Cooperative self-assembly between transcriptional regulators (TRs) and DNA cis-regulatory motifs (CRMs) coordinates precise gene expression in eukaryotes. Complexes of multivalent TRs enable the formation of highly specific regulatory connections between otherwise weakly-interacting, low-specificity molecular components and can be used to engineer regulatory specificity in synthetic gene circuits in yeast. Circuits composed of artificial zinc-finger TRs can be effectively insulated from aberrant misregulation of the host cell genome by relying on the collective action of multiple TRs to program functional circuit connections. Experiments and mathematical models demonstrate that assembly-mediated regulatory connections mitigate circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. This naturally-inspired approach offers a simple, generalizable means for building evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications.
Assemblies of cooperative TRs have also been found to facilitate the gene regulatory network rewiring that drives evolution. TRs that arise in new spatiotemporal regulatory contexts and cooperatively bind to ancestral TRs are stabilized and localized by their interaction, providing an opportunity for developing TR-CRM connections. The stability of the cooperative complex further allows for individual connections to be strengthened or weakened to meet regulatory requirements. Engineered cooperative regulation, paired with laboratory evolution experiments, offers a unique opportunity to elucidate the direct contribution of TR cooperativity to establishing new regulatory connections across evolutionary time. With design guided by a predictive theory of gene network evolution, cooperative circuits are poised for CRM evolution in longterm culture experiments at accelerated timescales compared to circuits without TR-TR cooperativity. In a cooperatively regulated circuit, random mutations to the DNA will be preserved if they initiate or improve a regulatory connection with a stabilized TR, and will be identified by DNA sequencing. This simplified synthetic approach in a controlled culture environment will elucidate the direct contribution of TR-TR cooperativity to network rewiring and evolution.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/46370 |
| Date | 16 June 2023 |
| Creators | Bragdon, Meghan Dorothy-Jean |
| Contributors | Khalil, Ahmad |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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