Predictive shifts in free energy couple mutations to their phenotypic consequences.

Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find that the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions.

Countermovement jump variables not tensiomyography can distinguish between sprint and endurance focused track cyclists.

This study investigated the reliability and discriminative ability of tensiomyography and countermovement jump variables as measures of a muscles contractile properties in a cohort of elite endurance and sprint track cyclists. Tensiomyography was performed on the vastus lateralis (VL) and rectus femoris (RF) muscles in sprint track cyclists (N = 8) and endurance track cyclists (N = 8). Additionally, the participants completed a countermovement jump on a force plate. Tensiomyography measurements obtained from the RF displayed greater reliability (ICC = 0.879-0.997) than VL (ICC = 0.746-0.970). Radial muscle belly displacement (Dm), contraction time (Tc) and delay time (Td) demonstrated the most reliable TMG measurements. Only two variables displayed acceptable coefficient of variation (RF Td = 8.89, VL Td = 6.88), other variables presented as unacceptable. The TMG variables were unable to discriminate between endurance and sprint track cyclists whilst the CMJ variables could. Due to the high variability in measurements and its inability to distinguish between sprint and endurance based track cyclists TMG should be used cautiously in this athlete population and if available the CMJ is a more appropriate assessment of leg muscle function.

Crystal structure of the lambda repressor and a model for pairwise cooperative operator binding.

Bacteriophage lambda has for many years been a model system for understanding mechanisms of gene regulation. A 'genetic switch' enables the phage to transition from lysogenic growth to lytic development when triggered by specific environmental conditions. The key component of the switch is the cI repressor, which binds to two sets of three operator sites on the lambda chromosome that are separated by about 2,400 base pairs (bp). A hallmark of the lambda system is the pairwise cooperativity of repressor binding. In the absence of detailed structural information, it has been difficult to understand fully how repressor molecules establish the cooperativity complex. Here we present the X-ray crystal structure of the intact lambda cI repressor dimer bound to a DNA operator site. The structure of the repressor, determined by multiple isomorphous replacement methods, reveals an unusual overall architecture that allows it to adopt a conformation that appears to facilitate pairwise cooperative binding to adjacent operator sites.

In Vitro Generation of Megakaryocytes from Engineered Mouse Embryonic Stem Cells.

The in vitro differentiation of pluripotent stem cells into desired lineages enables mechanistic studies of cell transitions into more mature states that can provide insights into the design principles governing cell fate control. We are interested in reprogramming pluripotent stem cells with synthetic gene circuits to drive mouse embryonic stem cells (mESCs) down the hematopoietic lineage for the production of megakaryocytes, the progenitor cells for platelets. Here, we describe the methodology for growing and differentiating mESCs, in addition to inserting a transgene to observe its expression throughout differentiation. This entails four key methods: (1) growing and preparing mouse embryonic fibroblasts for supporting mESC growth and expansion, (2) growing and preparing OP9 feeder cells to support the differentiation of mESCs, (3) the differentiation of mESCs into megakaryocytes, and (4) utilizing an integrase-mediated docking site to insert transgenes for their stable integration and expression throughout differentiation. Altogether, this approach demonstrates a streamline differentiation protocol that emphasizes the reprogramming potential of mESCs that can be used for future mechanistic and therapeutic studies of controlling cell fate outcomes.

Advancing in vivo reprogramming with synthetic biology.

Reprogramming cells will play a fundamental role in shaping the future of cell therapies by developing new strategies to engineer cells for improved performance and higher-order physiological functions. Approaches in synthetic biology harness cells' natural ability to sense diverse signals, integrate environmental inputs to make decisions, and execute complex behaviors based on the health of the organism or tissue. In this review, we highlight strategies in synthetic biology to reprogram cells, and discuss how recent approaches in the delivery of modified mRNA have created new opportunities to alter cell function in vivo. Finally, we discuss how combining concepts from synthetic biology and the delivery of mRNA in vivo could provide a platform for innovation to advance in vivo cellular reprogramming.

In vitro generation of megakaryocytes from engineered mouse embryonic stem cells.

The in vitro differentiation of pluripotent stem cells into desired lineages enables mechanistic studies of cell transitions into more mature states that can provide insights into the design principles governing cell fate control. We are interested in reprogramming pluripotent stem cells with synthetic gene circuits to drive mouse embryonic stem cells (mESCs) down the hematopoietic lineage for the production of megakaryocytes, the progenitor cells for platelets. Here, we describe the methodology for growing and differentiating mESCs, in addition to inserting a transgene to observe its expression throughout differentiation. This entails four key methods: (1) growing and preparing mouse embryonic fibroblasts for supporting mESC growth and expansion, (2) growing and preparing OP9 feeder cells to support the differentiation of mESCs, (3) the differentiation of mESCs into megakaryocytes, and (4) utilizing an integrase mediated docking site to insert transgenes for their stable integration and expression throughout differentiation. Altogether, this approach demonstrates a streamline differentiation protocol that emphasizes the reprogramming potential of mESCs that can be used for future mechanistic and therapeutic studies of controlling cell fate outcomes.

