Diese Arbeit wurde von der Deutschen Forschungsgemeinschaft (DFG) und der Exzellenzinitiative der deutschen Bundesregierung und der Länder gefördert.

1. Die Vernetzung von Pflanzenmaterial <ol><li> Ernte-1-2g Arabidopsis Blättern (6 bis 10cm Rosette Durchmesser) in einem 50-ml Plastikröhrchen und füllen bis zu 40ml mit Vernetzung Puffer. </li><li> Stellen Sie sicher, Blätter bleiben eingetaucht, zum Beispiel durch Füllung eine maßgeschneiderte Filterschwamm oben rechts Puffer Oberfläche. Dann legen Sie die Röhrchen in einem Exsikkator. </li><li> Vakuum für 10 Minuten zu infiltrieren. Zu jedem Röhrchen add 2,5 ml 2M Glycin, um die Vernetzung zu stoppen und umzukehren Rohre gleich Dispersion von Glycin zu rechtfertigen. </li><li> Vacuum weitere 5min zu infiltrieren und zu verwerfen Flüssigkeit während der Ernte lässt auf ein Sieb. </li><li> Wash Blätter zweimal mit 1 l Wasser in ein Becherglas, dann trockene Blätter gründlich mit Küchenpapier. </li><li> Sammeln Sie Laub in einem frischen Kunststoffrohr und frieren in flüssigem Stickstoff. Shop Blätter bei -80 ° C bis Chromatin isoliert. </li></ol> 2. Isolierung von Chromatin <ol><li> Blätter wurden mit Mörtel und Stempel, die in flüssigem Stickstoff bis zur Verwendung aufbewahrt wurden Boden. </li><li> Transfer-Pulver zu einem 50-mL Plastikröhrchen und suspendieren in 30 mL Extraktionspuffer Nr. 1. </li><li> Inkubieren 15min bei 4 ° C auf einem Überkopfschüttler. </li><li> Filtrat Suspension durch vier Schichten Miracloth in ein frisches 50-mL Plastikröhrchen. </li><li> Spin für 20min bei 2.800 xg bei 4 ° C. </li><li> Überstand entfernen und Suspend-Pellet mit einem Pinsel in 1ml Extraktionspuffer # 2. Fügen Sie zunächst ein kleines Volumen von Puffer Nr. 2, dann auszusetzen Pellet mit einem Pinsel, und fügen Sie dann den Rest des Puffers. </li><li> Transfer-Suspension auf eine 1,5 ml Mikrozentrifugenröhrchen und Spin für 10min bei 12.000 xg bei 4 ° C. </li><li> In der Zwischenzeit bereiten 2ml-Mikrozentrifugenröhrchen mit 1,5 ml Extraktionspuffer Nr. 3. </li><li> Entfernen Sie aus dem gesponnen Rohr (Schritt 2,7) Überstand und Pellet in auszusetzen 300μL Extraktionspuffer Nr. 3. Fügen Sie zunächst ein kleines Volumen von Puffer Nr. 3, auszusetzen Pellet mit einem Pinsel, und fügen Sie dann die restlichen Puffer. </li><li> Sorgfältig pipettieren des suspendierten Pellets (Schritt 2,9) auf der Oberseite des 1,5 ml Extraktionspuffer Nr. 3 in Schritt 2,8 zubereitet. Achten Sie darauf, die beiden Phasen nicht zu vermischen. Spin für 1h bei 16.000 xg bei 4 ° C. </li><li> Überstand entfernen und Suspend Chromatin Pellet mit einem Pinsel in 300μL der Beschallung Puffer. </li><li> Ultraschallbad Chromatin mit einer Sonotrode, oder in einem Ultraschallbad, um eine DNA-Größe von ca. 400bp. Als Beschallung, stellen Sie sicher, Chromatin nicht über ca. 30 ° C erhitzt werden, um wärmeempfindlichen Vernetzungen zu schützen. Die Dauer der Beschallung muss experimentell bestimmt werden, da es deutlich variiert zwischen den verschiedenen Ultraschall-Geräten. Zur Orientierung sind die Einstellungen für zwei verschiedene Geräte zur Verfügung: </li></ol> Für Beschallung mit einem Bandelin Marke Sonotrode aussetzen Chromatin in 0,6 ml Mikrozentrifugenröhrchen fünfmal bis 20 Ausbrüche mit den Einstellungen 25% Leistung, Zyklus = 50. Während dabei kühl Probe nach 20 th platzt in ein Eisbad. Für Beschallung mit einem Diagenode Bioruptor Ultraschallbad, füllen Bad mit Wasser und Eis und beschallen zehnmal für 30 Sekunden in 1,5 ml Mikrozentrifugenröhrchen mit der Einstellung &quot;hoch&quot;. Nach 30 Sekunden, kühlen Proben für weitere 30 Sekunden. <ol start="13"><li> Spin beschallten Probe für 5 min bei 16.000 g bei 4 ° C zu fällen ungelöstes Material. Der Überstand kann direkt für die Immunpräzipitation