Serial ChIP as a tool to investigate the co-localization or exclusion of proteins on plant genes

<p>Abstract</p> <p>Background</p> <p>Establishing transcriptional regulatory networks that include protein-protein and protein-DNA interactions has become a key component to better understanding many fundamental biological processes. Although a variety of techniques are available to expose protein-protein and protein-DNA interactions, unequivocally establishing whether two proteins are targeted together to the same promoter or DNA molecule poses a very challenging endeavour. Yet, the recruitment of multiple regulatory proteins simultaneously to the same promoter provides the basis for combinatorial transcriptional regulation, central to the transcriptional regulatory network of eukaryotes. The serial ChIP (sChIP) technology was developed to fill this gap in our knowledge, and we illustrate here its application in plants.</p> <p>Results</p> <p>Here we describe a modified sChIP protocol that provides robust and quantitative information on the co-association or exclusion of DNA-binding proteins on particular promoters. As a proof of principle, we investigated the association of histone H3 protein variants with modified tails (H3K9ac and H3K9me2) with <it>Arabidopsis </it>RNA polymerase II (RNPII) on the promoter of the constitutively expressed <it>actin </it>gene (At5g09810), and the trichome-expressed <it>GLABRA3 </it>(<it>GL3</it>) gene. As anticipated, our results show a strong positive correlation between H3K9ac and RNPII and a negative correlation between H3K9me2 and RNPII on the actin gene promoter. Our findings also establish a weak positive correlation between both H3K9ac and H3K9me2 and RNPII on the <it>GL3 </it>gene promoter, whose expression is restricted to a discrete number of cell types. We also describe mathematical tools that allow the easy interpretation of sChIP results.</p> <p>Conclusion</p> <p>The sChIP method described here provides a reliable tool to determine whether the tethering of two proteins to the same DNA molecule is positively or negatively correlated. With the increasing need for establishing transcriptional regulatory networks, this modified sChIP method is anticipated to provide an excellent way to explore combinatorial gene regulation in eukaryotes.</p

Respuesta temprana de neurospora crassa a la inhibición parcial de la síntesis de proteínas

La inhibición parcial de la síntesis proteica porcicloheximida o ayuno total de nutrientes en Neurospora crassa,produce la inducción de varios genes. Se clonaron dos de ellos, cuyos transcriptos aparecenluego de apenas media hora de agregado el inhibidor. Seidentificaron asimismo, los productos de traducción de estosgenes y se demostró que la aparición de los mRNAs correspondientesestá acompañadade su traducción. Las proteínas de 27 kD y 31 kD están entre las sintetizadas en mayor abundancialuego de la inhibición parcial de la sintesis proteica ycorresponden a los productos de los genes clonados. Estos genes serían inducibles por otras situacionescapaces de inhibir la síntesis proteica, como el agregado depuromicina o el ayuno por 12 horas en buffer fosfato. No serían,sin embargo, inducibles por shock térmico. Uno de los genes se expresaría en forma constitutivaen una cepa que carece de la enzima ornitina decarboxilasa,cuando crece en presencia de espermidina. El clon genómicoque contiene a este gen completo fue secuenciado totalmente.Fil: Grotewold, Erich. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Light-induced morphological alteration in anthocyanin-accumulating vacuoles of maize cells

