Systematic and synthetic approaches to rewire regulatory networks

Abstract

Microbial gene regulatory networks are composed of cis- and trans-components that in concert act to control essential and adaptive cellular functions. Regulatory components and interactions evolve to adopt new configurations through mutations and network rewiring events, resulting in novel phenotypes that may benefit the cell. Advances in high-throughput DNA synthesis and sequencing have enabled the development of new tools and approaches to better characterize and perturb various elements of regulatory networks. Here, we highlight key recent approaches to systematically dissect the sequence space of cis-regulatory elements and trans-regulators as well as their inter-connections. These efforts yield fundamental insights into the architecture, robustness, and dynamics of gene regulation and provide models and design principles for building synthetic regulatory networks for a variety of practical applications.

Keywords: Cis-regulatory elements; DNA synthesis; Gene regulation; Regulatory network rewiring; Trans-regulatory proteins.

Figure 1. Approaches to systematically dissect regulatory network architectures and processes

(a) Systematic analysis of cis-regulatory elements can be performed through in silico library design, in vitro DNA synthesis, and in vivo characterization by various methods including DNA-seq, RNA-seq, and FACS-seq. Detailed analyses of high-throughput datasets yield new regulatory motifs and transcriptomic information (b) Modulating gene expression by altering trans-regulator capacities via deep mutagenesis, domain swapping, and heterologous expression. Mutagenesis of native regulators yields variants with altered cis-element specificity and strength. Domain swapping generates chimeric regulators with altered binding profiles to native regulators. Heterologous expression of foreign regulators can activate cis-elements that are not normal targets of native regulators. (c) Rewiring regulatory networks through combinatorial libraries that alternatively assign cis-elements and trans-regulators with new connections to explore new network architectures. Two regulatory networks are shown, wild type (WT) and rewired network (RN). In the rewired network, global regulators (R1–R3) are assigned to different target genes (dashed lines) compared to their wild-type targets (solid lines). Various characterization tools for phenotyping or network analysis can be utilized to assess network architecture, dynamics, and performance.