Synthetic approaches to study transcriptional networks and noise in mammalian systems

Abstract

Synthetic biology aims to build new functional organisms and to rationally re-design existing ones by applying the engineering principle of modularity. Apart from building new life forms to perform technical applications, the approach of synthetic biology is useful to dissect complex biological phenomena into simple and easy to understand synthetic modules. Synthetic gene networks have been successfully implemented in prokaryotes and lower eukaryotes, with recent approaches moving ahead towards the mammalian environment. However, synthetic circuits in higher eukaryotes present a more challenging scenario, since its reliability is compromised because of the strong stochastic nature of transcription. Here, the authors review recent approaches that take advantage of the noisy response of synthetic regulatory circuits to learn key features of the complex machinery that orchestrates transcription in higher eukaryotes. Understanding the causes and consequences of biological noise will allow us to design more reliable mammalian synthetic circuits with revolutionary medical applications.

Fig. 1

Synthetic network to study transcriptional bursting in mammalian cells a Diagram of the synthetic construct developed by Raj et al. [12]. The cell line CHO was engineered to express the tetracycline‐controlled Tet‐off transactivator tTA (a fusion of the E. coli TetR repressor and the Herpes simplex VP16) which binds to the tet operator sequence and promotes the transcription of the YFP. The antibiotic doxycycline, which prevents tTA to bind to the operator sequence, is added to the growth medium to regulate the level of produced mRNA. The probe binding sequence detected by fluorescent in situ hybridisation FISH provides single molecule sensitivity b Typical output of transcriptional burst in the mRNA production

Fig. 2

Mammalian synthetic negative feedback loop with variable delay a Diagram of the synthetic construct developed by Swinburne et al. [24]. An oscillatory behaviour is achieved as a result of an imposed delay in an autoinhibitory system. TetR binds to the tet‐O sequence and inhibits the transcription of the YFP that is used to monitor the network dynamics. A couple of exogenous intronic sequences are inserted after the first intron of the promoter (1 kb). The modular intron sequences introduced were either 7 or 16 kb long, providing three different types of cells with the same coding region b Mathematical modelling of the synthetic network include explicit simulations of the RNA polymerase II transcription elongation, that was forced to move along vector spaces equivalent to either 3, 10 or 19 kb. Simulated polymerases transcribed at constant velocities sampled from a Gaussian distribution with a mean velocity of 1 kb/min and a σ = 0.255 kb/min. First column represents protein output for different gene length. Oscillations varied with gene length showing that transcriptional bursting is promoted by long polymers chains. As shown in the centre and right columns, time between pulses and protein levels, respectively, depicted a broader distributions of values when increasing gene length, proving that congestion events produce large heterogeneities in the system dynamics

Fig. 3

Simulations of polymerase traffic jams with different velocity distributions for a 10 kb gene a For standard deviation σ = 0, polymerases transcribe and terminate at a constant rate leading to regular oscillations b When σ = 0.44 kb/min, the assignation of random broad velocities produces irregular pulses and long pauses. The slow polymerase object creates a congestion where the pause is correlated with the amount of polymerases terminating at the same time

Fig. 4

Synthetic positive feedback loop leading to a bimodal and hysteretic expression pattern a Scheme of the circuit implemented by Kramer et al. in CHO cells [40]. The tetracycline dependent transactivator (tTA) induces the hybrid promoter tet ₀₇ − ETR₈ − P _hCMVmin driving its own expression as well as the expression of SEAP (human placental secreted alkaline phosphatase), used to quantify the expression of tTA. The constitutively expressed transrepressor E‐KRAB (a fusion of the E. coli macrolide resistance operon repressor E and the human trans‐silencing domain KRAB) binds to the ETR₈ region of the promoter in a fashion modulated by the antibiotic erythromycin (EM) who inhibits the transrepressor capacity by binding also to the hybrid promoter. In the absence of antibiotics the E‐KRAB action dominates over the tTA function resulting in the suppression of the positive feedback loop b Profile of the gene expression provided by the synthetic arrangement. For the same stimulus two possible responses are possible (bistability) and the signal intensity to switch from one state to another depends on the initial state (hysteresis)