BACKGROUND: Plant pigmentation is affected by a variety of factors. Light, an important plant developmental signal, influences the accumulation of anthocyanins primarily through the activation of the transcription factors that regulate the flavonoid biosynthetic pathway. In this study, we utilized maize Black Mexican Sweet (BMS) cells expressing the R and C1 regulators of anthocyanin biosynthesis from a light-insensitive promoter as a means to investigate the existence of additional levels of control of pigmentation by light. RESULTS: BMS cells expressing the R and C1 regulators from the CaMV 35S constitutive promoter accumulate anthocyanins when grown in complete darkness, suggesting that the transcription factors R and C1 are sufficient for the transcription of the genes corresponding to the structural enzymes of the pathway, with no requirement for additional light-induced regulators. Interestingly, light induces a "darkening" in the color of the purple anthocyanin pigmentation of transgenic BMS cells expressing R and C1. This change in the pigment hue is not associated with a variation in the levels or types of anthocyanins present, or with an alteration of the transcript levels of several flavonoid biosynthetic genes. However, cytological observations show that light drives unexpected changes in the morphology and distribution of the anthocyanins-containing vacuolar compartments. CONCLUSION: By uncoupling the effect of light on anthocyanin accumulation, we have found light to induce the fusion of anthocyanin-containing vacuoles, the coalescence of anthocyanic vacuolar inclusion (AVI)-like structures contained, and the spread of anthocyanins from the inclusions into the vacuolar sap. Similar light-induced alterations in vacuolar morphology are also evident in the epidermal cells of maize floral whorls accumulating anthocyanins. Our findings suggest a novel mechanism for the action of light on the vacuolar storage of anthocyanin

Engineering phenolics metabolism in the grasses using transcription factors

The economical competitiveness of agriculture-derived biofuels can be significantly enhanced by increasing biomass/acre yields and by furnishing the desired carbon balance for facilitating liquid fuel production (e.g., ethanol) or for high-energy solid waste availability to be used as biopower (e.g., for electricity production). Biomass production and carbon balance are tightly linked to the biosynthesis of phenolic compounds, which are found in crops and in agricultural residues either as lignins, as part of the cell wall, or as soluble phenolics which play a variety of functions in the biology of plants. The grasses, in particular maize, provide the single major source of agricultural biomass, offering significant opportunities for increasing renewable fuel production. Our laboratory has pioneered the use of transcription factors for manipulating plant metabolic pathways, an approach that will be applied here towards altering the composition of phenolic compounds in maize. Previously, we identified a small group of ten maize R2R3-MYB transcription factors with all the characteristics of regulators of different aspects of phenolic biosynthesis. Here, we propose to investigate the participation of these R2R3-MYB factors in the regulation of soluble and insoluble maize phenolics, using a combination of over-expression and down-regulation of these transcription factors in transgenic maize cultured cells and in maize plants. Maize cells and plants altered in the activity of these regulatory proteins will be analyzed for phenolic composition by targeted metabolic profiling. Specifically, we will I) Investigate the effect of gain- and loss-of-function of a select group of R2R3-MYB transcription factors on the phenolic composition of maize plants and II) Identify the biosynthetic genes regulated by each of the selected R2R3-MYB factors. While a likely outcome of these studies are transgenic maize plants with altered phenolic composition, this research will significantly contribute to understanding how different branches of the phenolic biosynthetic grid are regulated. Given the conservation of the selected regulators in other grasses, results derived from this project are likely to provide important tools for the manipulation of phenolic compounds in other emerging biomass producers (e.g., switchgrass or miscanthus), either through conventional breeding techniques (e.g., marker-assisted breeding) or by using transgenic approaches

Emergence of switch-like behavior in a large family of simple biochemical networks

Bistability plays a central role in the gene regulatory networks (GRNs) controlling many essential biological functions, including cellular differentiation and cell cycle control. However, establishing the network topologies that can exhibit bistability remains a challenge, in part due to the exceedingly large variety of GRNs that exist for even a small number of components. We begin to address this problem by employing chemical reaction network theory in a comprehensive in silico survey to determine the capacity for bistability of more than 40,000 simple networks that can be formed by two transcription factor-coding genes and their associated proteins (assuming only the most elementary biochemical processes). We find that there exist reaction rate constants leading to bistability in ~90% of these GRN models, including several circuits that do not contain any of the TF cooperativity commonly associated with bistable systems, and the majority of which could only be identified as bistable through an original subnetwork-based analysis. A topological sorting of the two-gene family of networks based on the presence or absence of biochemical reactions reveals eleven minimal bistable networks (i.e., bistable networks that do not contain within them a smaller bistable subnetwork). The large number of previously unknown bistable network topologies suggests that the capacity for switch-like behavior in GRNs arises with relative ease and is not easily lost through network evolution. To highlight the relevance of the systematic application of CRNT to bistable network identification in real biological systems, we integrated publicly available protein-protein interaction, protein-DNA interaction, and gene expression data from Saccharomyces cerevisiae, and identified several GRNs predicted to behave in a bistable fashion.Comment: accepted to PLoS Computational Biolog

