Ce travail a été financé par la Fondation allemande des sciences (DFG) et l&#39;Initiative d&#39;excellence des gouvernements fédéral allemand et l&#39;Etat.

1. La réticulation de la matière végétale <ol><li> Récolte 1-2g de feuilles d&#39;Arabidopsis (6 à 10cm de diamètre rosette) dans un tube en plastique de 50 ml et compléter à 40 mL avec le tampon de réticulation. </li><li> Assurez-vous que les feuilles restent immergés, par exemple en farce d&#39;un droit adapté filtre éponge dessus de la surface du tampon. Ensuite, placez les tubes dans un dessiccateur. </li><li> Vide infiltrer pour 10min. Pour chaque tube 2,5 ml de glycine 2M pour arrêter de réticulation, et inverser les tubes pour justifier la dispersion égale de la glycine. </li><li> Vide d&#39;infiltrer pour une autre 5min et jeter fluide pendant la récolte des feuilles sur un tamis. </li><li> Laver les feuilles à deux reprises avec 1 litre d&#39;eau dans un récipient en verre, puis le laisse sécher avec des serviettes en papier. </li><li> Ramasser les feuilles dans un tube en plastique douce et geler dans l&#39;azote liquide. Magasin de feuilles à -80 ° C jusqu&#39;à ce que l&#39;isolement chromatine. </li></ol> 2. Isolement de la chromatine <ol><li> Feuilles ont été broyées avec du mortier et pistil qui ont été conservés dans l&#39;azote liquide jusqu&#39;à leur utilisation. </li><li> Transfert de poudre pour un tube en plastique de 50 ml et de suspendre dans 30mL de tampon d&#39;extraction n ° 1. </li><li> Incuber pendant 15min à 4 ° C sur un agitateur vertical. </li><li> Filtrat à travers la suspension de quatre couches de miracloth dans un tube en plastique de 50 ml fraîche. </li><li> Spin pour 20min à 2800 xg à 4 ° C. </li><li> Retirer le surnageant et suspendre culot avec un pinceau dans le tampon d&#39;extraction 1ml # 2. Ajouter d&#39;abord un petit volume de tampon n ° 2, puis de suspendre culot avec un pinceau, puis ajoutez le reste de la mémoire tampon. </li><li> Transfert de suspension pour un tube de micro et de 1,5 ml de spin pour 10min à 12.000 xg à 4 ° C. </li><li> Pendant ce temps, préparez-2mL microcentrifugeuse tubes contenant 1,5 ml de tampon d&#39;extraction n ° 3. </li><li> Retirer le surnageant du tube filé (étape 2.7) et de suspendre le culot dans 300μL de tampon d&#39;extraction n ° 3. Ajouter d&#39;abord un petit volume de tampon n ° 3, suspendre culot avec un pinceau, puis ajoutez le tampon restant. </li><li> Soigneusement pipette le culot en suspension (étape 2.9) sur le dessus de la mémoire tampon de 1,5 ml d&#39;extraction n ° 3 préparé à l&#39;étape 2.8. Assurez-vous que les deux phases se mélangent pas. Spin pour 1h à 16 000 xg à 4 ° C. </li><li> Retirer le surnageant et suspendre la chromatine culot avec un pinceau dans 300μL de tampon de sonication. </li><li> Soniquer chromatine avec une sonotrode, ou dans un bain à ultrasons, à une taille d&#39;ADN d&#39;environ 400bp. Lorsque sonication, assurez-vous que la chromatine ne sera pas chauffé au-dessus d&#39;environ 30 ° C pour protéger sensibles à la chaleur réticule. La durée de la sonication doit être déterminée expérimentalement, car il varie significativement entre les appareils sonication différents. Pour obtenir des conseils, des paramètres pour deux appareils différents sont prévus: </li></ol> Pour sonication avec une marque de Bandelin sonotrode chromatine exposer dans le tube 0.6ml microcentrifugeuse cinq fois à 20 coups avec les paramètres de 25% de puissance, cycle = 50. Ce faisant, l&#39;échantillon fraîche après chaque 20 ème éclate dans un bain de glace. Pour sonication avec un bain à ultrasons Bioruptor Diagenode, remplissez salle de bain avec eau et la glace et soniquer dix fois pendant 30 secondes en microtubes de 1,5 ml avec réglage «élevé». Après toutes les 30 secondes, les échantillons au frais pendant 30 secondes. <ol start="13"><li> Spin soniqué échantillon pour 5min à 16.000 g à 4 ° C pour précipiter matière non dissoute. Le surnageant peut être directement utilisé pour l&#39;immunoprécipitation. Alternativement, les échantillons peuvent être un choc congelés dans l&#39;azote liquide et stockés à -80 ° C jusqu&#39;à utilisation ultérieure. </li></ol> 3. Immunoprécipitation <ol><li> Préparer microtubes de 2 ml contenant la protéine A 40μL. Agarose et 1,8 ml d&#39;anticorps contraignant tampon Ajouter 200 ul de la préparation de chromatine (étape 2.13). </li><li> Incuber les tubes pour 1h sur un agitateur vertical. </li><li> Jeter la protéine A billes d&#39;agarose et l&#39;utilisation surnageant pour immunoprécipitation. </li><li> Supprimer une partie aliquote 40μL-pour déterminer la concentration de chromatine (&#39;input&#39;). </li><li> Ajouter 30μL protéines-A. Agarose et le montant de la modification d&#39;anticorps spécifique qui est indiqué dans le tableau ci-dessous des réactifs et équipements spécifiques à la suspension chromatine soniquée 400μL </li><li> Incuber pendant 2.5h ou toute la nuit sur un agitateur vertical à 4 ° C. </li><li> Isoler perles pour 2min à 440 x g. </li><li> Lavez perles culot avec 900μL de chaque tampon de lavage (voir la liste ci-dessous). Pour chaque tampon, perles incubation dans le tampon pendant 10 minutes sur un agitateur vertical à 4 ° C, puis de spin pour 2min à 440 xg, éliminer le surnageant et ajouter le tampon suivant. <ol><li> Faible tampon de lavage du sel </li><li> Haute tampon de lavage du sel </li><li> LiCl tampon de lavage </li><li> TE tampon de lavage </li></ol></li><li> Après le lavage final, enlever complètement toute tampon restant. </li></ol> 4. De-réticulation des histones et l&#39;ADN, et la purification de l&#39;ADN précipité <ol><li> Ajouter 100 pi de tampon de-réticulation au culot d&#39;agarose et de l&#39;échantillon «d&#39;entrée» du 40μl de l&#39;étape 3.4, mélanger pendant 5min sur un vortex, les échantillons de spin, et incubateursBate à 65 ° C pendant la nuit. </li><li> Purifier l&#39;ADN avec un ADN commercial ou kit de purification de PCR. </li><li> Typiquement 5uL de la dilution 1:05 de l&#39;éluat de la colonne finale sont suffisants pour effectuer conventionnelles spécifiques de locus en temps réel RT-PCR quantitative (RT-qPCR). </li></ol> 5. Table des tampons <table border="1"><tbody><tr><td colspan="2"> Mémoire tampon de réticulation </td></tr><tr><td> Butyrate de sodium </td><td> 10mM </td></tr><tr><td> Saccharose </td><td> 400mm </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10mM </td></tr><tr><td> β-mercaptoéthanol </td><td> 5mM </td></tr><tr><td> Formaldéhyde </td><td> 3% (v / v) </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampon d&#39;extraction n ° 1 </td></tr><tr><td> Butyrate de sodium </td><td> 10mM </td></tr><tr><td> Saccharose </td><td> 400mm </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10mM </td></tr><tr><td> β-mercaptoéthanol </td><td> 5mM </td></tr><tr><td> Inhibiteur de protéase complète </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampon d&#39;extraction n ° 2 </td></tr><tr><td> Butyrate de sodium </td><td> 10mM </td></tr><tr><td> Saccharose </td><td> 250mm </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10mM </td></tr><tr><td> β-mercaptoéthanol </td><td> 5mM </td></tr><tr><td> MgCl 2 </td><td> 10mM </td></tr><tr><td> Triton X-100 </td><td> 1% (p / v) </td></tr><tr><td> Inhibiteur de protéase complète </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampon d&#39;extraction n ° 3 </td></tr><tr><td> Butyrate de sodium </td><td> 10mM </td></tr><tr><td> Saccharose </td><td> 1,7 M </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10mM </td></tr><tr><td> β-mercaptoéthanol </td><td> 5mM </td></tr><tr><td> MgCl 2 </td><td> 2mM </td></tr><tr><td> Triton X-100 </td><td> 0,15% (p / v) </td></tr><tr><td> Inhibiteur de protéase complète </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampon de sonication </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 25mM </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 5mM </td></tr><tr><td> SDS </td><td> 0,5% (p / v) </td></tr><tr><td> Inhibiteur de protéase complète </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Buffer fixation des anticorps </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 50mM </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 1mM </td></tr><tr><td> NaCl </td><td> 150 mM </td></tr><tr><td> Triton X-100 </td><td> 0,1% (p / v) </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Faible tampon de lavage du sel </td></tr><tr><td> NaCl </td><td> 150 mM </td></tr><tr><td> SDS </td><td> 0,1% (p / v) </td></tr><tr><td> Triton X-100 </td><td> 1% (p / v) </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 2mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 20 mM </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Haute tampon de lavage du sel </td></tr><tr><td> NaCl </td><td> 500mm </td></tr><tr><td> SDS </td><td> 0,1% (p / v) </td></tr><tr><td> Triton X-100 </td><td> 1% (p / v) </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 2mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 20 mM </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> LiCl tampon de lavage </td></tr><tr><td> Le chlorure de lithium </td><td> 250mm </td></tr><tr><td> Nonidet-P40 </td><td> 1% (v / v) </td></tr><tr><td> Désoxycholate de sodium </td><td> 1% (p / v) </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 1mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 20 mM </td></tr></tbody></table><table border="1"><tbody><tr><td> TE tampon de lavage </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 10mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 1mM </td></tr></tbody></table><table border="1"><tbody><tr><td> Buffer Decrosslinking </td></tr><tr><td> Tris-HCl, pH 6,8 </td><td> 62.5mm </td></tr><tr><td> NaCl </td><td> 200 mM </td></tr><tr><td> SDS </td><td> 2% (p / v) </td></tr><tr><td> TNT </td><td> 10mM </td></tr></tbody></table> 6. Les résultats représentatifs: L&#39;expression du gène de la défense d&#39;Arabidopsis thaliana codant pour une β PR2-1,3-glucanase ayant une activité antimicrobienne 12, a été induite par benzothiadiazole (BTH), un analogue synthétique de l&#39;acide salicylique, hormone végétale 3. La figure 1 montre que l&#39;activation du PR2 expression est associée à une augmentation de l&#39;acétylation des résidus lysine des histones H3 et H4 sur le promoteur PR2. Comme la densité diminue au cours de 13 nucléosome activation des gènes, les valeurs ont été normalisées acétylation des histones pour le signal obtenu avec un anticorps à une région invariante de 3 histones. <img src="/files/ftp_upload/3096/3096fig1.jpg" alt="Figure 1"/> Figure 1 BTH induit l&#39;acétylation des histones sur le promoteur PR2 chez Arabidopsis. Les plantes ont été traitées avec 100 microns BTH ou un contrôle de poudre mouillable. Après 72h, les feuilles ont été recueillies et de la chromatine a été isolé. Le signal obtenu après précipitation avec l&#39;acétylation des anticorps spécifiques (comme indiqué dans la figure) a été normalisé au signal obtenu par précipitation avec un anticorps à un domaine invariant de l&#39;histone H3. Chromatine précipité a été quantifiée par RT-qPCR. Les données sont normalisées pour les niveaux modification des histones, en l&#39;absence de traitement BTH.

