rates in a country. Since the testing capacity of a country is limited by its medical resources, we investigated if a cost-benefit trade-off can be designed connecting testing rate and extent of unlocking. We present a strategy to optimize this trade-off in a country specific manner by providing a quantitative estimate of testing and quarantine rates required to allow different extents of unlocks while aiming to maintain [Formula: see text]. We further show that a small fraction of superspreaders can dramatically increase the number of infected individuals even during strict lockdowns by strengthening the positive feedback loop driving infection spread. Harnessing the benefit of optimized country-specific testing rates would critically require minimizing the movement of these superspreaders via strict social distancing norms, such that the positive feedback driven switch-like exponential spread phase of infection can be avoided/delayed.

Design of a MAPK signalling cascade balances energetic cost versus accuracy of information transmission.

Cellular processes are inherently noisy, and the selection for accurate responses in presence of noise has likely shaped signalling networks. Here, we investigate the trade-off between accuracy of information transmission and its energetic cost for a mitogen-activated protein kinase (MAPK) signalling cascade. Our analysis of the pheromone response pathway of budding yeast suggests that dose-dependent induction of the negative transcriptional feedbacks in this network maximizes the information per unit energetic cost, rather than the information transmission capacity itself. We further demonstrate that futile cycling of MAPK phosphorylation and dephosphorylation has a measurable effect on growth fitness, with energy dissipation within the signalling cascade thus likely being subject to evolutionary selection. Considering optimization of accuracy versus the energetic cost of information processing, a concept well established in physics and engineering, may thus offer a general framework to understand the regulatory design of cellular signalling systems.

The p53-MDM2 network: from oscillations to apoptosis.

The p53 protein is well-known for its tumour suppressor function. The p53-MDM2 negative feedback loop constitutes the core module of a network of regulatory interactions activated under cellular stress. In normal cells, the level of p53 proteins is kept low by MDM2, i.e. MDM2 negatively regulates the activity of p53. In the case of DNA damage, the p53-mediated pathways are activated leading to cell cycle arrest and repair of the DNA. If repair is not possible due to excessive damage, the p53-mediated apoptotic pathway is activated bringing about cell death. In this paper, we give an overview of our studies on the p53-MDM2 module and the associated pathways from a systems biology perspective. We discuss a number of key predictions, related to some specific aspects of cell cycle arrest and cell death, which could be tested in experiments.

Evolutionary Remodeling of Bacterial Motility Checkpoint Control.

Regulatory networks play a central role in the relationship between genotype and phenotype in all organisms. However, the mechanisms that underpin the evolutionary plasticity of these networks remain poorly understood. Here, we used experimental selection for enhanced bacterial motility in a porous environment to explore the adaptability of one of the most complex networks known in bacteria. We found that the resulting phenotypic changes are mediated by adaptive mutations in several functionally different proteins, including multiple components of the flagellar motor. Nevertheless, this evolutionary adaptation could be explained by a single mechanism, namely remodeling of the checkpoint regulating flagellar gene expression. Supported by computer simulations, our findings suggest that the specific "bow-tie" topology of the checkpoint facilitates evolutionary tuning of the cost-benefit trade-off between motility and growth. We propose that bow-tie regulatory motifs, which are widespread in cellular networks, play a general role in evolutionary adaptation.

Gene copy number and cell cycle arrest.

The cell cycle is an orderly sequence of events which ultimately lead to the division of a single cell into two daughter cells. In the case of DNA damage by radiation or chemicals, the damage checkpoints in the G1 and G2 phases of the cell cycle are activated. This results in an arrest of the cell cycle so that the DNA damage can be repaired. Once this is done, the cell continues with its usual cycle of activity. We study a mathematical model of the DNA damage checkpoint in the G2 phase which arrests the transition from the G2 to the M (mitotic) phase of the cell cycle. The tumor suppressor protein p53 plays a key role in activating the pathways leading to cell cycle arrest in mammalian systems. If the DNA damage is severe, the p53 proteins activate other pathways which bring about apoptosis, i.e., programmed cell death. Loss of the p53 gene results in the proliferation of cells containing damaged DNA, i.e., in the growth of tumors which may ultimately become cancerous. There is some recent experimental evidence which suggests that the mutation of a single copy of the p53 gene (in the normal cell each gene has two identical copies) is sufficient to trigger the formation of tumors. We study the effect of reducing the gene copy number of the p53 and two other genes on cell cycle arrest and obtain results consistent with experimental observations.

Noise characteristics of feed forward loops.

A prominent feature of gene transcription regulatory networks is the presence in large numbers of motifs, i.e., patterns of interconnection, in the networks. One such motif is the feed forward loop (FFL) consisting of three genes X, Y and Z. The protein product x of X controls the synthesis of protein product y of Y. Proteins x and y jointly regulate the synthesis of z proteins from the gene Z. The FFLs, depending on the nature of the regulating interactions, can be of eight different types which can again be classified into two categories: coherent and incoherent. In this paper, we study the noise characteristics of FFLs using the Langevin formalism and the Monte Carlo simulation technique based on the Gillespie algorithm. We calculate the variances around the mean protein levels in the steady states of the FFLs and find that, in the case of coherent FFLs, the most abundant FFL, namely, the type-1 coherent FFL, is the least noisy. This is shown to be true for all parameter values when the FFLs operate above their thresholds of activation/repression. In the case of incoherent FFLs, no such general conclusion can be shown. The results suggest possible relationships between noise, functionality and abundance.

