Mutual repressor switch

We designed a genetic switch based on a pair of TAL repressors repressing each other as demonstrated before for the classical genetic toggle switch (Gardner et al., 2000). We tested five induction systems for mammalian cells (described in Methods) and adapted three of them for regulation of TAL regulators in the switch. Experimental results demonstrated low stability of the states of the mutual repressor toggle switch. Modeling analysis indicated that the mutual repressor switch is highly intolerant to leaky gene expression and that it requires high cooperativity of repressors in order to exhibit bistability.

Design of the mutual repressor switch

Figure 1.Scheme of a mutual repressor switch. The switch is composed of two repressors that repress transcription of each other through binding to the DNA operator upstream of the promoters, similar as in the original bistable toggle switch (Gardner et al., 2000). .

Initially we decided to construct the designed switch based on TAL repressors using the same type of topology as the classical toggle switch which was based on bacterial repressors, consisting of two repressors that mutually repress each other (Figure 1). We reasoned that a symmetric state in such a system should be inherently unstable and the system should achieve one of the two stable states when one repressor overpowers the other (either due to stochastic events or due to the exogenous input signal).

Toggle switches based on prokaryotic binding domains (e.g. TetR and LacI repressors) have been previously used both in bacteria (Gardner et al., 2000) and in mammalian cells, where a moderate dynamic range (the ratio of expression of a reporter protein between its OFF and ON state) was demonstrated (Kramer et al., 2004). The basic switch was further improved by the addition of RNA silencing elements (Greber et al., 2008).

Figure 2. Components of the mutual repressor switch based on TAL repressors. The system consists of sixoperons - two operons that express different TAL repressors from constitutive promoters with TAL binding sites ensure mutual repression (TALA binding sites [A] on the construct expressing TALB repressor and vice versa), two operons that express TAL repressors from inducible promoters, and twooperons that constitutively express inducer-dependent transcription factors. .

In our switch we planned to use different TALs as DNA binding domains, with a Kruppel-associated box (KRAB) domain as the repressor domain. Our goal was to enable design and introduction of multiple orthogonal switches into the same cell to perform complex logic operations.

Control over the switch is exerted by activating transcription of identical types of TAL repressors under the control of inducible promoters (Figure 2). Because TAL repressors are expected to have very similar properties, we expected them to be balanced better than two different types of naturally occurring repressors, such as for example the TetR or LacI repressor. The ability to prepare a large number of orthogonal TAL repressors could also support function of many orthogonal switches within a single cell.

Reporters. To enable monitoring the level of expression of each TAL repressor and thereby the state of the switch, we linked the TAL repressor with a unique fluorescent reporter - mCitrine or mNeptune (Figure 2). TAL repressor and fluorescent reporter were connected by an intermediate t2A sequence, causing the ribosome to skip the formation of a peptide bond during protein translation within the t2A sequence, producing the repressor and reporter as separate proteins in equimolar amounts (recently also used by Garg et al., 2012; Kim et al., 2011; de Felipe et al., 2006). We also tested our system using a luciferase reporter.

Inducers. For the external regulation of the epigenetic state of the system we introduced plasmids for transcriptional factors that are regulated by small inducer molecules (pristinamycin, doxycycline, erythromycin, rapamycine or ponasterone). We modified the induction systems so that the addition of inducers triggers expression of TAL repressors or activators that can be pluggedin to regulate the TAL-based switch (Figure 3).

The system should assume one of the two stable states. In state 1 TALA:KRAB is produced, which represses the expression of TALB:KRAB. By the addition of inducer 2, transcription of TALB:KRAB is induced and as TALB:KRAB accumulates, it reduces transcription of TALA:KRAB, relieving in a double negative feedback loop the inhibition of its own expression from the constitutive promoter. When the switch is in the state 2, where TALB:KRAB is expressed and TALA:KRAB is repressed, the external signal can be withdrawn and the switch should remain stable in this state. The state of the switch can be reversed again any time by another pulse of an inductor switching the system tothe opposite state.