<ol>
	<li>Eberharter, A., Becker, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation:+a+switch+between+repressive+and+permissive+chromatin.">Histone acetylation: a switch between repressive and permissive chromatin.</a> EMBO rep. 3, 224-229 (2002).</li><li>Ruthenburg, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylation+of+lysine+4+on+histone+H3:+intricacy+of+writing+and+reading+a+single+epigenetic.">Methylation of lysine 4 on histone H3: intricacy of writing and reading a single epigenetic.</a> 25, 15-30 (2007).</li><li>Jaskiewicz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modification+acts+as+a+memory+for+systemic+acquired+resistance+in+the+plant+stress+response.">Chromatin modification acts as a memory for systemic acquired resistance in the plant stress response.</a> EMBO rep. 12, 50-55 (2011).</li><li>Ng, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordered+histone+modifications+are+associated+with+transcriptional+poising+and+activation+of+the+phaseolin+promoter.">Ordered histone modifications are associated with transcriptional poising and activation of the phaseolin promoter.</a> Plant Cell. 18, 119-132 (2006).</li><li>Offermann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illumination+is+necessary+and+sufficient+to+induce+histone+acetylation+independent+of+transcriptional+activity+at+the+C4-specific+phosphoenolpyruvate+carboxylase+promoter+in+maize.">Illumination is necessary and sufficient to induce histone acetylation independent of transcriptional activity at the C4-specific phosphoenolpyruvate carboxylase promoter in maize.</a> Plant Physiol. 141, 1078-1088 (2006).</li><li>Deng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+histone+acetyltransferase+AtHAC1+in+the+regulation+of+flowering+time+via+repression+of+FLOWERING+LOCUS+C+in+Arabidopsis.">Involvement of the histone acetyltransferase AtHAC1 in the regulation of flowering time via repression of FLOWERING LOCUS C in Arabidopsis.</a> Plant Physiol. 143, 1660-1668 (2007).</li><li>Benhamed, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+GCN5,+HD1,+and+TAF1/HAF2+interact+to+regulate+histone+acetylation+required+for+light-responsive+gene+expression.">Arabidopsis GCN5, HD1, and TAF1/HAF2 interact to regulate histone acetylation required for light-responsive gene expression.</a> Plant Cell. 18, 2893-2903 (2006).</li><li>Bowler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+techniques+for+plant+cells.">Chromatin techniques for plant cells.</a> Plant Journal. 39, 776-789 (2004).</li><li>Orlando, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+chromosomal+proteins+in+vivo+by+formaldehyde+decrosslinked-chromatin+immunoprecipitation.">Mapping chromosomal proteins in vivo by formaldehyde decrosslinked-chromatin immunoprecipitation.</a> Trends Biochem. 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Aucun conflit d&#39;intérêt déclaré.

