Cooperative assembly confers regulatory specificity and long-term genetic circuit stability [RNA-seq]

In eukaryotes, links in gene regulatory networks are often maintained through cooperative self-assembly between transcriptional regulators (TR) and DNA cis-regulatory motifs, a strategy widely thought to enable highly specific regulatory connections to be formed between otherwise weakly-interacting, low-specificity molecular components. Here, we directly test whether this regulatory strategy can be used to engineer regulatory specificity in synthetic gene circuits constructed in yeast. We show that circuits composed of artificial zinc-finger TRs can be effectively insulated from aberrant misregulation of the host cell genome by using cooperative multivalent TR assemblies to program circuit connections. As we demonstrate in experiments and mathematical models, assembly-mediated regulatory connections enable mitigation of circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. Our 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.在真核生物中，基因调控网络的连接通常通过转录调控因子（transcriptional regulators, TR）与DNA顺式调控基序（cis-regulatory motifs）之间的协同自组装得以维持。这一策略被广泛认为能够在原本相互作用较弱、特异性较低的分子组分之间，构建高度特异性的调控连接。本研究直接检验了该调控策略是否可用于在酿酒酵母中构建的人工基因回路中，实现调控特异性的工程化改造。我们证实，通过协同多价转录调控因子组装体程序化设计回路连接，可使由人工锌指转录调控因子构成的基因回路有效免受宿主细胞基因组的异常失调干扰。正如我们通过实验与数学模型所证明的那样，组装介导的调控连接能够缓解回路引发的适应性缺陷，使得基因回路在长期连续培养过程中保持遗传与功能稳定性。我们这一受自然启发的研究方法，为构建进化稳健的基因回路提供了一种简洁且可推广的途径，这类回路可拓展适配多种宿主生物与应用场景。