EGFR signaling activates intestinal stem cells by promoting mitochondrial biogenesis and ß-oxidation.

EGFR-RAS-ERK signaling promotes growth and proliferation in many cell types, and genetic hyperactivation of RAS-ERK signaling drives many cancers. Yet, despite intensive study of upstream components in EGFR signal transduction, the identities and functions of downstream effectors in the pathway are poorly understood. In Drosophila intestinal stem cells (ISCs), the transcriptional repressor Capicua (Cic) and its targets, the ETS-type transcriptional activators Pointed (pnt) and Ets21C, are essential downstream effectors of mitogenic EGFR signaling. Here, we show that these factors promote EGFR-dependent metabolic changes that increase ISC mass, mitochondrial growth, and mitochondrial activity. Gene target analysis using RNA and DamID sequencing revealed that Pnt and Ets21C directly upregulate not only DNA replication and cell cycle genes but also genes for oxidative phosphorylation, the TCA cycle, and fatty acid beta-oxidation. Metabolite analysis substantiated these metabolic functions. The mitochondrial transcription factor B2 (mtTFB2), a direct target of Pnt, was required and partially sufficient for EGFR-driven ISC growth, mitochondrial biogenesis, and proliferation. MEK-dependent EGF signaling stimulated mitochondrial biogenesis in human RPE-1 cells, indicating the conservation of these metabolic effects. This work illustrates how EGFR signaling alters metabolism to coordinately activate cell growth and cell division.

Structural analysis of lac repressor bound to allosteric effectors.

The lac operon is a model system for understanding how effector molecules regulate transcription and are necessary for allosteric transitions. The crystal structures of the lac repressor bound to inducer and anti-inducer molecules provide a model for how these small molecules can modulate repressor function. The structures of the apo repressor and the repressor bound to effector molecules are compared in atomic detail. All effectors examined here bind to the repressor in the same location and are anchored to the repressor through hydrogen bonds to several hydroxyl groups of the sugar ring. Inducer molecules form a more extensive hydrogen-bonding network compared to anti-inducers and neutral effector molecules. The structures of these effector molecules suggest that the O6 hydroxyl on the galactoside is essential for establishing a water-mediated hydrogen bonding network that bridges the N-terminal and C-terminal sub-domains. The altered hydrogen bonding can account in part for the different structural conformations of the repressor, and is vital for the allosteric transition.

Tuning Transcriptional Regulation through Signaling: A Predictive Theory of Allosteric Induction.

Allosteric regulation is found across all domains of life, yet we still lack simple, predictive theories that directly link the experimentally tunable parameters of a system to its input-output response. To that end, we present a general theory of allosteric transcriptional regulation using the Monod-Wyman-Changeux model. We rigorously test this model using the ubiquitous simple repression motif in bacteria by first predicting the behavior of strains that span a large range of repressor copy numbers and DNA binding strengths and then constructing and measuring their response. Our model not only accurately captures the induction profiles of these strains, but also enables us to derive analytic expressions for key properties such as the dynamic range and [EC50]. Finally, we derive an expression for the free energy of allosteric repressors that enables us to collapse our experimental data onto a single master curve that captures the diverse phenomenology of the induction profiles.

Author Correction: An Autogenously Regulated Expression System for Gene Therapeutic Ocular Applications.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Prevalence of the EH1 Groucho interaction motif in the metazoan Fox family of transcriptional regulators.

