Shadow Enhancers Foster Robustness of Drosophila Gastrulation

SummaryCritical developmental control genes sometimes contain “shadow” enhancers that can be located in remote positions, including the introns of neighboring genes [1]. They nonetheless produce patterns of gene expression that are the same as or similar to those produced by more proximal primary enhancers. It was suggested that shadow enhancers help foster robustness in gene expression in response to environmental or genetic perturbations [2, 3]. We critically tested this hypothesis by employing a combination of bacterial artificial chromosome (BAC) recombineering and quantitative confocal imaging methods [2, 4]. Evidence is presented that the snail gene is regulated by a distal shadow enhancer located within a neighboring locus. Removal of the proximal primary enhancer does not significantly perturb snail function, including the repression of neurogenic genes and formation of the ventral furrow during gastrulation at normal temperatures. However, at elevated temperatures, there is sporadic loss of snail expression and coincident disruptions in gastrulation. Similar defects are observed at normal temperatures upon reductions in the levels of Dorsal, a key activator of snail expression (reviewed in [5]). These results suggest that shadow enhancers represent a novel mechanism of canalization whereby complex developmental processes “bring about one definite end-result regardless of minor variations in conditions” [6]

Precisely timed regulation of enhancer activity defines the binary expression pattern of Fushi tarazu in the Drosophila embryo

The genes that drive development each typically have many different enhancers. Properly coordinating the action of these different enhancers is crucial to correctly specifying cell-fate decisions, yet it remains poorly understood how their activity is choregraphed in time. To shed light on this question, we used recently developed single-cell live imaging tools to dissect the regulation of Fushi tarazu (Ftz) in Drosophila melanogaster embryos. Ftz is a transcription factor that is expressed in asymmetric stripes by two distinct enhancers: autoregulatory and zebra. The anterior edge of each stripe needs to be sharply defined to specify essential lineage boundaries. Here, we tracked how boundary cells commit to either a high-Ftz or low-Ftz fate by measuring Ftz protein traces in real time and simultaneously quantifying transcription from the endogenous locus and individual enhancers. This revealed that the autoregulatory enhancer does not establish this fate choice. Instead, it perpetuates the decision defined by zebra. This is contrary to the prevailing view that autoregulation drives the fate decision by causing bi-stable Ftz expression. Furthermore, we showed that the autoregulatory enhancer is not activated based on a Ftz-concentration threshold but through a timing-based mechanism. We hypothesize that this is regulated by several ubiquitously expressed pioneer-like transcription factors, which have recently been shown to act as timers in the embryo. Our work provides new insight into how precisely timed enhancer activity can directly regulate the dynamics of gene regulatory networks, which may be a general mechanism for ensuring that embryogenesis runs like clockwork

Quantum Transition State Theory for proton transfer reactions in enzymes

We consider the role of quantum effects in the transfer of hyrogen-like species in enzyme-catalysed reactions. This study is stimulated by claims that the observed magnitude and temperature dependence of kinetic isotope effects imply that quantum tunneling below the energy barrier associated with the transition state significantly enhances the reaction rate in many enzymes. We use a path integral approach which provides a general framework to understand tunneling in a quantum system which interacts with an environment at non-zero temperature. Here the quantum system is the active site of the enzyme and the environment is the surrounding protein and water. Tunneling well below the barrier only occurs for temperatures less than a temperature $T_0$ which is determined by the curvature of potential energy surface near the top of the barrier. We argue that for most enzymes this temperature is less than room temperature. For physically reasonable parameters quantum transition state theory gives a quantitative description of the temperature dependence and magnitude of kinetic isotope effects for two classes of enzymes which have been claimed to exhibit signatures of quantum tunneling. The only quantum effects are those associated with the transition state, both reflection at the barrier top and tunneling just below the barrier. We establish that the friction due to the environment is weak and only slightly modifies the reaction rate. Furthermore, at room temperature and for typical energy barriers environmental degrees of freedom with frequencies much less than 1000 cm$^{-1}$ do not have a significant effect on quantum corrections to the reaction rate.Comment: Aspects of the article are discussed at condensedconcepts.blogspot.co

Long-Range Intra-Protein Communication Can Be Transmitted by Correlated Side-Chain Fluctuations Alone

