Modular cis-regulatory organization of Endo16, a gut-specific gene of the sea urchin embryo

The Endo16 gene of Strongylocentrotus purpuratus is expressed at the blastula stage of embryogenesis throughout the vegetal plate, at the gastrula stage in the whole of the archenteron and in postgastrular stages only in the midgut. We showed earlier that a 2300 bp upstream sequence suffices to faithfully recreate this pattern of expression when fused to a CAT reporter gene. Here we define the functional organization of this cis-regulatory domain, which includes over thirty high specificity binding sites, serviced by at least thirteen different putative transcription factors, in addition to >20 sites for a factor commonly found in the regulatory sequences of other sea urchin genes as well (SpGCF1). The Endo16 cis-regulatory domain consists of several different functional elements, or modules, each containing one or two unique DNA-binding factor target sites, plus sites for factors binding in other modules as well. Modular regulatory function was defined in experiments in which regions of the cis-regulatory DNA containing specific clusters of sites were tested in isolation, combined with one another, or by selective deletion, and the effects on expression of the CAT reporter were determined by whole-mount in situ hybridization or CAT enzyme activity measurements. The most proximal module (A) is mainly responsible for early embryonic expression, and module A alone suffices to locate expression in the vegetal plate and archenteron. The adjacent module (B) is responsible for a steep postgastrular rise in expression, when the gene is transcribed only in the midgut and, prior to this module B alone also suffices to promote expression in the vegetal plate and archenteron. The most distal module, G, acts as a booster for either A or B modules. However, no combination of A, B and G modules generates vegetal plate or gut expression exclusively. Ectopic expression of A-, B- and G-CAT fusion constructs occurs in the adjacent (veg1-derived) ectoderm and in skeletogenic mesenchyme cells. For expression to be confined to endoderm requires negative regulatory functions mediated by modules E, F and DC. Modules E and F each repress ectopic expression specifically in veg1 ectoderm. Module DC represses ectopic expression specifically in skeletogenic mesenchyme. Expression of some Endo16 constructs is dramatically increased by treatment with LiCl, which expands the territory in which the endogenous Endo16 gene is expressed at the expense of veg1 ectoderm. The same modules that act to repress ectopic expression in untreated embryos are required for enhanced expression of constructs after LiC1 treatment. Furthermore, both the negative spatial control functions and response to LiC1 require the presence of module A. The total regulatory requirements of the Endo16 gene during embryogenesis can be expressed in terms of the positive and negative functions of the individual modules and the interactions between modules that are identified in this study

Cis-regulatory logic in the endo16 gene: switching from a specification to a differentiation mode of control

The endo16 gene of Strongylocentrotus purpuratus encodes a secreted protein of the embryonic and larval midgut. The overall functional organization of the spatial and temporal control system of this gene are relatively well known from a series of earlier cis-regulatory studies. Our recent computational model for the logic operations of the proximal region of the endo16 control system (Module A) specifies the function of interactions at each transcription factor target site of Module A. Here, we extend sequence level functional analysis to the adjacent cis-regulatory region, Module B. The computational logic model is broadened to include B/A interactions as well as other Module B functions. Module B drives expression later in development and its major activator is responsible for a sharp, gut-specific increase in transcription after gastrulation. As shown earlier, Module B output undergoes a synergistic amplification that requires interactions within Module A. The interactions within Module B that are required to generate and transmit its output to Module A are identified. Logic considerations predicted an internal cis-regulatory switch by which spatial control of endo16 expression is shifted from Module A (early) to Module B (later). This prediction was confirmed experimentally and a distinct set of interactions in Module B that mediate the switch function was demonstrated. The endo16 computational model now provides a detailed explanation of the information processing functions executed by the cis-regulatory system of this gene throughout embryogenesis. Early in development the gene participates in the specification events that define the endomesoderm; later it functions as a gut-specific differentiation gene. The cis-regulatory switch mediates this functional change

Quantitative functional interrelations within the cis-regulatory system of the S. purpuratus Endo16 gene