Figure 3. Designed states of the mutual repressor switch. (A) Addition of inducer 1 triggers dissociation of its cognate repressor from its DNA-binding site. Consequently TALA:KRAB is expressed and inhibits state 2, thus causing the switch to switch to state 1. (B) When the inducer is removed, the inducible repressor binds back to its DNA-binding site, but TALA:KRAB is still expressed from the other plasmid and maintains state 1. (C) and (D) depict the mutual repressor switch switched to state two.

Results

Induction systems

To test the expression of TAL repressors from our induction systems, cells were transfected with a reporter plasmid, an inducible TAL repressor plasmid and an inducer-dependent transcription effector (e.g. E:KRAB, PIP:KRAB). Because the transcription effector represses the expression of the TAL repressor which in turn represses the expression of the reporter, we refer to it as second level logic. We have optimized the amount of plasmids required for the induction from the pristinamycin and erythromycin systems (Figure 4).

Figure 4. Function of induction systems driving transcription of TAL repressors as the input for TAL-based switches. (A) Erythromycin induces expression of TALA:KRAB which in turn represses a reporter. HEK293T cells were transfected with a transfection control (20 ngmCherry), 25 ng of a reporter plasmid (mCitrine under control of the TALA operator) and different amounts of pPCMV_[ETR]_TALA:KRAB and pPCMV_E:KRAB. Two hours post-transfection the cells were stimulated with 2 µg/ml erythromycin. Transfection with TALA:KRAB under a constitutive promoter was used for the experiment control. Fluorescence was measured 3 days post transfection. (B) Pristinamycin induces expression of TALB:KRAB which in turn represses a reporter. HEK293T cells were transfected with transfection control (10 ng Renilla luciferase), a reporter plasmid (10 ng of fLuciferase under control of the TALB operator) and different amounts of PCMV_[PIR]_TALB:KRAB and PIP:KRAB. Two hours post transfection the cells were stimulated with 2 µg/ml pristinamycin. Dual luciferase assay was performed threedays post-transfection. Schemes of erythromycin (A) and pristinamycin (B) induction systems are depicted below the graphs.

Mutual repressor switch

HEK293T cells were transfected with combinations of plasmids encoding the mutual repressor switch and reporters. Initially we tested the system with the addition of one of the TAL repressors expressed constitutively (an internal, constitutive signal) and a luciferase reporter for the opposite TAL. The experiment showed that the second level logic required for the switch works as designed (Figure 5).

We also tested the performance of the cellular logic using flow cytometry to measure the number of cells exhibiting fluorescence of each reporter (mCitrine or mNeptune), with different plasmids. The results additionally confirmed the second level logic with constitutively expressed TAL repressors (Figure 6). Unfortunately, we could not detect the mNeptune reporter with the cytometer due to its fluorescence characteristics, but we could detect it using confocal microscopy.

Figure 5. Constitutively expressed TAL repressor controls the switch which in turn represses a reporter. HEK293T cells were transfected with transfection control (5 ng Renilla luciferase), the mutual repressor switch (20 ng each) , 10 ng of fLuciferase reporter under control of the TALB operon, and different amounts of TALA:KRAB under a constitutive promoter. TALA:KRAB represses the expression of TALB:KRAB from the switch, relieving the repression of the reporter. Dual luciferase assay was performed 4 days post-transfection.

Figure 6. Constitutive TAL repressors drive the switch into the desired state. Numbers of HEK293T cells positive for expression of reporter mCitrine were recorded with flow cytometry. Legend: 1. control - nontransfected cells; 2. cells transfected with p[B]_PCMV_TALA:KRAB_mCitrine (100 ng); 3. cells transfected with p[B]_PCMV_TALA:KRAB_mCitrine and p[A]_PCMV_TALB:KRAB_mNeptune (100 ng each); 4. cells transfected as in 3 with the addition of pPCMV_TALA:KRAB (80 ng); 5. cells transfected as in 3 with the addition of pPCMV_TALB:KRAB (80 ng). The cells were analyzed by flow cytometrythreedays after transfection.