Covalent attachment of the plant natural product naringenin to small glass and ceramic beads

BACKGROUND: Natural products have numerous medicinal applications and play important roles in the biology of the organisms that accumulate them. Few methods are currently available for identifying proteins that bind to small molecules, therefore the discovery of cellular targets for natural products with pharmacological activity continues to pose a significant challenge in drug validation. Similarly, the identification of enzymes that participate in the biosynthesis or modification of natural products remains a formidable bottleneck for metabolic engineering. Flavonoids are one large group of natural products with a diverse number of functions in plants and in human health. The coupling of flavonoids to small ceramic and glass beads provides a first step in the development of high-throughput, solid-support base approaches to screen complex libraries to identify proteins that bind natural products. RESULTS: The utilization of small glass and ceramic beads as solid supports for the coupling of small molecules was explored. Initial characterization of the beads indicated uniform and high capacity loading of amino groups. Once the beads were deemed adequate for the linking of small molecules by the coupling of NHS-fluorescein followed by microscopy, chemical hydrolysis and fluorometry, the flavonoid naringenin was modified with 1,4-dibromobutane, followed by the attachment of aminopropyltriethoxysilane. After NMR structural confirmation, the resulting 7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin was attached to the ceramic beads. CONCLUSION: Our results demonstrate that ceramic and glass beads provide convenient solid supports for the efficient and facile coupling of small molecules. We succeeded in generating naringenin-coupled ceramic and glass beads. We also developed a convenient series of steps that can be applied for the solid-support coupling of other related flavonoids. The availability of solid-support coupled naringenin opens up new opportunities for the identification of flavonoid-binding proteins

Sub-cellular trafficking of phytochemicals explored using auto-fluorescent compounds in maize cells

BACKGROUND: Little is known regarding the trafficking mechanisms of small molecules within plant cells. It remains to be established whether phytochemicals are transported by pathways similar to those used by proteins, or whether the expansion of metabolic pathways in plants was associated with the evolution of novel trafficking pathways. In this paper, we exploited the induction of green and yellow auto-fluorescent compounds in maize cultured cells by the P1 transcription factor to investigate their targeting to the cell wall and vacuole, respectively. RESULTS: We investigated the accumulation and sub-cellular localization of the green and yellow auto-fluorescent compounds in maize BMS cells expressing the P1 transcription factor from an estradiol inducible promoter. We established that the yellow fluorescent compounds accumulate inside the vacuole in YFBs that resemble AVIs. The green fluorescent compounds accumulate initially in the cytoplasm in large spherical GFBs. Cells accumulating GFBs also contain electron-dense structures that accumulate initially in the ER and which later appear to fuse with the plasma membrane. Structures resembling the GFBs were also observed in the periplasmic space of plasmolized cells. Ultimately, the green fluorescence accumulates in the cell wall, in a process that is insensitive to the Golgi-disturbing agents BFA and monensin. CONCLUSIONS: Our results suggest the presence of at least two distinct trafficking pathways, one to the cell wall and the other to the vacuole, for different auto-fluorescent compounds induced by the same transcription factor in maize BMS cells. These compartments represent two of the major sites of accumulation of phenolic compounds characteristic of maize cells. The secretion of the green auto-fluorescent compounds occurs by a pathway that does not involve the TGN, suggesting that it is different from the secretion of most proteins, polysaccharides or epicuticular waxes. The yellow auto-fluorescent compounds accumulate in a vacuolar compartment, in structures that resemble the AVIs present in many cells accumulating anthocyanins. Together, our studies suggest that the accumulation of auto-fluorescent compounds can provide a powerful tool to dissect the trafficking of phytochemicals, knowledge necessary for the efficient engineering of plant metabolism

