Narendra Maheshri

Noise can induce bimodality in positive transcriptional feedback loops without bistability.

Bursty, Infrequent Noise In gene regulatory networks, positive feedback loops can give rise to bistability and hysteresis in gene expression, thereby allowing switching mechanisms and memory effects. To and Maheshri (p. 1142; see the Perspective by Levens and Gupta) eschew the commonly held idea that sigmoidal promoter responses are required to achieve a steady-state bimodal response in a positive feedback loop. Instead, using a model and data from an experiment, they favor noisy gene expression and multiple, noncooperative transcription factor binding as an explanation for the bimodal response, and they expect that similar noisy systems are widespread in biology. Noise induced by multiple binding sites, rather than deterministic bistability, may cause bimodal gene expression in yeast. Transcriptional positive-feedback loops are widely associated with bistability, characterized by two stable expression states that allow cells to respond to analog signals in a digital manner. Using a synthetic system in budding yeast, we show that positive feedback involving a promoter with multiple transcription factor (TF) binding sites can induce a steady-state bimodal response without cooperative binding of the TF. Deterministic models of this system do not predict bistability. Rather, the bimodal response requires a short-lived TF and stochastic fluctuations in the TF’s expression. Multiple binding sites provide these fluctuations. Because many promoters possess multiple binding sites and many TFs are unstable, positive-feedback loops in gene regulatory networks may exhibit bimodal responses, but not necessarily because of deterministic bistability, as is commonly thought.

Detection of single-molecule H2O2 signalling from epidermal growth factor receptor using fluorescent single-walled carbon nanotubes

An emerging concept in cell signalling is the natural role of reactive oxygen species such as hydrogen peroxide (H2O2) as beneficial messengers in redox signalling pathways. The nature of H2O2 signalling is confounded, however, by difficulties in tracking it in living systems, both spatially and temporally, at low concentrations. Here, we develop an array of fluorescent single-walled carbon nanotubes that can selectively record, in real time, the discrete, stochastic quenching events that occur as H2O2 molecules are emitted from individual human epidermal carcinoma cells stimulated by epidermal growth factor. We show mathematically that such arrays can distinguish between molecules originating locally on the cell membrane from other contributions. We find that epidermal growth factor induces 2 nmol H2O2 locally over a period of 50 min. This platform promises a new approach to understanding the signalling of reactive oxygen species at the cellular level.

A regulatory role for repeated decoy transcription factor binding sites in target gene expression.

Tandem repeats of DNA that contain transcription factor (TF) binding sites could serve as decoys, competitively binding to TFs and affecting target gene expression. Using a synthetic system in budding yeast, we demonstrate that repeated decoy sites inhibit gene expression by sequestering a transcriptional activator and converting the graded dose–response of target promoters to a sharper, sigmoidal‐like response. On the basis of both modeling and chromatin immunoprecipitation measurements, we attribute the altered response to TF binding decoy sites more tightly than promoter binding sites. Tight TF binding to arrays of contiguous repeated decoy sites only occurs when the arrays are mostly unoccupied. Finally, we show that the altered sigmoidal‐like response can convert the graded response of a transcriptional positive‐feedback loop to a bimodal response. Together, these results show how changing numbers of repeated TF binding sites lead to qualitative changes in behavior and raise new questions about the stability of TF/promoter binding.

Epigenetic and conventional regulation is distributed among activators of FLO11 allowing tuning of population-level heterogeneity in its expression.

Epigenetic switches encode their state information either locally, often via covalent modification of DNA or histones, or globally, usually in the level of a trans-regulatory factor. Here we examine how the regulation of cis-encoded epigenetic switches controls the extent of heterogeneity in gene expression, which is ultimately tied to phenotypic diversity in a population. We show that two copies of the FLO11 locus in Saccharomyces cerevisiae switch between a silenced and competent promoter state in a random and independent fashion, implying that the molecular event leading to the transition occurs locally at the promoter, in cis. We further quantify the effect of trans regulators both on the slow epigenetic transitions between a silenced and competent promoter state and on the fast promoter transitions associated with conventional regulation of FLO11. We find different classes of regulators affect epigenetic, conventional, or both forms of regulation. Distributing kinetic control of epigenetic silencing and conventional gene activation offers cells flexibility in shaping the distribution of gene expression and phenotype within a population.

