Noise-induced switches in network systems of the genetic toggle switch

Background  

Bistability, the capacity to achieve two distinct stable steady states in response to a set of external stimuli, arises within biological systems ranging from the λ phage switch in bacteria to cellular signal transduction pathways in mammalian cells. On the other hand, more and more experimental evidence in the form of bimodal population distribution has indicated that noise plays a very important role in the switching of bistable systems. However, the physiological mechanism underling noise-induced switching behaviors remains to be fully understood.     

Streptogramin-based gene regulation systems for mammalian cells

Here we describe repressible (PipOFF) as well as inducible (PipON) systems for regulated gene expression in mammalian cells, based on the repressor Pip (pristinamycin-induced protein), which is encoded by the streptogramin resistance operon of Streptomyces coelicolor. Expression of genes placed under control of these systems was responsive to clinically approved antibiotics belonging to the streptogramin group (pristinamycin, virginiamycin, and Synercid). The versatility of these systems was demonstrated by streptogramin-regulated expression of mouse erythropoietin (EPO), human placental secreted alkaline phosphatase (SEAP), or green fluorescent protein (GFP) in diverse cell lines (BHK, CHO, HeLa, and mouse myoblasts). Analysis of isogenic constructs in CHO cells demonstrated the PipOFF system gave lower background and higher induction ratios than the widely used tetracycline-repressible (TetOFF) expression systems. The streptogramin-based expression technology was functionally compatible with the TetOFF system, thus enabling the selective use of different antibiotics to independently control two different gene activities in the same cell.     

Potential energy landscape and robustness of a gene regulatory network: toggle switch

Finding a multidimensional potential landscape is the key for addressing important global issues, such as the robustness of cellular networks. We have uncovered the underlying potential energy landscape of a simple gene regulatory network: a toggle switch. This was realized by explicitly constructing the steady state probability of the gene switch in the protein concentration space in the presence of the intrinsic statistical fluctuations due to the small number of proteins in the cell. We explored the global phase space for the system. We found that the protein synthesis rate and the unbinding rate of proteins to the gene were small relative to the protein degradation rate; the gene switch is monostable with only one stable basin of attraction. When both the protein synthesis rate and the unbinding rate of proteins to the gene are large compared with the protein degradation rate, two global basins of attraction emerge for a toggle switch. These basins correspond to the biologically stable functional states. The potential energy barrier between the two basins determines the time scale of conversion from one to the other. We found as the protein synthesis rate and protein unbinding rate to the gene relative to the protein degradation rate became larger, the potential energy barrier became larger. This also corresponded to systems with less noise or the fluctuations on the protein numbers. It leads to the robustness of the biological basins of the gene switches. The technique used here is general and can be applied to explore the potential energy landscape of the gene networks.     

Enhanced dynamic range in a genetically encoded Ca sensor

Genetically encoded fluorescence resonance energy transfer (FRET) indicators are powerful tools for real-time detection of second messenger molecules and activation of signal proteins. However, these fluorescent protein-based sensors typically display marginal FRET efficiency. To improve their FRET efficiency for optical imaging and screening, we developed a number of fluorescent protein mutants based on cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). To improve FRET ratios, which were initially within a narrow dynamic range, we used DNA shuffling to develop a new FRET pair called 3xCFP/Venus. The optimized 3xCFP/Venus pair exhibited higher FRET ratios than CyPet/YPet, which has one of the greatest dynamic ranges of protein-based FRET pairs. We converted this FRET pair to a Ca²⁺ FRET indicators using circular permutation Venus (cpVenus) linked with 3xCFP to form 3xCFP/cpVenus, which displayed an ∼11-fold change in dynamic range in response to Ca²⁺ binding. The enhanced dynamic range for Ca²⁺ concentration detection using 3xCFP/cpVenus was confirmed in PC12 cells using previously established indicators (TN-XXL, ECFP/cpCitrine). To our knowledge, this FRET pair displays the largest dynamic range so far among genetically-encoded sensors, and can be used for sensitive FRET detection.     