The capacity for multistability in small gene regulatory networks

<p>Abstract</p> <p>Background</p> <p>Recent years have seen a dramatic increase in the use of mathematical modeling to gain insight into gene regulatory network behavior across many different organisms. In particular, there has been considerable interest in using mathematical tools to understand how multistable regulatory networks may contribute to developmental processes such as cell fate determination. Indeed, such a network may subserve the formation of unicellular leaf hairs (trichomes) in the model plant <it>Arabidopsis thaliana</it>.</p> <p>Results</p> <p>In order to investigate the capacity of small gene regulatory networks to generate multiple equilibria, we present a chemical reaction network (CRN)-based modeling formalism and describe a number of methods for CRN analysis in a parameter-free context. These methods are compared and applied to a full set of one-component subnetworks, as well as a large random sample from 40,680 similarly constructed two-component subnetworks. We find that positive feedback and cooperativity mediated by transcription factor (TF) dimerization is a requirement for one-component subnetwork bistability. For subnetworks with two components, the presence of these processes increases the probability that a randomly sampled subnetwork will exhibit multiple equilibria, although we find several examples of bistable two-component subnetworks that do not involve cooperative TF-promoter binding. In the specific case of epidermal differentiation in <it>Arabidopsis</it>, dimerization of the GL3-GL1 complex and cooperative sequential binding of GL3-GL1 to the CPC promoter are each independently sufficient for bistability.</p> <p>Conclusion</p> <p>Computational methods utilizing CRN-specific theorems to rule out bistability in small gene regulatory networks are far superior to techniques generally applicable to deterministic ODE systems. Using these methods to conduct an unbiased survey of parameter-free deterministic models of small networks, and the <it>Arabidopsis </it>epidermal cell differentiation subnetwork in particular, we illustrate how future experimental research may be guided by network structure analysis.</p

Flavonols protect Arabidopsis plants against UV-B deleterious effects

Flavonols are synthesized by flavonol synthase (FLS) enzymes (Martens et al., 2010). These compounds absorb UV-B light in the 280?320 nm region, and their concentration increases in plants exposed to environmental abiotic and biotic stresses, including UV-B; consequently, flavonols are thought to act as UV-B filters (Agati et al., 2011). It has been also suggested that these metabolites function as reactive oxygen species (ROS) scavengers, as they contain an OH- group in the 3-position of the flavonoid skeleton, which allows them to chelate metals, inhibiting the formation of free radicals and ROS accumulation, once formed (Agati et al., 2009). For these reasons, it has been suggested that flavonols play uncharacterized roles in UV responses (Verdan et al., 2011). Nevertheless, despite the fact that the role of flavonols in UV-B protection has been inferred; the protection conferred by flavonols on the target sites of UV-B-damage has not been directly proven in planta. Recently, we demonstrated that maize FLS1 (ZmFLS1) complements the flavonol deficiency of the Arabidopsis fls1 mutant and decreases its high level of anthocyanins, characteristic of this mutant plant (Falcone Ferreyra et al., 2010). In order to demonstrate that flavonols protect plants against UV-B damage, we generated Arabidopsis transgenic plants overexpressing the maize FLS1 cDNA (35S:ZmFLS1), and evaluated different responses of these transgenic plants against UV-B damage.Fil: Emiliani, Julia.Fil: Grotewold, Erich.Fil: Falcone Ferreyra, María Lorena.Fil: Casati, Paula