Embryonic expression of the Endo16 gene of Strongylocentrotus purpuratus is controlled by interactions with at least 13 different DNA-binding factors. These interactions occur within a cis-regulatory domain that extends about 2300 bp upstream from the transcription start site. A recent functional characterization of this domain reveals six different subregions, or cis-regulatory modules, each of which displays a specific regulatory subfunction when linked with the basal promoter and in some cases various other modules (C.-H. Yuh and E. Davidson (1996) Development 122, 1069-1082). In the present work, we analyzed quantitative time-course measurements of the CAT enzyme output of embryos bearing expression constructs controlled by various Endo16 regulatory modules, either singly or in combination. Three of these modules function positively in that, in isolation, each is capable of promoting expression in vegetal plate and adjacent cell lineages, though with different temporal profiles of activity. Models for the mode of interaction of the three positive modules with one another were tested by assuming mathematical relations that would generate, from the measured single module time courses, the experimentally observed profiles of activity obtained when the relevant modules are physically linked in the same construct. The generated and observed time functions were compared, and the differences were minimized by least squares adjustment of a scale parameter. When the modules were tested in context of the endogenous promoter region, one of the positive modules (A) was found to increase the output of the others (B and G), by a constant factor. In contrast, a solution in which the time-course data of modules A and B are multiplied by one another was required for the interrelations of the positive modules when a minimal SV40 promoter was used. One interpretation is that, in this construct, each module independently stimulates the basal transcription complex. We used a similar approach to analyze the repressive activity of the three Endo16 cis-regulatory modules that act negatively in controlling spatial expression. The evidence obtained confirms that the repressive modules act only by affecting the output of module A (C.-H. Yuh and E. Davidson (1996) Development 122, 1069-1082). A new hierarchical model of the cis-regulatory system was formulated in which module A plays a central integrating role, and which also implies specific functions for certain DNA-binding sites within the basal promoter fragment of the gene. Additional kinetic experiments were then carried out, and key aspects of the model were confirmed

Time-lapse seismic monitoring of subsurface fluid flow

Time-lapse seismic monitoring repeats 3D seismic imaging over a reservoir to map fluid movements in a reservoir. During hydrocarbon production, the fluid saturation, pressure, and temperature of a reservoir change, thereby altering the acoustic properties of the reservoir. Time-lapse seismic analysis can illuminate these dynamic changes of reservoir properties, and therefore has strong potential for improving reservoir management. However, the response of a reservoir depends on many parameters and can be diffcult to understand and predict. Numerical modeling results integrating streamline fluid flow simulation, rock physics, and ray-Born seismic modeling address some of these problems. Calculations show that the sensitivity of amplitude changes to porosity depend on the type of sediment comprising the reservoir. For consolidated rock, high-porosity models show larger amplitude changes than low porosity models. However, in an unconsolidated formation, there is less consistent correlation between amplitude and porosity. The rapid time-lapse modeling schemes also allow statistical analysis of the uncertainty in seismic response associated with poorly known values of reservoir parameters such as permeability and dry bulk modulus. Results show that for permeability, the maximum uncertainties in time-lapse seismic signals occur at the water front, where saturation is most variable. For the dry bulk-modulus, the uncertainty is greatest near the injection well, where the maximum saturation changes occur. Time-lapse seismic methods can also be applied to monitor CO2 sequestration. Simulations show that since the acoustic properties of CO2 are very different from those of hydrocarbons and water, it is possible to image CO2 saturation using seismic monitoring. Furthermore, amplitude changes after supercritical fluid CO2 injection are larger than liquid CO2 injection. Two seismic surveys over Teal South Field, Eugene Island, Gulf of Mexico, were acquired at different times, and the numerical models provide important insights to understand changes in the reservoir. 4D seismic differences after cross-equalization show that amplitude dimming occurs in the northeast and brightening occurs in the southwest part of the field. Our forward model, which integrates production data, petrophysicals, and seismic wave propagation simulation, shows that the amplitude dimming and brightening can be explained by pore pressure drops and gas invasion, respectively