Étapes les plus critiques dans le protocole sont la réticulation de l&#39;ADN et les histones, le type et la quantité de matériau de feuilles, broyage de la matière, et la sonication de la chromatine. Pour une discussion plus détaillée de ces questions, voir refs. 9 et 10. Sonication et réticulation: Il est important de perturber la chromatine pendant la sonication à des fragments d&#39;environ 400bp. Sonication exhaustive va détruire la chromatine en perturbant l&#39;ADN et les histones, alors que la sonication insuffisante va laisser des fragments de chromatine longtemps. Ceux-ci affectent la spécificité de la méthode, parce que les modifications des histones distale du locus testés ne peuvent être discriminés par des modifications locales. L&#39;efficacité sonication est mieux testée par 20 pi de la chromatine collecte des échantillons avant et après traitement par ultrasons, de réticulation de l&#39;ADN et les histones, isoler l&#39;ADN et de déterminer la longueur de l&#39;ADN cisaillé par électrophorèse sur gel. RNAse devraient être ajoutés aux échantillons avant l&#39;électrophorèse, parce que la préparation de chromatine contient encore de grandes quantités d&#39;ARN à ce stade de la purification qui pourraient interférer avec la visualisation de l&#39;ADN fragmenté. Les échantillons sont utiles pour immunoprécipitation de la chromatine, si l&#39;ADN de la non-cisaillée chromatine est plus long que 10kbp, tandis que l&#39;ADN de la chromatine cisaillé formes d&#39;un frottis avec une intensité maximale du signal autour de 400bp de l&#39;ADN. Nous utilisons régulièrement de 3% (v / v) de formaldéhyde réticulation chromatine dans des feuilles intactes, mais 1% (v / v) de formaldéhyde pourrait être suffisant dans la plupart des cas. Dans nos mains, les concentrations de formaldéhyde permettent des temps de sonication plus courtes et les signaux de fond dans des réactions PCR conséquente. Cependant, des quantités insuffisantes de formaldéhyde aboutissent souvent à faible reproductibilité du signal PCR entre expériences indépendantes. Ceci pourrait être dû à la pénétration insuffisante de feuilles avec du formaldéhyde à des concentrations faibles. Assurez-vous d&#39;échanger votre stock de formaldéhyde souvent que le composé a tendance à se polymériser avec le temps. Les solutions de formaldéhyde ne sont stables que pendant un mois environ, et cette période est à peine prolongée par une stabilisation du méthanol. Il est conseillé de tester différentes concentrations de formaldéhyde lors de la création des conditions expérimentales. Montant de la matière végétale et de rectification: Il est important d&#39;infiltrer de faibles quantités de matière végétale dans un grand volume de tampon pour éviter les feuilles collent les unes aux autres. Feuille coller aboutit souvent à une infiltration insuffisante de formaldéhyde. Par ailleurs, du matériel végétal trop de bloquer les pores du tissu miracloth. L&#39;efficacité du broyage dépend considérablement de l&#39;aide d&#39;un mortier et un pilon avec une coupe parfaite. Nous avons régulièrement moudre avec un liquide refroidi à l&#39;azote pilon et un mortier en l&#39;absence de l&#39;azote liquide supplémentaire de trois fois pendant 50 secondes jusqu&#39;à ce que le matériel est broyé en une fine poudre.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> Protéine A-agarose </td><td> Roche </td><td> 11 134 515 001 </td><td> dépend de la classe d&#39;anticorps </td></tr><tr><td> Protéine G-agarose </td><td> Roche </td><td> 11 243 233 001 </td><td> dépend de la classe d&#39;anticorps </td></tr><tr><td> Anti-hyperacetyl-H4 </td><td> Millipore </td><td> 06-946 </td><td> nous avons utilisé 5uL </td></tr><tr><td> Anti-acétyl-H4K5 </td><td> Millipore </td><td> 07-327 </td><td> nous avons utilisé 5uL </td></tr><tr><td> Anti-acétyl-H4K8 </td><td> Millipore </td><td> 07-328 </td><td> nous avons utilisé 5uL </td></tr><tr><td> Anti-acétyl-H4K12 </td><td> Millipore </td><td> 07-595 </td><td> nous avons utilisé 5uL </td></tr><tr><td> Anti-acétyl-H4K16 </td><td> Millipore </td><td> 07-329 </td><td> nous avons utilisé 5uL </td></tr><tr><td> Anti-acétyl-H4K18 </td><td> Millipore </td><td> 07-354 </td><td> nous avons utilisé 1 microlitre </td></tr><tr><td> Anti-acétyl-H3K9 </td><td> Millipore </td><td> 07-352 </td><td> nous avons utilisé 5uL </td></tr><tr><td> Anti-acétyl-H3K14 </td><td> Millipore </td><td> 07-353 </td><td> nous avons utilisé 1 microlitre </td></tr><tr><td> Anti-triméthyl-H3K4 </td><td> Diagenode </td><td> PAB-003-50 </td><td> nous avons utilisé 5uL </td></tr><tr><td> Anti-diméthyl-H3K4 </td><td> Millipore </td><td> 07-030 </td><td> nous avons utilisé 5uL </td></tr><tr><td> Anti-monométhyl-H3K4 </td><td> Abcam </td><td> ab8895 </td><td> 2,5-10 ul </td></tr><tr><td> Anti-H3 </td><td> Abcam </td><td> ab1791 </td><td> nous avons utilisé pour vérifier la densité 1 microlitre d&#39;histone </td></tr><tr><td> Bioruptor </td><td> Diagenode </td><td> UCD-200 à </td><td></td></tr><tr><td> Sonotrode MS72 </td><td> Bandelin </td><td></td><td></td></tr><tr><td> Miracloth </td><td> Calbiochem </td><td> 475855 </td><td></td></tr><tr><td> Inhibiteur de protéase complète </td><td> Roche </td><td> 11836145001 </td><td></td></tr></tbody></table>

