An Optogenetic Platform for Real-Time, Single-Cell Interrogation of Stochastic Transcriptional Regulation

Transcription is a highly regulated and inherently stochastic process. The complexity of signal transduction and gene regulation makes it challenging to analyze how the dynamic activity of transcriptional regulators affects stochastic transcription. By combining a fast-acting, photo-regulatable transcription factor with nascent RNA quantification in live cells and an experimental setup for precise spatiotemporal delivery of light inputs, we constructed a platform for the real-time, single-cell interrogation of transcription in Saccharomyces cerevisiae. We show that transcriptional activation and deactivation are fast and memoryless. By analyzing the temporal activity of individual cells, we found that transcription occurs in bursts, whose duration and timing are modulated by transcription factor activity. Using our platform, we regulated transcription via light-driven feedback loops at the single-cell level. Feedback markedly reduced cell-to-cell variability and led to qualitative differences in cellular transcriptional dynamics. Our platform establishes a flexible method for studying transcriptional dynamics in single cells

Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest—of either synthetic or biological origin—is modified at its 3′-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide–alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2′-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2′-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules

Optogenetic Regulation and Interrogation of Gene Expression Variability

Gene expression can exhibit substantial cell-to-cell variability, which partly stems from stochasticity inherent to the transcription process. Nevertheless, its precise regulation is crucial for most biological processes as well as in biotechnological applications. Many of the steps involved in transcriptional regulation are highly dynamic. For example, a variety of transcription factors (TFs) exhibit a pulsatile pattern of activity. However, the complexity of signal transduction and gene regulation hampers our ability to analyze how the dynamic activity of TFs affects transcription and cellular heterogeneity. In this thesis, we establish a synthetic biology approach that makes use of a fast-acting, light-responsive (optogenetic) TF to quantitatively study multiple aspects of transcriptional regulation. To this end, we first implement and thoroughly characterize an optogenetic gene expression system based on the synthetic TF VP-EL222 in S. cerevisiae (chapter 2). We then compare how constant and pulsatile input signals affect gene expression. We find that pulse-width-modulation (PWM), meaning that the duration of input pulses is modulated to regulate gene expression levels, results in the coordinated expression of genes whose promoters respond differentially to constant input signals. We further show that pulsatile TF regulation can reduce cell-to-cell variability in protein expression and that expression mean and variability can be independently tuned by adjusting the frequency of input signals. This phenomenon is then employed to quantify the phenotypic consequence of cell-to-cell variability in metabolic enzyme expression. Both mathematical modeling and experiments indicate that the observed variability reduction largely stems from the attenuation of heterogeneity arising from cell-to-cell differences in TF expression. Modeling further suggests that pulsatile inputs may reduce intrinsic variability that arises from the dynamic/bursty nature of transcription. We then combine the optogenetic TF with live-cell quantification of nascent RNA in order to study transcriptional regulation in more detail (chapter 3). We find that transcription in fact occurs in discontinuous bursts whose duration and timing are modulated by TF activity. By probing the system with pulsed TF inputs, we uncover that promoter activation is largely memoryless and that bursts are terminated upon TF unbinding. Based on these results, we propose a mechanistic model of transcriptional bursting based on TF binding and a rate limiting step, which we interpret as chromatin remodeling, that quantitatively reproduces our experimental observations. Our results demonstrate the merit of using easily controllable synthetic systems to gain new insight into fundamental biological processes. The knowledge gained by this approach may be applied to improve gene expression regulation in biotechnological and biomedical applications

Pulsatile inputs achieve tunable attenuation of gene expression variability and graded multi-gene regulation

Many natural transcription factors are regulated in a pulsatile fashion, but it remains unknown whether synthetic gene expression systems can benefit from such dynamic regulation. Here we find, using a fast-acting, optogenetic transcription factor in Saccharomyces cerevisiae, that dynamic pulsatile signals reduce cell-to-cell variability in gene expression. We then show that by encoding such signals into a single input, expression mean and variability can be independently tuned. Further, we construct a light-responsive promoter library and demonstrate how pulsatile signaling also enables graded multi-gene regulation at fixed expression ratios, despite differences in promoter dose-response characteristics. Pulsatile regulation can thus lead to beneficial functional behaviors in synthetic biological systems, which previously required laborious optimization of genetic parts or the construction of synthetic gene networks.ISSN:2041-172

Synthetic gene networks recapitulate dynamic signal decoding and differential gene expression

Cells live in constantly changing environments and employ dynamic signaling pathways to transduce information about the signals they encounter. However, the mechanisms by which dynamic signals are decoded into appropriate gene expression patterns remain poorly understood. Here, we devise networked optogenetic pathways that achieve dynamic signal processing functions that recapitulate cellular information processing. Exploiting light-responsive transcriptional regulators with differing response kinetics, we build a falling edge pulse detector and show that this circuit can be employed to demultiplex dynamically encoded signals. We combine this demultiplexer with dCas9-based gene networks to construct pulsatile signal filters and decoders. Applying information theory, we show that dynamic multiplexing significantly increases the information transmission capacity from signal to gene expression state. Finally, we use dynamic multiplexing for precise multidimensional regulation of a heterologous metabolic pathway. Our results elucidate design principles of dynamic information processing and provide original synthetic systems capable of decoding complex signals for biotechnological applications.ISSN:2405-472

Cell-in-the-loop pattern formation with optogenetically emulated cell-to-cell signaling

Designing and implementing synthetic biological pattern formation remains challenging due to underlying theoretical complexity as well as the difficulty of engineering multicellular networks biochemically. Here, we introduce a cell-in-the-loop approach where living cells interact through in silico signaling, establishing a new testbed to interrogate theoretical principles when internal cell dynamics are incorporated rather than modeled. We present an easy-to-use theoretical test to predict the emergence of contrasting patterns in gene expression among laterally inhibiting cells. Guided by the theory, we experimentally demonstrate spontaneous checkerboard patterning in an optogenetic setup, where cell-to-cell signaling is emulated with light inputs calculated in silico from real-time gene expression measurements. The scheme successfully produces spontaneous, persistent checkerboard patterns for systems of sixteen patches, in quantitative agreement with theoretical predictions. Our research highlights how tools from dynamical systems theory may inform our understanding of patterning, and illustrates the potential of cell-in-the-loop for engineering synthetic multicellular systems.ISSN:2041-172

Optogenetic closed-loop feedback control of the unfolded protein response optimizes protein production

In biotechnological protein production processes, the onset of protein unfolding at high gene expression levels leads to diminishing production yields and reduced efficiency. Here we show that in silico closed-loop optogenetic feedback control of the unfolded protein response (UPR) in S. cerevisiae clamps gene expression rates at intermediate near-optimal values, leading to significantly improved product titers. Specifically, in a fully-automated custom-built 1L-photobioreactor, we used a cybergenetic control system to steer the level of UPR in yeast to a desired set-point by optogenetically modulating the expression of α-amylase, a hard-to-fold protein, based on real-time feedback measurements of the UPR, resulting in 60% higher product titers. This proof-of-concept study paves the way for advanced optimal biotechnology production strategies that diverge from and complement current strategies employing constitutive overexpression or genetically hardwired circuits.ISSN:1096-7176ISSN:1096-718

Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells

The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.ISSN:2041-172

Principles of Systems Biology, No. 30

This month: two examples of door-opening, innovative microscopy (Garcia and also Benzinger et al.), expanding our functional knowledge of bacteria by over 10,000 genes (Deutschbauer), and probing how RNA structure dictates inclusion in liquid-like droplets in vivo (Langdon and Gladfelter)