Bio-logic: gene expression and the laws of combinatorial logic

Original article can be found at: http://www.mitpressjournals.org/ Copyright MIT Press DOI: 10.1162/artl.2008.14.1.121At the heart of the development of fertilized eggs into fully formed organisms and the adaptation of cells to changed conditions are genetic regulatory networks (GRNs). In higher multi-cellular organisms, signal selection and multiplexing is performed at the cis-regulatory domains of genes, where combinations of transcription factors (TFs) regulate the rates at which the genes are transcribed into mRNA. To be able to act as activators or repressors of gene transcription, TFs must first bind to target sequences on the regulatory domains. Two TFs that act in concert may bind entirely independently of each other, but more often binding of the first one will alter the affinity of the other for its binding site. This paper presents a systematic investigation into the effect of TF binding dependencies on the predicted regulatory function of this “bio-logic”. Four extreme scenarios, commonly used to classify enzyme activation and inhibition patterns, for the binding of two TFs were explored: independent (the TFs bind without affecting each other’s affinities), competitive (the TFs compete for the same binding site), ordered (the TFs bind in a compulsory order), and joint binding (the TFs either bind as a preformed complex, or binding of one is virtually impossible in the absence of the other). The conclusions are: 1) the laws of combinatorial logic hold only for systems with independently binding TFs; 2) systems formed according to the other scenarios can mimic the functions of their Boolean logical counterparts, but cannot be combined or decomposed in the same way; and 3) the continuously scaled output of systems consisting of competitively binding activators and repressors can be more robustly controlled than that of single TF or (quasi-) logical multi-TF systems. Keywords: Transcription regulation, Genetic regulatory networks, Enzyme kinetics, Combinatorial logic, Non-Boolean continuous logic, Modelling.Peer reviewe

Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin.

Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct territories. Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin

Evolutionary plasticity of developmental gene regulatory network architecture

Sea stars and sea urchins evolved from a last common ancestor that lived at the end of the Cambrian, approximately half a billion years ago. In a previous comparative study of the gene regulatory networks (GRNs) that embody the genomic program for embryogenesis in these animals, we discovered an almost perfectly conserved five-gene network subcircuit required for endoderm specification. We show here that the GRN structure upstream and downstream of the conserved network kernel has, by contrast, diverged extensively. Mesoderm specification is accomplished quite differently; the Delta–Notch signaling system is used in radically distinct ways; and various regulatory genes have been coopted to different functions. The conservation of the conserved kernel is thus the more remarkable. The results indicate types of network linkage subject to evolutionary change. An emergent theme is that subcircuit design may be preserved even while the identity of genes performing given roles changes because of alteration in their cis-regulatory control systems

Modular cis-regulatory organization of developmentally expressed genes: Two genes transcribed territorially in the sea urchin embryo, and additional examples

The cis-regulatory systems that control developmental expression of two sea urchin genes have been subjected to detailed functional analysis. Both systems are modular in organization: specific, separable fragments of the cis-regulatory DNA each containing multiple transcription factor target sites execute particular regulatory subfunctions when associated with reporter genes and introduced into the embryo. The studies summarized here were carried out on the CyIIIa gene, expressed in the embryonic aboral ectoderm and on the Endo16 gene, expressed in the embryonic vegetal plate, archenteron, and then midgut. The regulatory systems of both genes include modules that control particular aspects of temporal and spatial expression, and in both the territorial boundaries of expression depend on a combination of negative and positive functions. In both genes different regulatory modules control early and late embryonic expression. Modular cis-regulatory organization is widespread in developmentally regulated genes, and we present a tabular summary that includes many examples from mouse and Drosophila. We regard cis-regulatory modules as units of developmental transcription control, and also of evolution, in the assembly of transcription control systems

Short-scale turbulent fluctuations driven by the electron-temperature gradient in the national spherical torus experiment

Measurements with coherent scattering of electromagnetic waves in plasmas of the National Spherical Torus Experiment indicate the existence of turbulent fluctuations in the range of wave numbers k(perpendicular to)rho(e)=0.1-0.4, corresponding to a turbulence scale length nearly equal to the collisionless skin depth. Experimental observations and agreement with numerical results from a linear gyrokinetic stability code support the conjecture that the observed turbulence is driven by the electron-temperature gradient.X1155sciescopu

An Enhanced Nonlinear Critical Gradient for Electron Turbulent Transport due to Reversed Magnetic Shear

The first nonlinear gyrokinetic simulations of electron internal transport barriers (e-ITBs) in the National Spherical Torus Experiment show that reversed magnetic shear can suppress thermal transport by increasing the nonlinear critical gradient for electron-temperature-gradient-driven turbulence to three times its linear critical value. An interesting feature of this turbulence is nonlinearly driven off-midplane radial streamers. This work reinforces the experimental observation that magnetic shear is likely an effective way of triggering and sustaining e-ITBs in magnetic fusion devices.Comment: 4 pages, 5 figure