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Hysteresis in a synthetic mammalian gene network Kramer, B.P. and Fussenegger, M. 2005. Hysteresis in a synthetic mammalian gene network. Proc. Natl. Acad. Sci. 102: 9517-9522. Overview In 2005, the lab of Martin Fussenegger (at the Federal Institute of Technology in Zurich, Switzerland) published a paper on the construction a hysteretic switch in mammalian cells. This research can be viewed as an extension of the toggle switch that was constructed in the lab of J.J. Collins 5 years earlier. While the toggle switch construct was able to respond to two separate inputs in different ways, the hysteretic system can respond to a single input in different ways depending on the history of the cell. Using an autoregulatory positive feedback design, this system is a step in the direction of constructing a permanent memory circuit, which will be the topic of the next example paper. This work is also significant because of the use of a mammalian cell chassis, in which it is substantially more complicated to implement a genetic network than it is in E. coli. What is Hysteresis? Figure 1: A graphical depiction of hysteresis. "A system with hysteresis exhibits path-dependence, or 'rate-independent memory'. Consider a deterministic system with no hysteresis and no dynamics. In that case, we can predict the output of the system at some instant in time, given only the input to the system at that instant. If the system has hysteresis, then this is not the case; we can't predict the output without looking at the history of the input. In order to predict the output, we must look at the path that the input followed before it reached its current value. A system with hysteresis has memory" (Wikipedia). In Figure 1 on the right, hysteresis in demonstrated graphically. The arrows indicate the direction of movement from one state to another. For a system that exists in the low output state initially (red line), a relatively high level of input is required to induce a change to a high system output. For a system that exists in the high state initially (blue line), a relatively low level of input is required to induce a change to a low system output. In other words, more extreme amounts of input are required to move out of a state than are required to move into a state (meaning that the system resists a change of state). Specific Biological Design Figure 2 below shows the specific biological design that was used to construct a hysteretic gene network in mammalian cells. The system uses a type of autoregulatory positive feedback whereby an activator gene is downstream of its promoter, but in this case the activator is in competition for promoter binding with a repressor molecule. The activator molecule in this design is a fusion of the tetR gene and the VP16 transactivation domain. This protein binds to the tetO7 operator region of the hybrid promoter (PhCMVmin) and increases the promoter's affinity for RNA Polymerase, thus increasing promoter activity. The reporter gene in this design is secreted alkaline phosphatase (SEAP), a protein whose levels inside the cell can be easily measured without cell lysis. The SEAP gene is also downstream of the hybrid promoter (PhCMVmin) and is, therefore, transcribed at the same rate as the transactivator gene. On a separate plasmid, the E-KRAB repressor gene lies downstream of a constitutive (always on) promoter, PSV40. This repressor binds to the ETR8 operator region of the hybrid promoter (PhCMVmin) and blocks binding of RNA Polymerase to the promoter. The antibiotic erythromycin (EM) is able to bind to the E-KRAB repressor and prevent it from binding to the promoter. With no input of EM in the system, repressor binding will out-compete activator binding, and transcription downstream of the hybrid promoter will be blocked. As the concentration of EM in the system is increased, more promoter activity occurs and the system is able to toggle into the "SEAP on" state. In this state, an overabundance of activator in the system allows autoregulatory positive feedback to win out over repression from E-KRAB. Because of the competitive nature of promoter binding, the system resists a change in its stable steady state (whether that be from on to off or off to on) and, therefore, demonstrates hysteresis. This effectively means that a higher concentration of EM is required to change the system from off to on than is required to remain in the on state. Likewise, a lower concentration of EM is required to change the system from on to off than is required to remain in the off state. Figure 2: Biological design of a hysteretic switch in mammalian cells. Mathematical Modeling Results Conclusions