eingesetzt werden. Alternativ können die Proben Schock in flüssigem Stickstoff eingefroren und bei -80 ° C bis zur weiteren Verwendung werden. </li></ol> 3. Immunpräzipitation <ol><li> Bereiten Sie 2-mL Mikrozentrifugenröhrchen mit 40 ul Protein-A Agarose und 1,8 ml der Antikörper-Bindungspuffer. Add 200 ul des Chromatins Vorbereitung (Schritt 2,13). </li><li> Die Röhrchen für 1 Stunde auf einem Überkopfschüttler. </li><li> Discard-Protein-A Agarose-Beads und die Nutzung für die Immunpräzipitation Überstand. </li><li> Entfernen eines 40 ul-Aliquot auf Chromatin-Konzentration (&quot;Input&quot;) zu bestimmen. </li><li> Add 30μL Protein-A-Agarose und die Höhe der Änderung-spezifischen Antikörper, die in der unten stehenden Tabelle der spezifischen Reagenzien und Geräte für 400μL beschallt Chromatin Federung angegeben ist. </li><li> Inkubieren 2.5h oder über Nacht auf einem Überkopfschüttler bei 4 ° C. </li><li> Spin down Perlen für 2min bei 440 x g. </li><li> Wash Bead-Pellet mit 900μL von jedem Waschpuffer (siehe Liste unten). Für jeden Puffer inkubieren Perlen in den Puffer für 10min auf einem Überkopfschüttler bei 4 ° C, dann für 2min Spin bei 440 xg, Überstand verwerfen, und fügen Sie den nächsten Puffer. <ol><li> Low Salz Waschpuffer </li><li> Hohe Salz Waschpuffer </li><li> LiCl Waschpuffer </li><li> TE Waschpuffer </li></ol></li><li> Nach dem letzten Waschen vollständig entfernen Sie alle verbleibenden Puffer. </li></ol> 4. De-Vernetzung von Histonen und DNA, und Aufreinigung von DNA ausgefällt <ol><li> Add 100 &amp;mgr; von de-Vernetzung Puffer, um die Agarose Pellet und 40 ul &#39;Eingang&#39; Probe aus Schritt 3.4, Mix für 5min auf einem Vortex-Mixer, Spin Proben und InkubationDebatte bei 65 ° C über Nacht. </li><li> Purify DNA mit einem kommerziellen DNA oder PCR Purification Kit. </li><li> Typischerweise 5μl einer 1:5-Verdünnung der letzten Spalte Eluat sind ausreichend, um durchzuführen konventionellen Locus-spezifischen real-time quantitative RT-PCR (RT-qPCR). </li></ol> 5. Tabelle der Puffer <table border="1"><tbody><tr><td colspan="2"> Crosslinking-Puffer </td></tr><tr><td> Natriumbutyrat </td><td> 10 mM </td></tr><tr><td> Sucrose </td><td> 400mm </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10 mM </td></tr><tr><td> β-Mercaptoethanol </td><td> 5mM </td></tr><tr><td> Formaldehyd </td><td> 3% (v / v) </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Extraktionspuffer Nr. 1 </td></tr><tr><td> Natriumbutyrat </td><td> 10 mM </td></tr><tr><td> Sucrose </td><td> 400mm </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10 mM </td></tr><tr><td> β-Mercaptoethanol </td><td> 5mM </td></tr><tr><td> Komplette Protease Inhibitor </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Extraktionspuffer # 2 </td></tr><tr><td> Natriumbutyrat </td><td> 10 mM </td></tr><tr><td> Sucrose </td><td> 250 mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10 mM </td></tr><tr><td> β-Mercaptoethanol </td><td> 5mM </td></tr><tr><td> MgCl 2 </td><td> 10 mM </td></tr><tr><td> Triton X-100 </td><td> 1% (w / v) </td></tr><tr><td> Komplette Protease Inhibitor </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Extraktionspuffer # 3 </td></tr><tr><td> Natriumbutyrat </td><td> 10 mM </td></tr><tr><td> Sucrose </td><td> 1,7 Mio. </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10 mM </td></tr><tr><td> β-Mercaptoethanol </td><td> 5mM </td></tr><tr><td> MgCl 2 </td><td> 2mM </td></tr><tr><td> Triton X-100 </td><td> 0,15% (w / v) </td></tr><tr><td> Komplette Protease Inhibitor </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Beschallung Puffer </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% (w / v) </td></tr><tr><td> Komplette Protease Inhibitor </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Antikörper