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Cooperative assembly confers regulatory specificity and long-term genetic circuit stability [ChIP-Seq]

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE203144 | GEO | 2022/05/20

REPOSITORIES: GEO

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Project description:Human regulatory T cells (TR) cells have potential for the treatment of immune mediated diseases, such as graft versus host disease, but the anergic phenotype of these cells makes them difficult to expand in vitro. We have examined the requirements for growth and cytokine expression from highly purified human TR cells, and correlated these findings with the signal transduction events of these cells. We demonstrate that these cells do not proliferate or secrete IL-10 even in the presence of high doses of IL-2. Stimulation with a superagonistic anti-CD28 antibody (clone 9D4) and IL-2 partially reversed the proliferative defect, and this correlated with reversal of the defective calcium mobilization in these cells. Dendritic cells were effective at promoting TR cell proliferation, and under these conditions the proliferative capacity of TR cells was comparable to conventional CD4 lymphocytes. Blocking TGF-beta activity abrogated IL-10 expression from these cells, while addition of TGF-beta resulted in IL-10 production. These data demonstrate the ability of dendritic cells to provide proper costimulation to overcome the anergic phenotype of TR cells. In addition, these data demonstrate for the first time that TGF-beta is critical to enable TR cells to express IL-10. In order to confirm that our isolation strategy resulted in cells with characteristics of TR cells, we performed gene expression profiling of highly purified TR cells from two different individuals, which were compared to the gene expression profile of naÃ¯ve CD4+CD45RA+ cells. We first performed hierarchical clustering of the gene expression profiles of unactivated naÃ¯ve and TR cells from the two individuals. We show that the gene expression patterns of TR cells were highly similar between the two samples (correlation coefficient of 0.82), as were the gene expression profiles of naÃ¯ve CD4 cells between the two samples (correlation coefficient of 0.91). After averaging the gene expression signal from these two individuals, we compared the gene expression profile of unactivated TR cells with naÃ¯ve CD4 cells and show that TR cells over-express a variety of genes known to be associated with a TR phenotype (e.g. CTLA-4, IKZF2, IL-2RA). In addition, we also detected very high levels of expression of genes not typically associated with a TR phenotype, with the highest level of expression of IL-1 receptors in resting TR cells (Figure 2). These data confirm that the sorting strategy was effective at isolating T cells expressing a TR phenotype.

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Cooperative assembly confers regulatory specificity and long-term genetic circuit stability [ChIP-Seq]

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