Gene choice in cancer cells is exclusive in ion transport but concurrent in DNA replication.

Cancers share common cellular and physiological features. Little is known about whether distinctive gene expression patterns can be displayed at the single-cell level by gene families in cancer cells. The expression of gene homologs within a family can exhibit concurrence and exclusivity. Concurrence can promote all-or-none expression patterns of related genes and underlie alternative physiological states. Conversely, exclusive gene families express the same or similar number of homologs in each cell, allowing a broad repertoire of cell identities to be generated. We show that gene families involved in the cell-cycle and antigen presentation are expressed concurrently. Concurrence in the DNA replication complex MCM reflects the replicative status of cells, including cell lines and cancer-derived organoids. Exclusive expression requires precise regulatory mechanism, but cancer cells retain this form of control for ion homeostasis and extend it to gene families involved in cell migration. Thus, the cell adhesion-based identity of healthy cells is transformed to an identity based on migration in the population of cancer cells, reminiscent of epithelial-mesenchymal transition.

Polysome propensity and tunable thresholds in coding sequence length enable differential mRNA stability.

The half-life of mRNAs, as well as their translation, increases in proportion to the optimal codons, indicating a tight coupling of codon-dependent differential translation and degradation. Little is known about the regulation of this coupling. We found that the mRNA stability gain in yeast depends on the mRNA coding sequence length. Below a critical length, codon optimality fails to affect the stability of mRNAs although they can be efficiently translated into short peptides and proteins. Above this threshold length, codon optimality-dependent differential mRNA stability emerges in a switch-like fashion, which coincides with a similar increase in the polysome propensity of the mRNAs. This threshold length can be tuned by the untranslated regions (UTR). Some of these UTRs can destabilize mRNAs without reducing translation, which plays a role in controlling the amplitude of the oscillatory expression of cell cycle genes. Our findings help understand the translation of short peptides from noncoding RNAs and the translation by localized monosomes in neurons.

The life and death of RNA across temperatures.

Temperature is an environmental condition that has a pervasive effect on cells along with all the molecules and reactions in them. The mechanisms by which prototypical RNA molecules sense and withstand heat have been identified mostly in bacteria and archaea. The relevance of these phenomena is, however, broader, and similar mechanisms have been recently found throughout the tree of life, from sex determination in reptiles to adaptation of viral RNA polymerases, to genetic disorders in humans. We illustrate the temperature dependence of RNA metabolism with examples from the synthesis to the degradation of mRNAs, and review recently emerged questions. Are cells exposed to greater temperature variations and gradients than previously surmised? How do cells reconcile the conflicting thermal stability requirements of primary and tertiary structures of RNAs? To what extent do enzymes contribute to the temperature compensation of the reaction rates in mRNA turnover by lowering the energy barrier of the catalyzed reactions? We conclude with the ecological, forensic applications of the temperature-dependence of RNA degradation and the biotechnological aspects of mRNA vaccine production.

Determinants of the temperature adaptation of mRNA degradation.

The rate of chemical reactions increases proportionally with temperature, but the interplay of biochemical reactions permits deviations from this relation and adaptation. The degradation of individual mRNAs in yeast increased to varying degrees with temperature. We examined how these variations are influenced by the translation and codon composition of mRNAs. We developed a method that revealed the existence of a neutral half-life above which mRNAs are stabilized by translation but below which they are destabilized. The proportion of these two mRNA subpopulations remained relatively constant under different conditions, even with slow cell growth due to nutrient limitation, but heat shock reduced the proportion of translationally stabilized mRNAs. At the same time, the degradation of these mRNAs was partially temperature-compensated through Upf1, the mediator of nonsense-mediated decay. Compensation was also promoted by some asparagine and serine codons, whereas tyrosine codons promote temperature sensitization. These codons play an important role in the degradation of mRNAs encoding key cell membrane and cell wall proteins, which promote cell integrity.

Gene Families With Stochastic Exclusive Gene Choice Underlie Cell Adhesion in Mammalian Cells.

Exclusive stochastic gene choice combines precision with diversity. This regulation enables most T-cells to express exactly one T-cell receptor isoform chosen from a large repertoire, and to react precisely against diverse antigens. Some cells express two receptor isoforms, revealing the stochastic nature of this process. A similar regulation of odorant receptors and protocadherins enable cells to recognize odors and confer individuality to cells in neuronal interaction networks, respectively. We explored whether genes in other families are expressed exclusively by analyzing single-cell RNA-seq data with a simple metric. This metric can detect exclusivity independently of the mean value and the monoallelic nature of gene expression. Chromosomal segments and gene families are more likely to express genes concurrently than exclusively, possibly due to the evolutionary and biophysical aspects of shared regulation. Nonetheless, gene families with exclusive gene choice were detected in multiple cell types, most of them are membrane proteins involved in ion transport and cell adhesion, suggesting the coordination of these two functions. Thus, stochastic exclusive expression extends beyond the prototypical families, permitting precision in gene choice to be combined with the diversity of intercellular interactions.