Finally, in cells transfected with the mutual repressor switch without the induction system, we analyzed the expression of fluorescent reporters using confocal microscopy. In a bistable system we would expect each cell to drift stochastically into one or the other stable state, expressing only one of the reporters, because the activity of one repressor inhibits transcription of the other, including its reporter. Analysis of the images however indicated that on average 80% of the cells express both reporters simultaneously, suggesting that the mutual repressor switch does not exhibit a robust bistability (Figure 7).

Figure 7. The mutual repressor switch does not exhibit a bimodal distribution of fluorescence. HEK293T cells were transfected with transfection control (50 ngmCerulean), p[B]_PCMV_TALA:KRAB_mCitrine and p[A]_PCMV_TALB:KRAB_mNeptune (50 ng each) and visualized with confocal microscopy threedays post-transfection. Legend: mCitrine expressing cells depicted green (left); cells expressing mNeptune are marked red (middle) and overly of both images (right). Note: A high fraction of cells expressing both reporters is visible (yellow).

Modeling

We analyzed the expected performance of the designed TAL repressor-based switch using deterministic as well as stochastic simulation. This analysis (described in detail in the Modeling section) revealed that the switch would exhibit bistability only in case of high cooperativity of repressors with the non-linearity coefficient larger than approximately 2 (Figures 8 and 9), while the system does not exhibit bistability in case of a non-cooperative repressor binding and transcription (nonlinearity coefficient of 1). Since the crystal structure of a TAL effector bound to DNA indicates that the molecule binds as a monomer to the target DNA (Mak et al., 2012), it is safe to assume that binding of TAL repressors to the binding sites upstream of the promoter is independent and non-cooperative. However the cooperativity of the whole process, including not just binding, but also silencing of the heterochromatin, transcription, and translation, is important for the response. The concentration response of the TAL repressors suggested that the system has a low nonlinearity coefficient. Additionally the simulation indicated that the mutual repressor switch is very sensitive to leaky transcription (Figure 8).

Figure 8. The mutual repressor switch exhibited no bistability without cooperativity even with a relatively low expression leak (3% of production rates). For a detailed description, see the Modeling section.

Figure 9. The switch exhibited bistability at a nonlinearity coefficient of 3 and leaky expression equal to 0.05 (5% of protein production rates).

From the experimental and modeling results we can therefore conclude that a simple toggle switch design based on mutual TAL repressors is unstable due to low cooperativity. We thus had to design an improved switch to make the switch functional, achieve a higher dynamic range and increased robustness.

References

Gardner, T. S., Cantor, C. R. and Collins, J. J. (2000) Construction of a genetic toggle switch in Escherichia coli. Nature. 403, 339–342.

Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T. Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. Nucleic Acids Res. 40, 7584-7595.

Greber, D., Daoud-El-Baba, M., and Fussenegger, M. (2008) Intronically encoded siRNAs improve dynamic range of mammalian gene regulation systems and toggle switch. Nucleic Acids Res. 36, e101.

Kramer, B.P., Viretta, A.U., El-Baba, M.D., Aubel, D., Weber, W. and Fussenegger, M. (2004) An engineered epigenetic transgene switch in mammalian cells. Nat. biotechnol. 22, 867–870.

Mak, A. N., Bradley, P., Cernadas, R. A., Bogdanove, A. J., and Stoddard, B. L. (2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. Science 335, 716-719.

Kim, J. H., Lee, S.-R., Li, L.-H., Park, H.-J., Park, J.-H., Lee, K. Y., Kim, M.-K., et al. 2011. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PloS one 6,e18556.

de Felipe, P., Luke, G. a, Hughes, L. E., Gani, D., Halpin, C., Ryan, M. D. 2006. E unum pluribus: multiple proteins from a self-processing polyprotein. Trends in biotechnology 24,68–75.

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