detection of histone modifications in plant leaves

Structure de la chromatine est importante pour la régulation de l&#39;expression des gènes chez les eucaryotes. Dans ce processus, les modifications remodelage de la chromatine, la méthylation de l&#39;ADN, et covalente sur la queue amino-terminale des histones H3 et H4 jouent des rôles essentiels 1-2. Modifications des histones H3 et H4 comprennent la méthylation de la lysine et l&#39;arginine, l&#39;acétylation de la lysine, et la phosphorylation de 1-2 résidus sérine. Ces modifications sont liées soit à l&#39;activation des gènes, la répression, ou un état ​​apprêté du gène qui favorise l&#39;activation plus rapide et robuste d&#39;expression après la perception des signaux appropriés (microbe motifs moléculaires associés, la lumière, hormones, etc) 3-7. Ici, nous présentons une méthode pour la détection fiable et sensible de certaines modifications de la chromatine sur les gènes de la plante choisie. La technique est basée sur la réticulation du (modifié) histones et l&#39;ADN avec le formaldéhyde 8,9, l&#39;extraction et sonication de la chromatine, immunoprécipitation de la chromatine (ChIP) avec modification des anticorps spécifiques 9,10, de réticulation de l&#39;histone-ADN, et spécifiques du gène en temps réel de PCR quantitative. L&#39;approche s&#39;est avérée utile pour détecter les modifications des histones spécifiques associés à la photosynthèse en C 4 à 5,11 maïs et l&#39;immunité systémique en 3 Arabidopsis.

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Detection of Histone Modifications in Plant Leaves

Une approche fiable et utile pour détecter les modifications des histones sur les gènes de plantes spécifiques est décrite. L&#39;approche combine immunoprécipitation de la chromatine (ChIP) et en temps réel de PCR quantitative. Elle permet la détection des modifications des histones sur des gènes spécifiques avec un rôle dans divers processus physiologiques.

Détection des modifications des histones dans les feuilles des plantes

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

detection-of-histone-modifications-in-plant-leaves

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Biology

24.8K vues.  RWTH Aachen University. Une approche fiable et utile pour détecter les modifications des histones sur les gènes de plantes spécifiques est décrite. L'approche combine immunoprécipitation de la chromatine (ChIP) et en temps réel de PCR quantitative. Elle permet la détection des modifications des histones sur des gènes spécifiques avec un rôle dans divers processus physiologiques.

Vidéo: Detection of Histone Modifications in Plant Leaves

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

Détection des modifications post-traductionnelles sur les autochtones nucléosomes Intact par ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

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Biologie

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

Détection des interactions des protéines dans les plantes en utilisant une passerelle de complémentation par fluorescence bimoléculaire Compatible (BiFC) Système

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Profilage Glycan des polymères végétaux de la paroi cellulaire en utilisant Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Identification des modifications post-traductionnelles des protéines végétales Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

La technique d&#39;infiltration-centrifugation pour l&#39;extraction de apoplastique Fluid feuilles des plantes Utilisation<em&gt; Phaseolus vulgaris</em&gt;, Un exemple

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Résolution Quantification haut de Crystalline Cellulose Accumulation dans<em&gt; Arabidopsis</em&gt; Roots pour surveiller cellulaire spécifique d&#39;un tissu mural Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Haut-débit CRISPR Vector Construction et caractérisation de l&#39;ADN Modifications par génération de tomate Poilu Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Flux de travail complet pour l&#39;analyse des histones modifications post-traductionnelles utilisant la spectrométrie de masse Bottom-up: De Histone Extraction à l&#39;analyse des données

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Identification de nouveaux régulateurs de la transpiration végétale par le dépistage thermique à grande échelle à Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state.

Quantification au niveau global des modifications post-traductionnelles des histones dans un modèle de culture cellulaire 3D de tissu hépatique

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid ...