Tuning of mRNA stability through altering 3'-UTR sequences generates distinct output expression in a synthetic circuit driven by p53 oscillations.

Synthetic biological circuits that can generate outputs with distinct expression dynamics are useful for a variety of biomedical and industrial applications. We present a method to control output dynamics by altering output mRNA decay rates. Using oscillatory expression of the transcription factor p53 as the circuit regulator, we use two approaches for controlling target gene transcript degradation rates based on the output gene's 3'-untranslated region (3'-UTR): introduction of copies of destabilizing AU-rich elements into the 3'-UTR or swapping in naturally occurring 3'-UTRs conferring different transcript stabilities. As a proof of principle, we apply both methods to control the expression dynamics of a fluorescent protein and visualize the circuit output dynamics in single living cells. We then use the naturally occurring 3'-UTR approach to restore apoptosis in a tunable manner in a cancer cell line deficient for caspase-3 expression. Our method can be readily adapted to regulate multiple outputs each with different expression dynamics under the control of a single naturally occurring or synthetically constructed biological oscillator.

Protein stability of p53 targets determines their temporal expression dynamics in response to p53 pulsing.

In response to DNA damage, the transcription factor p53 accumulates in a series of pulses. While p53 dynamics play a critical role in regulating stress responses, how p53 pulsing affects target protein expression is not well understood. Recently, we showed that p53 pulses generate diversity in target mRNA expression dynamics; however, given that mRNA and protein expression are not necessarily well correlated, it remains to be determined how p53 pulses impact target protein expression. Using computational and experimental approaches, we show that target protein decay rates filter p53 pulses: Distinct target protein expression dynamics are generated depending on the relationship between p53 pulse frequency and target mRNA and protein stability. Furthermore, by mutating the targets MDM2 and PUMA to alter their stabilities, we show that downstream pathways are sensitive to target protein decay rates. This study delineates the mechanisms by which p53 dynamics play a crucial role in orchestrating the timing of events in the DNA damage response network.

Global Inhibition with Specific Activation: How p53 and MYC Redistribute the Transcriptome in the DNA Double-Strand Break Response.

In response to stresses, cells often halt normal cellular processes, yet stress-specific pathways must bypass such inhibition to generate effective responses. We investigated how cells redistribute global transcriptional activity in response to DNA damage. We show that an oscillatory increase of p53 levels in response to double-strand breaks drives a counter-oscillatory decrease of MYC levels. Using RNA sequencing (RNA-seq) of newly synthesized transcripts, we found that p53-mediated reduction of MYC suppressed general transcription, with the most highly expressed transcripts reduced to a greater extent. In contrast, upregulation of p53 targets was relatively unaffected by MYC suppression. Reducing MYC during the DNA damage response was important for cell-fate regulation, as counteracting MYC repression reduced cell-cycle arrest and elevated apoptosis. Our study shows that global inhibition with specific activation of transcriptional pathways is important for the proper response to DNA damage; this mechanism may be a general principle used in many stress responses.

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards.

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

p53 Pulses Diversify Target Gene Expression Dynamics in an mRNA Half-Life-Dependent Manner and Delineate Co-regulated Target Gene Subnetworks.

The transcription factor p53 responds to DNA double-strand breaks by increasing in concentration in a series of pulses of fixed amplitude, duration, and period. How p53 pulses influence the dynamics of p53 target gene expression is not understood. Here, we show that, in bulk cell populations, patterns of p53 target gene expression cluster into groups with stereotyped temporal behaviors, including pulsing and rising dynamics. These behaviors correlate statistically with the mRNA decay rates of target genes: short mRNA half-lives produce pulses of gene expression. This relationship can be recapitulated by mathematical models of p53-dependent gene expression in single cells and cell populations. Single-cell transcriptional profiling demonstrates that expression of a subset of p53 target genes is coordinated across time within single cells; p53 pulsing attenuates this coordination. These results help delineate how p53 orchestrates the complex DNA damage response and give insight into the function of pulsatile signaling pathways.

Using computational modeling and experimental synthetic perturbations to probe biological circuits.

This chapter describes approaches for using computational modeling of synthetic biology perturbations to analyze endogenous biological circuits, with a particular focus on signaling and metabolic pathways. We describe a bottom-up approach in which ordinary differential equations are constructed to model the core interactions of a pathway of interest. We then discuss methods for modeling synthetic perturbations that can be used to investigate properties of the natural circuit. Keeping in mind the importance of the interplay between modeling and experimentation, we next describe experimental methods for constructing synthetic perturbations to test the computational predictions. Finally, we present a case study of the p53 tumor-suppressor pathway, illustrating the process of modeling the core network, designing informative synthetic perturbations in silico, and testing the predictions in vivo.

Regulation of SREBP during hypoxia requires Ofd1-mediated control of both DNA binding and degradation.