BACKGROUND: The Fox gene family comprises a large and functionally diverse group of forkhead-related transcriptional regulators, many of which are essential for metazoan embryogenesis and physiology. Defining conserved functional domains that mediate the transcriptional activity of Fox proteins will contribute to a comprehensive understanding of the biological function of Fox family genes. RESULTS: Systematic analysis of 458 protein sequences of the metazoan Fox family was performed to identify the presence of the engrailed homology-1 motif (eh1), a motif known to mediate physical interaction with transcriptional corepressors of the TLE/Groucho family. Greater than 50% of Fox proteins contain sequences with high similarity to the eh1 motif, including ten of the nineteen Fox subclasses (A, B, C, D, E, G, H, I, L, and Q) and Fox proteins of early divergent species such as marine sponge. The eh1 motif is not detected in Fox proteins of the F, J, K, M, N, O, P, R and S subclasses, or in yeast Fox proteins. The eh1-like motifs are positioned C-terminal to the winged helix DNA-binding domain in all subclasses except for FoxG proteins, which have an N-terminal motif. Two similar eh1-like motifs are found in the zebrafish FoxQ1 and in FoxG proteins of sea urchin and amphioxus. The identification of eh1-like motifs by manual sequence alignment was validated by statistical analyses of the Swiss protein database, confirming a high frequency of occurrence of eh1-like sequences in Fox family proteins. Structural predictions suggest that the majority of identified eh1-like motifs are short alpha-helices, and wheel modeling revealed an amphipathicity that supports this secondary structure prediction. CONCLUSION: A search for eh1 Groucho interaction motifs in the Fox gene family has identified eh1-like sequences in greater than 50% of Fox proteins. The results predict a physical and functional interaction of TLE/Groucho corepressors with many members of the Fox family of transcriptional regulators. Given the functional importance of the eh1 motif in transcriptional regulation, our annotation of this motif in the Fox gene family will facilitate further study of the diverse transcriptional and regulatory roles of Fox family proteins.

X-ray structure of a maquette scaffold.

Maquettes are de novo designed mimicries of nature used to test the construction and engineering criteria of oxidoreductases. One type of scaffold used in maquette construction is a four-alpha-helical bundle. The sequence of the four-alpha-helix bundle maquettes follows a heptad repeat pattern typical of left-handed coiled-coils. Initial designs were molten globular due partly to the minimalist approach taken by the designers. Subsequent iterative redesign generated several structured scaffolds with similar heme binding properties. Variant [I(6)F(13)](2), a structured scaffold, was partially resolved with NMR spectroscopy and found to have a set of mobile inter-helical packing interfaces. Here, the X-ray structure of a similar peptide ([I(6)F(13)M(31)](2) i.e. ([CGGG EIWKL HEEFLKK FEELLKL HEERLKKM](2))(2) which we call L31M), has been solved using MAD phasing and refined to 2.8A resolution. The structure shows that the maquette scaffold is an anti-parallel four-helix bundle with "up-up-down-down" topology. No pre-formed heme-binding pocket exists in the protein scaffold. We report unexpected inter-helical crossing angles, residue positions and translations between the helices. The crossing angles between the parallel helices are -5 degrees rather than the expected +20 degrees for typical left-handed coiled-coils. Deviation of the scaffold from the design is likely due to the distribution and size of hydrophobic residues. The structure of L31M points out that four identical helices may interact differently in a bundle and heptad repeats with an alternating [HPPHHPP]/[HPPHHPH] (H: hydrophobic, P: polar) pattern are not a sufficient design criterion to generate left-hand coiled-coils.

The lac repressor.

Few proteins have had such a strong impact on a field as the lac repressor has had in Molecular Biology. Over 40 years ago, Jacob and Monod [Genetic regulatory mechanisms in the synthesis of proteins, J. Mol. Biol. 3 (1961) 318] proposed a model for gene regulation, which survives essentially unchanged in contemporary textbooks. It is a cogent depiction of how a set of 'structural' genes may be coordinately transcribed in response to environmental conditions and regulates metabolic events in the cell. In bacteria, the genes required for lactose utilization are negatively regulated when a repressor molecule binds to an upstream cis activated operator. The repressor and its operator together form a genetic switch, the lac operon. The switch functions when inducer molecules alter the conformation of the repressor in a specific manner. In the presence of a particular metabolite, the repressor undergoes a conformational change that reduces its affinity for the operator. The structures of the lac repressor and its complexes with operator DNA and effector molecules have provided a physical platform for visualizing at the molecular level the different conformations the repressor and the molecular basis for the switch. The structures of lac repressor, bound to its operator and inducer, have also been invaluable for interpreting a plethora of biochemical and genetic data.

An Autogenously Regulated Expression System for Gene Therapeutic Ocular Applications.

The future of treating inherited and acquired genetic diseases will be defined by our ability to introduce transgenes into cells and restore normal physiology. Here we describe an autogenous transgene regulatory system (ARES), based on the bacterial lac repressor, and demonstrate its utility for controlling the expression of a transgene in bacteria, eukaryotic cells, and in the retina of mice. This ARES system is inducible by the small non-pharmacologic molecule, Isopropyl ß-D-1-thiogalactopyranoside (IPTG) that has no off-target effects in mammals. Following subretinal injection of an adeno-associated virus (AAV) vector encoding ARES, luciferase expression can be reversibly controlled in the murine retina by oral delivery of IPTG over three induction-repression cycles. The ability to induce transgene expression repeatedly via administration of an oral inducer in vivo, suggests that this type of regulatory system holds great promise for applications in human gene therapy.

