Protocol: fine-tuning of a Chromatin Immunoprecipitation (ChIP) protocol in tomato

<p>Abstract</p> <p>Background</p> <p>Searching thoroughly for plant <it>cis</it>-elements corresponding to transcription factors is worthwhile to reveal novel gene activation cascades. At the same time, a great deal of research is currently focused on epigenetic events in plants. A widely used method serving both purposes is chromatin immunoprecipitation, which was developed for Arabidopsis and other plants but is not yet operational for tomato (<it>Solanum lycopersicum</it>), a model plant species for a group of economically important crops.</p> <p>Results</p> <p>We developed a chromatin immunoprecipitation protocol suitable for tomato by adjusting the parameters to optimise <it>in vivo </it>crosslinking, purification of nuclei, chromatin extraction, DNA shearing and precipitate analysis using real-time PCR. Results were obtained with two different antibodies, five control loci and two normalisation criteria.</p> <p>Conclusion</p> <p>Here we provide a chromatin immunoprecipitation procedure for tomato leaves that could be combined with high-throughput sequencing to generate a detailed map of epigenetic modifications or genome-wide nucleosome positioning data.</p

Atypical epigenetic mark in an atypical location: cytosine methylation at asymmetric (CNN) sites within the body of a non-repetitive tomato gene

Background: Eukaryotic DNA methylation is one of the most studied epigenetic processes, as it results in a direct and heritable covalent modification triggered by external stimuli. In contrast to mammals, plant DNA methylation, which is stimulated by external cues exemplified by various abiotic types of stress, is often found not only at CG sites but also at CNG (N denoting A, C or T) and CNN (asymmetric) sites. A genome-wide analysis of DNA methylation in Arabidopsis has shown that CNN methylation is preferentially concentrated in transposon genes and non-coding repetitive elements. We are particularly interested in investigating the epigenetics of plant species with larger and more complex genomes than Arabidopsis, particularly with regards to the associated alterations elicited by abiotic stress.Results: We describe the existence of CNN-methylated epialleles that span Asr1, a non-transposon, protein-coding gene from tomato plants that lacks an orthologous counterpart in Arabidopsis. In addition, to test the hypothesis of a link between epigenetics modifications and the adaptation of crop plants to abiotic stress, we exhaustively explored the cytosine methylation status in leaf Asr1 DNA, a model gene in our system, resulting from water-deficit stress conditions imposed on tomato plants. We found that drought conditions brought about removal of methyl marks at approximately 75 of the 110 asymmetric (CNN) sites analysed, concomitantly with a decrease of the repressive H3K27me3 epigenetic mark and a large induction of expression at the RNA level. When pinpointing those sites, we observed that demethylation occurred mostly in the intronic region.Conclusions: These results demonstrate a novel genomic distribution of CNN methylation, namely in the transcribed region of a protein-coding, non-repetitive gene, and the changes in those epigenetic marks that are caused by water stress. These findings may represent a general mechanism for the acquisition of new epialleles in somatic cells, which are pivotal for regulating gene expression in plants. © 2011 González et al; licensee BioMed Central Ltd.Fil:González, R.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Ricardi, M.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Iusem, N.D. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Predominantly Cytoplasmic Localization in Yeast of ASR1, a Non-Receptor Transcription Factor from Plants

The Asr gene family (named after abscisic acid, stress and ripening), currently classified as a novel group of the LEA superfamily, is exclusively present in the genomes of seed plants, except for the Brassicaceae family. It is associated with water-deficit stress and is involved in adaptation to dry climates. Motivated by separate reports depicting ASR proteins as either transcription factors or chaperones, we decided to determine the intracellular localization of ASR proteins. For that purpose, we employed an in vivo eukaryotic expression system, the heterologous model Saccharomyces cerevisiae, including wild type strains as well as mutants in which the variant ASR1 previously proved to be functionally protective against osmotic stress. Our methodology involved immunofluorescence-based confocal microscopy, without artificially altering the native structure of the protein under study. Results show that, in both normal and osmotic stress conditions, recombinant ASR1 turned out to localize mainly to the cytoplasm, irrespective of the genotype used, revealing a scattered distribution in the form of dots or granules. The results are discussed in terms of a plausible dual (cytoplasmic and nuclear) role of ASR proteins

Mitos y verdades del genoma humano

Nuestro genoma está formado por 1) componentes de función bioquímica conocida que ocupan un porcentaje minoritario de nuestra información genética y 2) una proporción muy elevada de elementos eventualmente móviles. La mayoría de éstos últimos se han convertido en fósiles moleculares, pero seguramente han tenido un rol en la plasticidad de nuestro genoma en el transcurrir de nuestra historia evolutiva, aunque pueden en el presente y en pequeña escala seguir teniendo efectos negativos en nuestra salud. La fracción del genoma interpretada como ocupada por “regiones intergénicas”, verdadero “DNA chatarra” si es que queremos seguir usando esta analogía, sigue disminuyendo a medida que se descubren cada vez más genes de ARN no codificante (localizados entre genes previamente conocidos) que ayudan a la célula a apagar genes circunstancialmente innecesarios. Todavía tenemos mucho que aprender sobre qué significan los tres mil millones de nucleótidos del Genoma Humano, sus modificaciones químicas adquiridas en cada tipo celular y tejido de acuerdo a estímulos externos (terreno de la epigenética), y la influencia en la expresión génica que ejerce la dinámica cromatina. Futuros avances en técnicas de Biología Molecular y en Bioinformática seguramente contribuirán al desafío, ayudando a profundizar en el conocimiento que tenemos sobre la evolución humana y a entender y prevenir enfermedades.Fil: Cramer, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina; Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Fisiologia, Biologia Molecular y Celular; Argentina;Fil: Iusem, Norberto Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina; Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Fisiologia, Biologia Molecular y Celular; Argentina

Guard cell-specific inhibition of Arabidopsis MPK3 expression causes abnormal stomatal responses to abscisic acid and hydrogen peroxide

MAP kinases have been linked to guard cell signalling. Arabidopsis thaliana MAP Kinase 3 (MPK3) is known to be activated by abscisic acid (ABA) and hydrogen peroxide (H2O2), which also control stomatal movements. We therefore studied the possible role of MPK3 in guard cell signalling through guard cell-specific antisense inhibition of MPK3 expression. Such transgenic plants contained reduced levels of MPK3 mRNA in the guard cells and displayed partial insensitivity to ABA in inhibition of stomatal opening, but responded normally to this hormone in stomatal closure. However, ABA-induced stomatal closure was reduced compared with controls when cytoplasmic alkalinization was prevented with sodium butyrate. MPK3 antisense plants were less sensitive to exogenous H2O2, both in inhibition of stomatal opening and in promotion of stomatal closure, thus MPK3 is required for the signalling of this compound. ABA-induced H2O2 synthesis was normal in these plants, indicating that MPK3 probably acts in signalling downstream of H2O2. These results provide clear evidence for the important role of MPK3 in the perception of ABA and H 2O2 in guard cells. © The Authors (2007).Fil:Gudesblat, G.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Iusem, N.D. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina