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BCL-3 attenuation of TNFA expression involves an incoherent feed-forward loop regulated by chromatin structure.

ABSTRACT: Induction of genes is rarely an isolated event; more typically occurring as part of a web of parallel interactions, or motifs, which act to refine and control gene expression. Here, we define an Incoherent Feed-forward Loop motif in which TNFα-induced NF-κB signalling activates expression of the TNFA gene itself and also controls synthesis of the negative regulator BCL-3. While sharing a common inductive signal, the two genes have distinct temporal expression profiles. Notably, while the TNFA gene promoter is primed to respond immediately to activated NF-κB in the nucleus, induction of BCL3 expression only occurs after a time delay of about 1h. We show that this time delay is defined by remodelling of the BCL3 gene promoter, which is required to activate gene expression, and characterise the chromatin delayed induction of BCL3 expression using mathematical models. The models show how a delay in inhibitor production effectively uncouples the rate of response to inflammatory cues from the final magnitude of inhibition. Hence, within this regulatory motif, a delayed (incoherent) feed-forward loop together with differential rates of TNFA (fast) and BCL3 (slow) mRNA turnover provide robust, pulsatile expression of TNFα . We propose that the structure of the BCL-3-dependent regulatory motif has a beneficial role in modulating expression dynamics and the inflammatory response while minimising the risk of pathological hyper-inflammation.

SUBMITTER: Walker T

PROVIDER: S-EPMC3794926 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:RNA-based regulators are promising tools for building synthetic biological systems that provide a powerful platform for achieving a complex regulation of transcription and translation. Recently, de novo-designed synthetic RNA regulators, such as the small transcriptional activating RNA (STAR), toehold switch (THS), and three-way junction (3WJ) repressor, have been utilized to construct RNA-based synthetic gene circuits in living cells. In this work, we utilized these regulators to construct type 1 incoherent feed-forward loop (IFFL) circuits in vivo and explored their dynamic behaviors. A combination of a STAR and 3WJ repressor was used to construct an RNA-only IFFL circuit. However, due to the fast kinetics of RNA-RNA interactions, there was no significant timescale difference between the direct activation and the indirect inhibition, that no pulse was observed in the experiments. These findings were confirmed with mechanistic modeling and simulation results for a wider range of conditions. To increase delay in the inhibition pathway, we introduced a protein synthesis process to the circuit and designed an RNA-protein hybrid IFFL circuit using THS and TetR protein. Simulation results indicated that pulse generation could be achieved with this RNA-protein hybrid model, and this was further verified with experimental realization in E. coli. Our findings demonstrate that while RNA-based regulators excel in speed as compared to protein-based regulators, the fast reaction kinetics of RNA-based regulators could also undermine the functionality of a circuit (e.g., lack of significant timescale difference). The agreement between experiments and simulations suggests that the mechanistic modeling can help debug issues and validate the hypothesis in designing a new circuit. Moreover, the applicability of the kinetic parameters extracted from the RNA-only circuit to the RNA-protein hybrid circuit also indicates the modularity of RNA-based regulators when used in a different context. We anticipate the findings of this work to guide the future design of gene circuits that rely heavily on the dynamics of RNA-based regulators, in terms of both modeling and experimental realization.

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BCL-3 attenuation of TNFA expression involves an incoherent feed-forward loop regulated by chromatin structure.

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