Construction of diverse adeno-associated viral libraries for directed evolution of enhanced gene delivery vehicles

Rational design of improved gene delivery vehicles is a challenging and potentially time-consuming process. As an alternative approach, directed evolution can provide a rapid and efficient means for identifying novel proteins with improved function. Here we describe a methodology for generating very large, random adeno-associated viral (AAV) libraries that can be selected for a desired function. First, the AAV2 cap gene is amplified in an error-prone PCR reaction and further diversified through a staggered extension process. The resulting PCR product is then cloned into pSub2 to generate a diverse (>106) AAV2 plasmid library. Finally, the AAV2 plasmid library is used to package a diverse pool of mutant AAV2 virions, such that particles are composed of a mutant AAV genome surrounded by the capsid proteins encoded in that genome, which can be used for functional screening and evolution. This procedure can be performed in approximately 2 weeks.

Computational and experimental analysis of DNA shuffling

We describe a computational model of DNA shuffling based on the thermodynamics and kinetics of this process. The model independently tracks a representative ensemble of DNA molecules and records their states at every stage of a shuffling reaction. These data can subsequently be analyzed to yield information on any relevant metric, including reassembly efficiency, crossover number, type and distribution, and DNA sequence length distributions. The predictive ability of the model was validated by comparison to three independent sets of experimental data, and analysis of the simulation results led to several unique insights into the DNA shuffling process. We examine a tradeoff between crossover frequency and reassembly efficiency and illustrate the effects of experimental parameters on this relationship. Furthermore, we discuss conditions that promote the formation of useless “junk” DNA sequences or multimeric sequences containing multiple copies of the reassembled product. This model will therefore aid in the design of optimal shuffling reaction conditions.

Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM.

A major hurdle to evolutionary engineering approaches for multigenic phenotypes is the ability to simultaneously modify multiple genes rapidly and selectively. Here, we describe a method for in vivo-targeted mutagenesis in yeast, targeting glycosylases to embedded arrays for mutagenesis (TaGTEAM). By fusing the yeast 3-methyladenine DNA glycosylase MAG1 to a tetR DNA-binding domain, we are able to elevate mutation rates >800 fold in a specific ∼20-kb region of the genome or on a plasmid that contains an array of tetO sites. A wide spectrum of transitions, transversions and single base deletions are observed. We provide evidence that TaGTEAM generated point mutations occur through error-prone homologous recombination (HR) and depend on resectioning and the error-prone polymerase Pol ζ. We show that HR is error-prone in this context because of DNA damage checkpoint activation and base pair lesions and use this knowledge to shift the primary mutagenic outcome of targeted endonuclease breaks from HR-independent rearrangements to HR-dependent point mutations. The ability to switch repair in this way opens up the possibility of using targeted endonucleases in diverse organisms for in vivo-targeted mutagenesis.

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

In Vivo Targeted Mutagenesis in Yeast Using TaGTEAM

A key step in evolutionary approaches to protein and cellular engineering is the creation of genetic diversity. In vitro mutagenesis followed by transformation of the appropriate genetic material allows control of which genetic elements to mutate, but diversity is limited by transformation efficiency. Moreover, the process may not scale easily when introducing larger portions of DNA. To overcome this limitation, we developed TaGTEAM (Targeting Glycosylases to Embedded Arrays for Mutagenesis), a method for in vivo mutagenesis restricted to a user-defined genomic region that is based on error-prone repair via homologous recombination. Here we describe detailed protocols for implementing TaGTEAM in yeast, suitability of the technique for various evolutionary engineering goals, and considerations for porting TaGTEAM to other fungal species and beyond.

Using the cre–lox system to randomize target gene expression states and generate diverse phenotypes

Modifying the expression of multiple genes enables both deeper understanding of their function and the engineering of complex multigenic cellular phenotypes. However, deletion or overexpression of multiple genes is typically laborious and involves multiple sequential genetic modifications. Here we describe a strategy to randomize the expression state of multiple genes in Saccharomyces cerevisiae using Cre–loxP recombination. By inserting promoters flanked by inverted loxP sites in front of a gene of interest we can randomly alter its expression by turning it OFF or ON, or between four distinct expression states. We show at least 6 genes can be randomized independently and argue that using orthogonal loxP sites should increase this number to at least 15. Finally, we show how combining this strategy with mating allows easy introduction of native regulation as an additional expression state and use this to probe the role of four different enzymes involved in base excision repair in tolerance to methyl methane sulfonate (MMS), a genotoxic DNA alkylating agent. The set of vectors developed here can be used to randomize the expression of both heterologous genes and endogenous genes, and could immediately prove useful for metabolic engineering in yeast. Because Cre–loxP recombination works in many organisms, this strategy should be readily extendable. Biotechnol. Bioeng. 2013;110: 2677–2686.