Phenotypic heterogeneity in mycobacterial stringent response.

BACKGROUND: A common survival strategy of microorganisms subjected to stress involves the generation of phenotypic heterogeneity in the isogenic microbial population enabling a subset of the population to survive under stress. In a recent study, a mycobacterial population of M. smegmatis was shown to develop phenotypic heterogeneity under nutrient depletion. The observed heterogeneity is in the form of a bimodal distribution of the expression levels of the Green Fluorescent Protein (GFP) as reporter with the gfp fused to the promoter of the rel gene. The stringent response pathway is initiated in the subpopulation with high rel activity. RESULTS: In the present study, we characterise quantitatively the single cell promoter activity of the three key genes, namely, mprA, sigE and rel, in the stringent response pathway with gfp as the reporter. The origin of bimodality in the GFP distribution lies in two stable expression states, i.e., bistability. We develop a theoretical model to study the dynamics of the stringent response pathway. The model incorporates a recently proposed mechanism of bistability based on positive feedback and cell growth retardation due to protein synthesis. Based on flow cytometry data, we establish that the distribution of GFP levels in the mycobacterial population at any point of time is a linear superposition of two invariant distributions, one Gaussian and the other lognormal, with only the coefficients in the linear combination depending on time. This allows us to use a binning algorithm and determine the time variation of the mean protein level, the fraction of cells in a subpopulation and also the coefficient of variation, a measure of gene expression noise. CONCLUSIONS: The results of the theoretical model along with a comprehensive analysis of the flow cytometry data provide definitive evidence for the coexistence of two subpopulations with overlapping protein distributions.

Optimized point dose measurement for monitor unit verification in intensity modulated radiation therapy using 6 MV photons by three different methodologies with different detector-phantom combinations: A comparative study.

The study was aimed to compare accuracy of monitor unit verification in intensity modulated radiation therapy (IMRT) using 6 MV photons by three different methodologies with different detector phantom combinations. Sixty patients were randomly chosen. Zero degree couch and gantry angle plans were generated in a plastic universal IMRT verification phantom and 30×30×30 cc water phantom and measured using 0.125 cc and 0.6 cc chambers, respectively. Actual gantry and couch angle plans were also measured in water phantom using 0.6 cc chamber. A suitable point of measurement was chosen from the beam profile for each field. When the zero-degree gantry, couch angle plans and actual gantry, couch angle plans were measured by 0.6 cc chamber in water phantom, the percentage mean difference (MD) was 1.35%, 2.94 % and Standard Deviation (SD) was 2.99%, 5.22%, respectively. The plastic phantom measurements with 0.125 cc chamber Semiflex ionisation chamber (SIC) showed an MD=4.21% and SD=2.73 %, but when corrected for chamber-medium response, they showed an improvement, with MD=3.38 % and SD=2.59 %. It was found that measurements with water phantom and 0.6cc chamber at gantry angle zero degree showed better conformity than other measurements of medium-detector combinations. Correction in plastic phantom measurement improved the result only marginally, and actual gantry angle measurement in a flat- water phantom showed higher deviation.

Positive feedback and noise activate the stringent response regulator rel in mycobacteria.

Phenotypic heterogeneity in an isogenic, microbial population enables a subset of the population to persist under stress. In mycobacteria, stresses like nutrient and oxygen deprivation activate the stress response pathway involving the two-component system MprAB and the sigma factor, SigE. SigE in turn activates the expression of the stringent response regulator, rel. The enzyme polyphosphate kinase 1 (PPK1) regulates this pathway by synthesizing polyphosphate required for the activation of MprB. The precise manner in which only a subpopulation of bacterial cells develops persistence, remains unknown. Rel is required for mycobacterial persistence. Here we show that the distribution of rel expression levels in a growing population of mycobacteria is bimodal with two distinct peaks corresponding to low (L) and high (H) expression states, and further establish that a positive feedback loop involving the mprAB operon along with stochastic gene expression are responsible for the phenotypic heterogeneity. Combining single cell analysis by flow cytometry with theoretical modeling, we observe that during growth, noise-driven transitions take a subpopulation of cells from the L to the H state within a "window of opportunity" in time preceding the stationary phase. It is these cells which adapt to nutrient depletion in the stationary phase via the stringent response. We find evidence of hysteresis in the expression of rel in response to changing concentrations of PPK1. Hysteresis promotes robustness in the maintenance of the induced state. Our results provide, for the first time, evidence that bistability and stochastic gene expression could be important for the development of "heterogeneity with an advantage" in mycobacteria and suggest strategies for tackling tuberculosis like targeting transitions from the low to the high rel expression state.