Tuning up Transcription Factors for Therapy.

The recent developments in the delivery and design of transcription factors put their therapeutic applications within reach, exemplified by cell replacement, cancer differentiation and T-cell based cancer therapies. The success of such applications depends on the efficacy and precision in the action of transcription factors. The biophysical and genetic characterization of the paradigmatic prokaryotic repressors, LacI and TetR and the designer transcription factors, transcription activator-like effector (TALE) and CRISPR-dCas9 revealed common principles behind their efficacy, which can aid the optimization of transcriptional activators and repressors. Further studies will be required to analyze the linkage between dissociation constants and enzymatic activity, the role of phase separation and squelching in activation and repression and the long-range interaction of transcription factors with epigenetic regulators in the context of the chromosomes. Understanding these mechanisms will help to tailor natural and synthetic transcription factors to the needs of specific applications.

Contribution of RNA Degradation to Intrinsic and Extrinsic Noise in Gene Expression.

Genetically identical cells contain variable numbers of molecules, even if the cells share the same environment. This stochastic variability is prominent when molecules have low abundance, which is the case for mRNA noise. Most studies focused on how transcription affects mRNA noise, and little is known about the role of RNA degradation. To discriminate the fluctuations in these processes during the decay of a pair of reporter mRNAs, we quantified the uncorrelated intrinsic and the correlated extrinsic noise using single-molecule RNA FISH. Intrinsic noise converges to the Poisson level during the decay. mRNAs that have a short half-life are more susceptible to extrinsic noise than stable mRNAs. However, the Xrn1 exonuclease and the NMD pathways, which degrade mRNAs rapidly, were found to have lower fluctuation, which mitigates the noise of the short-lived mRNAs. This permits low variability across the entire range of mRNA half-lives.

Synthetic Transcription Factors Switch from Local to Long-Range Control during Cell Differentiation.

Genes, including promoters and enhancers, are regulated by short- and long-range interactions in higher eukaryotes. It is unclear how mammalian gene expression subject to such a combinatorial regulation can be controlled by synthetic transcription factors (TF). Here, we studied how synthetic TALE transcriptional activators and repressors affect the expression of genes in a gene array during cellular differentiation. The protocadherin gene array is silent in mouse embryonic stem (ES) and neuronal progenitor cells. The TALE transcriptional activator recruited to a promoter activates specifically the target gene in ES cells. Upon differentiation into neuronal progenitors, the transcriptional regulatory logic changes: the same activator behaves like an enhancer, activating distant genes in a correlated, stochastic fashion. The long-range effect is reflected by the alterations in CpG methylation. Our findings reveal the limits of precision and the opportunities in the control of gene expression for TF-based therapies in cells of various differentiation stages.

Stochastic Gene Choice during Cellular Differentiation.

Genes in higher eukaryotes are regulated by long-range interactions, which can determine what combination of genes is expressed in a chromosomal segment. The choice of the genes can display exclusivity, independence, or co-occurrence. We introduced a simple measure to quantify this interdependence in gene expression and differentiated mouse embryonic stem cells to neurons to measure the single-cell expression of the gene isoforms in the protocadherin (Pcdh) cluster, a key component of neuronal diversity. As the neuronal progenitors mature into neurons, expression of the gene isoforms in the Pcdh array is initially concurrent. Even though the number of the expressed genes is increasing during differentiation, the expression shifts toward exclusivity. The expression frequency correlates highly with CTCF binding to the promoters and follows dynamically the changes in the binding during the differentiation. These findings aid in understanding the interplay between cellular differentiation and stochastic gene choice.

Impact of Methods on the Measurement of mRNA Turnover.

The turnover of the RNA molecules is determined by the rates of transcription and RNA degradation. Several methods have been developed to study RNA turnover since the beginnings of molecular biology. Here we summarize the main methods to measure RNA half-life: transcription inhibition, gene control, and metabolic labelling. These methods were used to detect the cellular activity of the mRNAs degradation machinery, including the exo-ribonuclease Xrn1 and the exosome. On the other hand, the study of the differential stability of mature RNAs has been hampered by the fact that different methods have often yielded inconsistent results. Recent advances in the systematic comparison of different method variants in yeast have permitted the identification of the least invasive methodologies that reflect half-lives the most faithfully, which is expected to open the way for a consistent quantitative analysis of the determinants of mRNA stability.

Multiplexed gene control reveals rapid mRNA turnover.