Cells adapt to changes in ambient oxygen by changing their gene expression patterns. In fission yeast, the sterol regulatory element-binding protein Sre1 is proteolytically cleaved under low oxygen, and its N-terminal segment (Sre1N) serves as a hypoxic transcription factor. When oxygen is present, the prolyl hydroxylase Ofd1 down-regulates Sre1N activity in two ways: first, by inhibiting its binding to DNA, and second, by accelerating its degradation. Here we use a mathematical model to assess what each of these two regulatory functions contributes to the hypoxic response of the cell. By disabling individual regulatory functions in the model, which would be difficult in vivo, we found that the Ofd1 function of inhibiting Sre1N binding to DNA is essential for oxygen-dependent Sre1N regulation. The other Ofd1 function of accelerating Sre1N degradation is necessary for the yeast to quickly turn off its hypoxic response when oxygen is restored. In addition, the model predicts that increased Ofd1 production at low oxygen plays an important role in the hypoxic response, and the model indicates that the Ofd1 binding partner Nro1 tunes the response to oxygen. This model quantifies our understanding of a novel oxygen-sensing mechanism that is widely conserved.

A framework for designing and analyzing binary decision-making strategies in cellular systems.

Cells make many binary (all-or-nothing) decisions based on noisy signals gathered from their environment and processed through noisy decision-making pathways. Reducing the effect of noise to improve the fidelity of decision-making comes at the expense of increased complexity, creating a tradeoff between performance and metabolic cost. We present a framework based on rate distortion theory, a branch of information theory, to quantify this tradeoff and design binary decision-making strategies that balance low cost and accuracy in optimal ways. With this framework, we show that several observed behaviors of binary decision-making systems, including random strategies, hysteresis, and irreversibility, are optimal in an information-theoretic sense for various situations. This framework can also be used to quantify the goals around which a decision-making system is optimized and to evaluate the optimality of cellular decision-making systems by a fundamental information-theoretic criterion. As proof of concept, we use the framework to quantify the goals of the externally triggered apoptosis pathway.

Identifying a static nonlinear structure in a biological system using noisy, sparse data.

When part of a biological system cannot be investigated directly by experimentation, we face the problem of structure identification: how can we construct a model for an unknown part of a mostly known system using measurements gathered from its input and output? This problem is especially difficult to solve when the measurements available are noisy and sparse, i.e. widely and unevenly spaced in time, as is common when measuring biological quantities at the cellular level. Here we present a procedure to identify a static nonlinearity embedded between two dynamical systems using noisy, sparse measurements. To reduce the level of error caused by measurement noise, we introduce the concept of weighted-sum predictability. If we make the input and output subsystems weighted-sum predictable and normalize the measurements to their weighted sum, we achieve better noise reduction than through normalizing to a loading control. We then interpolate the normalized measurements to obtain continuous input and output signals, with which we solve directly for the input-output characteristics of the unknown static nonlinearity. We demonstrate the effectiveness of this structure identification procedure by applying it to identify a model for ergosterol sensing by the proteins Sre1 and Scp1 in fission yeast. Simulations with this model produced outputs consistent with experimental observations. The techniques introduced here will provide researchers with a new tool by which biological systems can be identified and characterized.

Ergosterol regulates sterol regulatory element binding protein (SREBP) cleavage in fission yeast.

In fission yeast, the endoplasmic reticulum membrane-bound proteins Sre1 and Scp1, orthologs of mammalian sterol regulatory element binding protein (SREBP) and Scap, monitor sterol synthesis as an indirect measure of oxygen supply. When cellular oxygen levels are low, sterol synthesis is inhibited, and the Sre1-Scp1 complex responds by increasing transcription of genes required for adaptation to hypoxia. Sre1 and Scp1 are believed to detect a blockage in sterol synthesis by monitoring levels of particular sterols, but the evidence concerning which sterol signals this condition is unclear. Here, we demonstrate that Sre1-Scp1 senses ergosterol. Processing experimental data with a mathematical model of Sre1 and Scp1 function reveals a clear quantitative relationship between ergosterol concentration in the endoplasmic reticulum and Sre1 activation. Based on this relationship, we predict that the Sre1-Scp1 complex exists under "active" and "inactive" states and that the transition between these states is cooperatively mediated by ergosterol.

Bone tissue engineering: a review in bone biomimetics and drug delivery strategies.