Structure-function relationships in 3alpha-hydroxysteroid dehydrogenases: a comparison of the rat and human isoforms.

3alpha-Hydroxysteroid dehydrogenases (3alpha-HSDs) inactivate steroid hormones in the liver, regulate 5alpha-dihydrotestosterone (5alpha-DHT) levels in the prostate, and form the neurosteroid, allopregnanolone in the CNS. Four human 3alpha-HSD isoforms exist and correspond to AKR1C1-AKR1C4 of the aldo-keto reductase (AKR) superfamily. Unlike the related rat 3alpha-HSD (AKR1C9) which is positional and stereospecific, the human enzymes display varying ratios of 3-, 17-, and 20-ketosteroid reductase activity as well as 3alpha-, 17beta-, and 20alpha-hydroxysteroid oxidase activity. Their k(cat) values are 50-100-fold lower than that observed for AKR1C9. Based on their product profiles and discrete tissue localization, the human enzymes may regulate the levels of active androgens, estrogens, and progestins in target tissues. The X-ray crystal structures of AKR1C9 and AKR1C2 (human type 3 3alpha-HSD, bile acid binding protein and peripheral 3alpha-HSD) reveal that the AKR1C2 structure can bind steroids backwards (D-ring in the A-ring position) and upside down (beta-face inverted) relative to the position of a 3-ketosteroid in AKR1C9 and this may account for its functional plasticity. Stopped-flow studies on both enzymes indicate that the conformational changes associated with binding cofactor (the first ligand) are slow; they are similar in both enzymes but are not rate-determining. Instead the low k(cat) seen in AKR1C2 (50-fold less than AKR1C9) may be due to substrate "wobble" at the plastic active site.

Allostery and the lac Operon.

The ability to regulate gene expression is essential for controlling metabolic events in a cell. Proteins that function like molecular switches respond to fluctuations in the environment to maintain homeostasis. The operon model, proposed by Jacob and Monod, provides a cogent depiction for how gene expression is regulated. A molecular mechanism for the regulation followed shortly with the theory for allosteric transition. Over the past half-century, the details of the lac operon and the allosteric model have been tested using genetic, biochemical, and structural techniques. Remarkably, the principles originally put forward 50 years ago remain essentially unchanged. Models for the operon and the theory of allosteric transitions are two of the most profound discoveries of molecular biology.

A tale of two repressors.

Few proteins have had such a strong impact on a field, as the lac repressor and λ repressor have had in Molecular Biology in bacteria. The genes required for lactose utilization are negatively regulated; the lac repressor binds to an upstream operator blocking the transcription of the enzymes necessary for lactose utilization. A similar switch regulates the virus life cycle; λ repressor binds to an operator site and blocks transcription of the phage genes necessary for lytic development. It is now 50 years since Jacob and Monod first proposed a model for gene regulation, which survives essentially unchanged in contemporary textbooks. Jacob, F. & Monod, J. (1961). Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318-356. This model provides a cogent depiction of how a set of genes can be coordinately transcribed in response to environmental conditions and regulates metabolic events in the cell. A historical perspective that illustrates the role these two repressor molecules played and their contribution to our understanding of gene regulation is presented.

Thermodynamic analysis of mutant lac repressors.

The lactose (lac) repressor is an allosteric protein that can respond to environmental changes. Mutations introduced into the DNA binding domain and the effector binding pocket affect the repressor's ability to respond to its environment. We have demonstrated how the observed phenotype is a consequence of altering the thermodynamic equilibrium constants. We discuss mutant repressors, which (1) show tighter repression; (2) induce with a previously noninducing species, orthonitrophenyl-ß-D-galactoside; and (3) transform an inducible switch to one that is corepressed. The ability of point mutations to change multiple thermodynamic constants, and hence drastically alter the repressor's phenotype, shows how allosteric proteins can perform a wide array of similar yet distinct functions such as that exhibited in the Lac/Gal family of bacterial repressors.

Functional rules for lac repressor-operator associations and implications for protein-DNA interactions.

The Lac repressor has been used as a tool to understand protein-DNA recognition for many years. Recent experiments have demonstrated the ability of the Lac repressor to control gene expression in various eukaryotic systems, making the quest for an arsenal of protein-DNA binding partners desirable for potential therapeutic applications. Here, we present the results of the most exhaustive screen of Lac repressor-DNA binding partners to date, resulting in the elucidation of functional rules for Lac-DNA binding. Even within the confines of a single protein-DNA scaffold, modes of binding of different protein-DNA partners are sufficiently diverse so as to prevent elucidation of generalized rules for recognition for a single protein, much less an entire protein family.