The rates of mRNA synthesis and decay determine the mRNA expression level. The two processes are under coordinated control, which makes the measurements of these rates challenging, as evidenced by the low correlation among the methods of measurement of RNA half-lives. We developed a minimally invasive method, multiplexed gene control, to shut off expression of genes with controllable synthetic promoters. The method was validated by measuring the ratios of the nascent to mature mRNA molecules and by measuring the half-life with endogenous promoters that can be controlled naturally or through inserting short sequences that impart repressibility. The measured mRNA half-lives correlated highly with those obtained with the metabolic pulse-labeling method in yeast. However, mRNA degradation was considerably faster in comparison to previous estimates, with a median half-life of around 2 min. The half-life permits the estimation of promoter-dependent and promoter-independent transcription rates. The dynamical range of the promoter-independent transcription rates was larger than that of the mRNA half-lives. The rapid mRNA turnover and the broad adjustability of promoter-independent transcription rates are expected to have a major impact on stochastic gene expression and gene network behavior.

Measurement of In Vivo Protein Binding Affinities in a Signaling Network with Mass Spectrometry.

Protein interaction networks play a key role in signal processing. Despite the progress in identifying the interactions, the quantification of their strengths lags behind. Here we present an approach to quantify the in vivo binding of proteins to their binding partners in signaling-transcriptional networks, by the pairwise genetic isolation of each interaction and by varying the concentration of the interacting components over time. The absolute quantification of the protein concentrations was performed with targeted mass spectrometry. The strengths of the interactions, as defined by the apparent dissociation constants, ranged from subnanomolar to micromolar values in the yeast galactose signaling network. The weak homodimerization of the Gal4 activator amplifies the signal elicited by glucose. Furthermore, combining the binding constants in a feedback loop correctly predicted cellular memory, a characteristic network behavior. Thus, this genetic-proteomic binding assay can be used to faithfully quantify how strongly proteins interact with proteins, DNA and metabolites.

Measurement of bistability in a multidimensional parameter space.

Bistability plays an important role to generate two stable states for alternative cell fates, or to promote cellular diversity and cell cycle oscillations. Positive feedback loops are necessary for the existence of bistability and ultrasensitive reactions in the loops broaden the parameter range of bistability. The broader parameter range a system's bistability covers, the more robust the two states are. It is challenging to determine the bistable range of a parameter because noise and transient processes induce transitions between the two states. We found that a threshold of transition rates coincides with the bistability boundaries determined by the open-loop approach. With this threshold, we estimated the boundaries for various synthetic single-gene positive feedback loops in yeast in a two dimensional parameter space: the inducer concentration and promoter dynamic range. While the bistable range of inducer concentration was influenced by many factors, the promoter dynamic range was more informative. The narrowest promoter dynamic range at which bistability can emerge revealed whether the full potential of an ultrasensitive reaction, such as dimerization, is exploited in the feedback loop. The convenient control of basal expression to adjust the promoter dynamic range permits a practical and reliable comparison of robustness of related positive feedback loops.

An open-loop approach to calculate noise-induced transitions.

Bistability permits the co-existence of two distinct cell fates in a population of genetically identical cells. Noise induced transitions between two fates of a bistable system are difficult to calculate due to the intricate interplay between nonlinear dynamics and noise in bistable positive feedback loops. Here we opened multivariable feedback loops at the slowest variable to obtain the open-loop function and the fluctuations in the open-loop output. By the subsequent reclosing of the loop, we calculated the mean first passage time (MFPT) using the Fokker-Planck equation in good agreement with the exact stochastic simulation. When an external component interacts with a feedback component, it amplifies the extrinsic noise in the loop. Consequently, the open-loop function is shifted and the transition rates between the two states in the closed loop are increased. Despite this shift, the open-loop output reflects the system faithfully to predict the MFPT in the feedback loop. Therefore, the open-loop approach can help theoretical analysis. Furthermore, the measurement of the mean value, variance, and the reaction time-scale of the open-loop output permits the prediction of MFPT simply from experimental data, which underscores the practical value of the stochastic open-loop approach.

Contribution of Bistability and Noise to Cell Fate Transitions Determined by Feedback Opening.

Alternative cell fates represent a form of non-genetic diversity, which can promote adaptation and functional specialization. It is difficult to predict the rate of the transition between two cell fates due to the strong effect of noise on feedback loops and missing parameters. We opened synthetic positive feedback loops experimentally to obtain open-loop functions. These functions allowed us to identify a deterministic model of bistability by bypassing noise and the requirement to resolve individual processes in the loop. Combining the open-loop function with kinetic measurements and reintroducing the measured noise, we were able to predict the transition rates for the feedback systems without parameter tuning. Noise in gene expression was the key determinant of the transition rates inside the bistable range. Transitions between two cell fates were also observed outside of the bistable range, evidenced by bimodality and hysteresis. In this case, a slow transient process was the rate-limiting step in the transitions. Thus, feedback opening is an effective approach to identify the determinants of cell fate transitions and to predict their rates.

Protein Dimerization Generates Bistability in Positive Feedback Loops.

Bistability plays an important role in cellular memory and cell-fate determination. A positive feedback loop can generate bistability if it contains ultrasensitive molecular reactions. It is often difficult to detect bistability based on such molecular mechanisms due to its intricate interaction with cellular growth. We constructed transcriptional feedback loops in yeast. To eliminate growth alterations, we reduced the protein levels of the transcription factors by tuning the translation rates over two orders of magnitude with designed RNA stem loops. We modulated two ultrasensitive reactions, homodimerization and the cooperative binding of the transcription factor to the promoter. Either of them is sufficient to generate bistability on its own, and when acting together, a particularly robust bistability emerges. This bistability persists even in the presence of a negative feedback loop. Given that protein homodimerization is ubiquitous, it is likely to play a major role in the behavior of regulatory networks.

Identification of optimal parameter combinations for the emergence of bistability.

Bistability underlies cellular memory and maintains alternative differentiation states. Bistability can emerge only if its parameter range is either physically realizable or can be enlarged to become realizable. We derived a general rule and showed that the bistable range of a reaction parameter is maximized by a pair of other parameters in any gene regulatory network provided they satisfy a general condition. The resulting analytical expressions revealed whether or not such reaction pairs are present in prototypical positive feedback loops. They are absent from the feedback loop enclosed by protein dimers but present in both the toggle-switch and the feedback circuit inhibited by sequestration. Sequestration can generate bistability even at narrow feedback expression range at which cooperative binding fails to do so, provided inhibition is set to an optimal value. These results help to design bistable circuits and cellular reprogramming and reveal whether bistability is possible in gene networks in the range of realistic parameter values.

Quantification of pre-mRNA escape rate and synergy in splicing.

Splicing reactions generally combine high speed with accuracy. However, some of the pre-mRNAs escape the nucleus with a retained intron. Intron retention can control gene expression and increase proteome diversity. We calculated the escape rate for the yeast PTC7 intron and pre-mRNA. This prediction was facilitated by the observation that splicing is a linear process and by deriving simple algebraic expressions from a model of co- and post-transcriptional splicing and RNA surveillance that determines the rate of the nonsense-mediated decay (NMD) of the pre-mRNAs with the retained intron. The escape rate was consistent with the observed threshold of splicing rate below which the mature mRNA level declined. When an mRNA contains multiple introns, the outcome of splicing becomes more difficult to predict since not only the escape rate of the pre-mRNA has to be considered, but also the possibility that the splicing of each intron is influenced by the others. We showed that the two adjacent introns in the SUS1 mRNA are spliced cooperatively, but this does not counteract the escape of the partially spliced mRNA. These findings will help to infer promoter activity and to predict the behavior of and to control splicing regulatory networks.

Myoblasts inhibit prostate cancer growth by paracrine secretion of tumor necrosis factor-α.

PURPOSE: Myoblasts can form muscle fibers after transplantation. Therefore, they are envisioned as a treatment for urinary incontinence after radical prostatectomy. However, to our knowledge the safety of this treatment and the interaction of myoblasts with any remaining neighboring cancer are unknown. We investigated the interactions between myoblasts and prostate carcinoma cells in vitro and in vivo. MATERIALS AND METHODS: Myoblasts isolated from the rectus abdominis were used in a series of co-culture experiments with prostate cancer cells and subcutaneously co-injected in vivo. Cell proliferation, cell cycle arrest and apoptosis of cancer in co-culture with myoblasts were assessed. Tumor volume and metastasis formation were evaluated in a mouse model. Tissue specific markers were assessed by immunohistochemistry, fluorescence activated cell sorting analysis, Western blot and real-time quantitative polymerase chain reaction. RESULTS: Myoblasts in proximity to tumor provided paracrine tumor necrosis factor-α to their microenvironment, decreasing the tumor growth of all prostate cancer cell lines examined. Co-culture experiments revealed induction of cell cycle arrest, tumor death by apoptosis and increased myoblast differentiation. This effect was largely blocked by tumor necrosis factor-α inhibition. The same outcome was noted in a mouse model, in which co-injected human myoblasts also inhibited the tumor growth and metastasis formation of all prostate cancer cell lines evaluated. CONCLUSIONS: Myoblasts restrict prostate cancer growth and limit metastasis formation by paracrine tumor necrosis factor-α secretion in vitro and in vivo.