Orthogonal control of mean and variability of endogenous genes in a human cell line

Abstract

Stochastic fluctuations at the transcriptional level contribute to isogenic cell-to-cell heterogeneity in mammalian cell populations. However, we still have no clear understanding of the repercussions of this heterogeneity, given the lack of tools to independently control mean expression and variability of a gene. Here, we engineer a synthetic circuit to modulate mean expression and heterogeneity of transgenes and endogenous human genes. The circuit, a Tunable Noise Rheostat (TuNR), consists of a transcriptional cascade of two inducible transcriptional activators, where the output mean and variance can be modulated by two orthogonal small molecule inputs. In this fashion, different combinations of the inputs can achieve the same mean but with different population variability. With TuNR, we achieve low basal expression, over 1000-fold expression of a transgene product, and up to 7-fold induction of the endogenous gene NGFR. Importantly, for the same mean expression level, we are able to establish varying degrees of heterogeneity in expression within an isogenic population, thereby decoupling gene expression noise from its mean. TuNR is therefore a modular tool that can be used in mammalian cells to enable direct interrogation of the implications of cell-to-cell variability.

Independent control of mean and noise by convolution of gene expression distributions

“…Independent control of mean and noise in a single strain requires manipulation of two separate processes impacting protein copy number 36 . This result has been demonstrated by combining two small-molecule responsive regulators in a cascade 37 – 40 , altering both the frequency and bias of promoter state switching 41 , tuning both transcription and mRNA degradation rates 42 , or using a time-varying input to independently control promoter activation frequency and transcription rate 43 .…”

Changes in Biomechanical Properties of A375 Cells Due to the Silencing of TMSB4X Expression Are Not Directly Correlated with Alterations in Their Stemness Features

“…This research field is gaining more and more attention recently and focuses on how fluctuations in transcription result in different phenotypes and cell behavior. Splendid work published by Bonny and colleagues [89] presents an elegant system to study phenotypic heterogeneity, i.e., Tunable Noise Rheostat (TuNR). Using it on the PC9 cell line of human lung adenocarcinoma, the researchers proved varying degrees of gene expression heterogeneity within this cell line.…”

Population Design for Synthetic Gene Circuits

“…However, we could not achieve higher values of the CV , most likely because variability reduction is directly linked to repression strength: increasing repression would decrease cell-to-cell variability as well as mean expression of the repressed component. To weaken or eliminate this link between mean and variability, one may need to consider more complex topologies [3]. Note also that we limited our analysis to a small number of dimensions.…”