Reconstructing the single-cell-level behavior of a toggle switch from population-level measurements

Single-cell-level behaviors of cells are typically inferred from ensemble measurements. However, such inferences implicitly assume a biological version of ergodicity: the percentage of cells in a state is identical to the probability to find a cell in that state. While the ergodicity does not always hold, it has been rarely tested. Here, we reveal that the ergodicity does not necessarily hold even for simple toggle switches and that apparent stabilities of the switches are due to a balance between single-cell-level biased stabilities and growth rates differences. Therefore, verification of the ergodicity and reconstructing single-cell-level behaviors are crucial for understanding intracellular systems.     

Let me count the ways: mechanisms of gene regulation by miRNAs and siRNAs

The downregulation of gene expression by miRNAs and siRNAs is a complex process involving both translational repression and accelerated mRNA turnover, each of which appears to occur by multiple mechanisms. Moreover, under certain conditions, miRNAs are also capable of activating translation. A variety of cellular proteins have been implicated in these regulatory mechanisms, yet their exact roles remain largely unresolved.     

Activation of the mammalian immune system by siRNAs

Inhibition of gene expression through RNA interference (RNAi) is emerging as a powerful experimental tool for gene function and target validation studies. The potential uses of this technology seem unlimited, extending to the prevention and therapy of human diseases. However, recent work demonstrating that there are unanticipated, different nonspecific effects associated with the use of small interfering RNAs in mammals has raised concerns about the safe use of RNAi in vivo. These nonspecific effects include activation of the immune system, potentially harming the individual. The application of screening assays for nonspecific activation of both innate and acquired immunity will be necessary for further development of RNAi as a therapeutic tool.     

Inheritance and state switching of genetic toggle switch in different culture growth phases

Using the gene engineering methods, one can construct simple artificial gene networks with two stable functioning regimes (bistable genetic systems). Such genetic systems make it possible for cells with identical genotype to inherit two alternative phenotypes. The toggle switch is just one of the types of bistable genetic systems. In this work, we investigate the inheritance and switching of toggle switch functioning regimes in the cells at different culture growth phases. It is shown that during transition into the stationary growth phase the inheritance of stable states is disturbed and variations in the toggle-switching rate are more possible in different cells. Also, simultaneous expression of two genes of the system has been experimentally modelled. According to our results, the culture growth phase in this period determines later on the ratio between cell phenotypes in a population.     

Antagonistic control of a dual-input mammalian gene switch by food additives

Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni, which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies.     

Artificial mammalian gene regulation networks-novel approaches for gene therapy and bioengineering

Recently developed strategies for targeted molecular interventions in mammalian cells have created novel opportunities in biotechnological and biomedical research with huge economic and therapeutic impact: the design of mammalian cells with desired phenotypes for biopharmaceutical manufacturing, tissue engineering and gene therapy. These advances have been enabled by constructing artificial gene regulation systems with control modalities similar to those evolved in key regulatory networks of mammalian cells. This review highlights recurring cellular regulation strategies and artificial gene regulation technology currently in use for rational reprogramming of cellular key events including metabolism, growth, differentiation and cell death to achieve sophisticated bioprocess and therapeutic goals.     

Functional comparison of single- and double-stranded siRNAs in mammalian cells

The concept of small interfering RNA (siRNA) has been extended to include not only short double-stranded RNA of 19-25bp, but also single-stranded antisense RNA of the same length, since such single-stranded antisense siRNAs were recently found to be able to inhibit gene expression as well. We made comprehensive comparison of double- and single-stranded siRNA functions in RNA interference (RNAi), targeting multiple sites and different mRNAs, measuring RNAi effects at different time-points and in different cell lines, and examining response curves. Duplex siRNAs were found to be more potent than single-stranded antisense siRNAs. This was verified by the observation that single-stranded antisense siRNAs, which were inefficient in some cases when used alone, could be rescued from inefficiency by sequentially transfecting with the sense siRNAs. This result suggests that the structural character of siRNA molecules might be a more important determinant of siRNA efficiency than the cellular persistence of them.