Allosteric regulation is a key component of cellular communication, but the way in which information is passed from one site to another within a folded protein is not often clear. While backbone motions have long been considered essential for long-range information conveyance, side-chain motions have rarely been considered. In this work, we demonstrate their potential utility using Monte Carlo sampling of side-chain torsional angles on a fixed backbone to quantify correlations amongst side-chain inter-rotameric motions. Results indicate that long-range correlations of side-chain fluctuations can arise independently from several different types of interactions: steric repulsions, implicit solvent interactions, or hydrogen bonding and salt-bridge interactions. These robust correlations persist across the entire protein (up to 60 Å in the case of calmodulin) and can propagate long-range changes in side-chain variability in response to single residue perturbations

Long-Range Intra-Protein Communication Can Be Transmitted by Correlated Side-Chain Fluctuations Alone

Allosteric regulation is a key component of cellular communication, but the way in which information is passed from one site to another within a folded protein is not often clear. While backbone motions have long been considered essential for long-range information conveyance, side-chain motions have rarely been considered. In this work, we demonstrate their potential utility using Monte Carlo sampling of side-chain torsional angles on a fixed backbone to quantify correlations amongst side-chain inter-rotameric motions. Results indicate that long-range correlations of side-chain fluctuations can arise independently from several different types of interactions: steric repulsions, implicit solvent interactions, or hydrogen bonding and salt-bridge interactions. These robust correlations persist across the entire protein (up to 60 Å in the case of calmodulin) and can propagate long-range changes in side-chain variability in response to single residue perturbations

LlamaTags: A Versatile Tool to Image Transcription Factor Dynamics in Live Embryos.

Embryonic cell fates are defined by transcription factors that are rapidly deployed, yet attempts to visualize these factors in vivo often fail because of slow fluorescent protein maturation. Here, we pioneer a protein tag, LlamaTag, which circumvents this maturation limit by binding mature fluorescent proteins, making it possible to visualize transcription factor concentration dynamics in live embryos. Implementing this approach in the fruit fly Drosophila melanogaster, we discovered stochastic bursts in the concentration of transcription factors that are correlated with bursts in transcription. We further used LlamaTags to show that the concentration of protein in a given nucleus heavily depends on transcription of that gene in neighboring nuclei; we speculate that this inter-nuclear signaling is an important mechanism for coordinating gene expression to delineate straight and sharp boundaries of gene expression. Thus, LlamaTags now make it possible to visualize the flow of information along the central dogma in live embryos

Distance dependence of mutual information in different NMR models of barstar.

<p>The average excess mutual information per residue pair is plotted here for various atomic interactions, binned according to the - inter-residue distance, for the crystal structure (1a19 <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002168#pcbi.1002168-Ratnaparkhi1" target="_blank">[47]</a>) and four NMR model structures (1btb <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002168#pcbi.1002168-Lubienski1" target="_blank">[43]</a>) of barstar, using the full LJ+IS+HBSB potential. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002168#s4" target="_blank">Methods</a> for details.</p

Mutual information of residue pairs in calmodulin.

<p>The mutual information, , associated with side-chain fluctuations of residue pairs in calmodulin. Plots (b)–(f) display the mutual information signal∶noise ratio, (upper left triangles) and the excess mutual information (lower right triangles), as indicated in (a). The - and -axes run over labels, and respectively, of residues in the amino acid sequence, excluding those lacking rotameric freedom in our model. Scale bars for the signal∶noise ratio and the excess mutual information are presented on the top and bottom left, respectively. Results are shown for the following combinations of interactions: (b) repulsive sterics (S), (c) implicit solvent (IS) (d) Lennard-Jones (LJ) interaction comprising repulsive sterics and van der Waals attractions, (e) hydrogen bonding and salt-bridges (HBSB), and (f) the full potential (LJ+HBSB+IS). Residue 30K, which we scrutinize in detail later (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002168#pcbi-1002168-g005" target="_blank">Fig. 5</a>), is highlighted in (f) for reference.</p

Structural representations of extended crystalline calmodulin.

<p>The crystal structure (a) and contact map (b) of calcium-bound calmodulin (3cln <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002168#pcbi.1002168-Babu1" target="_blank">[39]</a>). The calcium ions are shown in yellow, and several residues are labeled in both panels for reference. The distance between each pair of atoms is indicated by color (see scale bar) in (b), where - and -axes run over residue labels. The residue labeling corresponds to the full sequence, however residues that do not possess torsional degrees of freedom in our model (A, G, P, and all residues bound to the calcium ions) are excluded from the contact map.</p