Critical-sized defects in bone, whether induced by primary tumor resection, trauma, or selective surgery have in many cases presented insurmountable challenges to the current gold standard treatment for bone repair. The primary purpose of a tissue-engineered scaffold is to use engineering principles to incite and promote the natural healing process of bone which does not occur in critical-sized defects. A synthetic bone scaffold must be biocompatible, biodegradable to allow native tissue integration, and mimic the multidimensional hierarchical structure of native bone. In addition to being physically and chemically biomimetic, an ideal scaffold is capable of eluting bioactive molecules (e.g., BMPs, TGF-betas, etc., to accelerate extracellular matrix production and tissue integration) or drugs (e.g., antibiotics, cisplatin, etc., to prevent undesired biological response such as sepsis or cancer recurrence) in a temporally and spatially controlled manner. Various biomaterials including ceramics, metals, polymers, and composites have been investigated for their potential as bone scaffold materials. However, due to their tunable physiochemical properties, biocompatibility, and controllable biodegradability, polymers have emerged as the principal material in bone tissue engineering. This article briefly reviews the physiological and anatomical characteristics of native bone, describes key technologies in mimicking the physical and chemical environment of bone using synthetic materials, and provides an overview of local drug delivery as it pertains to bone tissue engineering is included.

Nanostructured tantala as a template for enhanced osseointegration.

The goal of current dental and orthopedic biomaterials research is to design implants that induce controlled and guided tissue growth, and rapid healing. In addition to acceleration of normal wound healing phenomena, these implants should result in the formation of a characteristic interfacial layer with adequate biomechanical properties. To achieve these goals, however, a better understanding of events at the bone-material interface is needed, as well as the development of new materials and approaches that promote osseointegration. Here we present novel nanostructured nanoarrays from tantala that can promote cell adhesion and differentiation. Our results suggest that tantala nanotube arrays enhance osteoblast cell adhesion, proliferation and differentiation. The routes of fabrication of tantala nanotube arrays are flexible and cost-effective, enabling realization of desired platform topologies on existing non-planar orthopedic implants.

Biocompatibility and mesenchymal stem cell response to poly(epsilon-caprolactone) nanowire surfaces for orthopedic tissue engineering.

Concerns over utilizing autogenous cancellous bone grafts (such as donor-site morbidity, increased surgical time/complication rate, and restricted availability) as the gold-standard treatment for critical-sized defects in bone have motivated the development of a wide variety of sophisticated synthetic bone scaffolds in recent years. In this work, a novel solvent-free template synthesis technique was utilized to fabricate poly(epsilon-caprolactone) (PCL) nanowire surfaces as a building block for the development of three-dimensional bone scaffolds. Bone marrow-derived mesenchymal stem cells (MSCs) were used to characterize the short- and long-term in vitro biocompatibility and cellular response to these surfaces. A 4-week study in rats was conducted to assess in vivo biocompatibility as well. Short-term in vitro studies revealed that PCL nanowire surfaces enhanced MSC response in terms of survivability, viability, cytoskeleton changes, and morphology as compared with control surfaces (smooth PCL and polystyrene). In long-term in vitro studies, nanowire surfaces induced a rapid production of bone extracellular matrix by differentiated MSCs as indicated by accelerated calcium phosphate mineralization, and osteocalcin and osteopontin production. In vivo studies and histological analysis confirmed that nanowire surfaces are biocompatible. Preliminary biodegradation studies were conducted and indicated that rate of PCL biodegradation can, to some extent, be controlled through the inclusion of nanowires and ester-degrading enzymes. In addition to demonstrating enhanced short- and long-term MSC response to PCL nanowire surfaces, this work presents a simple technique for solvent-free fabrication and bioactive molecule encapsulation of biocompatible, biodegradable three-dimensional bone scaffold components and warrants further investigation.

Biodegradable poly(epsilon-caprolactone) nanowires for bone tissue engineering applications.

Critical-sized defects in bone, whether caused by cancer tumor resection, trauma, or selective surgery have in many cases presented insurmountable challenges to the current gold-standard treatment for bone repair. The primary purpose of a tissue-engineered scaffold is to incite and promote the natural healing process of bone, which does not occur in critical-sized defects. In this work, a solvent-free template synthesis technique was utilized to fabricate uniform arrays of substrate-bound poly(epsilon-caprolactone) (PCL) nanowires. Biodegradation of PCL nanowire surfaces was characterized using scanning electron microscopy (SEM) and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Rat bone marrow-derived mesenchymal stem cells (MSCs) were employed to assess short-term biocompatibility and long-term bioactivity of nanowire surfaces. Short-term cell studies indicated that PCL nanowire surfaces supported enhanced cell adhesion and viability compared with control surfaces. MSCs seeded on nanowire surfaces also displayed increased levels of alkaline phosphatase (ALP) after 1, 2, and 3 weeks in culture. Calcium-phosphate mineralization was substantially accelerated on nanowire surfaces compared to control surfaces as indicated through calcium staining, von Kossa staining, SEM, and electron dispersive spectroscopy (EDS). Increased levels of inter- and extracellular levels of osteocalcin and osteopontin were observed on nanowire surfaces using immunofluorescence techniques after 3 weeks of culture. Considering the simplicity of the presented fabrication technique, capacity for solvent-free encapsulation of bioactive molecules or particles, and enhanced MSC performance on nanowire surfaces, this work presents an excellent foundation for the development of 3-D scaffolds for bone tissue regeneration.