No abstract

SummaryIn multicellular organisms, cells actively sense, respond to, and control their own population density. Synthetic mammalian quorum sensing circuits could provide insight into principles of population control and improve cell therapies. However, a key challenge is avoiding their inherent sensitivity to “cheater” mutations that evade control. Here, we repurposed the plant hormone auxin to enable orthogonal mammalian cell-cell communication and quorum sensing. Further, we show that a “paradoxical” circuit design, in which auxin stimulates and inhibits net cell growth at different concentrations, achieves population control that is robust to cheater mutations, controlling growth for 43 days of continuous culture. By contrast, a non-paradoxical control circuit limited growth but was susceptible to mutations. These results establish a foundation for future cell therapies that can respond to and control their own population sizes.

Methylation of cytosines in CG dinucleotides (CpGs) within promoters has been shown to lead to gene silencing in mammals in natural contexts. Recently, engineered recruitment of methyltransferases (DNMTs) at specific loci was shown to be sufficient to silence synthetic and endogenous gene expression through this mechanism. A critical parameter for DNA methylation-based silencing is the distribution of CpGs within the target promoter. However, how the number or density of CpGs in the target promoter affects the dynamics of silencing by DNMT recruitment has remained unclear. Here, we constructed a library of promoters with systematically varying CpG content, and analyzed the rate of silencing in response to recruitment of DNMT. We observed a tight correlation between silencing rate and CpG content. Further, methylation-specific analysis revealed a constant accumulation rate of methylation at the promoter after DNMT recruitment. We identified a single CpG site between TATA box and transcription start site (TSS) that accounted for a substantial part of the difference in silencing rates between promoters with differing CpG content, indicating that certain residues play disproportionate roles in controlling silencing. Together, these results provide a library of promoters for synthetic epigenetic and gene regulation applications, as well as insights into the regulatory link between CpG content and silencing rate.

Methylation of cytosines in CG dinucleotides (CpGs) within promoters has been shown to lead to gene silencing in mammals in natural contexts. Recently, engineered recruitment of methyltransferases (DNMTs) at specific loci was shown to be sufficient to silence synthetic and endogenous gene expression through this mechanism. A critical parameter for DNA methylation-based silencing is the distribution of CpGs within the target promoter. However, how the number or density of CpGs in the target promoter affects the dynamics of silencing by DNMT recruitment has remained unclear. Here we constructed a library of promoters with systematically varying CpG content, and analyzed the rate of silencing in response to recruitment of DNMT. We observed a tight correlation between silencing rate and CpG content. Further, methylation-specific analysis revealed a constant accumulation rate of methylation at the promoter after DNMT recruitment. We identified a single CpG site between TATA box and transcription start site (TSS) that accounted for a substantial part of the difference in silencing rates between promoters with differing CpG content, indicating that certain residues play disproportionate roles in controlling silencing. Together, these results provide a library of promoters for synthetic epigenetic and gene regulation applications, as well as insights into the regulatory link between CpG content and silencing rate.