Bindungspuffer </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% (w / v) </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Low Salz Waschpuffer </td></tr><tr><td> NaCl </td><td> 150 mM </td></tr><tr><td> SDS </td><td> 0,1% (w / v) </td></tr><tr><td> Triton X-100 </td><td> 1% (w / 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"> Hohe Salz Waschpuffer </td></tr><tr><td> NaCl </td><td> 500 mM </td></tr><tr><td> SDS </td><td> 0,1% (w / v) </td></tr><tr><td> Triton X-100 </td><td> 1% (w / 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 Waschpuffer </td></tr><tr><td> Lithiumchlorid </td><td> 250 mM </td></tr><tr><td> Nonidet-P40 </td><td> 1% (v / v) </td></tr><tr><td> Natriumdesoxycholat </td><td> 1% (w / 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 Waschpuffer </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 10 mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 1mM </td></tr></tbody></table><table border="1"><tbody><tr><td> Decrosslinking Puffer </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% (w / v) </td></tr><tr><td> DTT </td><td> 10 mM </td></tr></tbody></table> 6. Repräsentative Ergebnisse: Die Expression des Arabidopsis thaliana PR2 Verteidigung Gen für ein β-1,3-Glucanase mit antimikrobieller Wirkung 12, wurde von Benzothiadiazol (BTH), ein synthetisches Analogon des Pflanzenhormon Salicylsäure 3 induziert. Abbildung 1 zeigt, dass die Aktivierung von PR2 Ausdruck ist mit einem Anstieg bei der Acetylierung von Lysin-Resten auf Histon-assoziierten H3 und H4 auf den PR2 Promotor. Da Nukleosomen Dichte nimmt während Genaktivierung 13 wurden Histonacetylierung Werte für das Signal mit einem Antikörper gegen eine Invariante Region von Histon 3 erhaltenen normalisiert. <img src="/files/ftp_upload/3096/3096fig1.jpg" alt="Abbildung 1"/> Abbildung 1 BTH induziert Histon-Acetylierung auf die PR2-Promotors in Arabidopsis. Die Pflanzen wurden mit 100 &amp;mgr; BTH oder ein Spritzpulver Kontrolle behandelt. Nach 72h wurden Blätter gesammelt und Chromatin isoliert wurde. Das Signal nach der Fällung mit Acetylierung-spezifischen Antikörpern (wie in der Abbildung angegeben) erhalten wurde, um das Signal durch Fällung mit einem Antikörper erhalten, um eine Invariante Domain von Histon H3 normalisiert. Gefällte Chromatin wurde mittels RT-qPCR quantifiziert. Die Daten werden standardisiert für Histonmodifikationen Ebenen in der Abwesenheit von BTH-Behandlung.

<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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Keine Interessenskonflikte erklärt.

Die meisten kritischen Schritte in dem Protokoll sind die Vernetzung von DNA und Histonen, die Art und Menge der Blattmaterial, Schleifen des Materials und die Beschallung von Chromatin. Für eine detailliertere Erörterung dieser Probleme finden Sie unter refs. 9 und 10. Beschallung und Vernetzung: Es ist wichtig, Chromatin während der Beschallung auf Fragmente von ca. 400bp zu stören. Dank intensiver Beschallung wird Chromatin durch Unterbrechung der DNA und Histonen zu zerstören, während unzureichende Beschallung lange Chromatinfragmenten verlassen wird. Diese beeinflussen die Spezifität der Methode, weil Histon-Modifikationen distal von den getesteten locus keine lokalen Änderungen benachteiligt werden. Beschallung Effizienz am besten durch das Sammeln von 20 &amp;mgr; l Chromatin von Proben vor und nach Beschallung, de-Vernetzung DNA und Histonen, Isolierung der DNA und die Bestimmung der Länge der DNA geschert durch Agarosegelelektrophorese überprüft. RNAse sollte, um die Proben vor der Elektrophorese hinzugefügt werden, da das Chromatin Vorbereitung enthält noch große Mengen an RNA in diesem Stadium der Reinigung, die mit der Visualisierung von fragmentierten DNA stören könnten. Die Proben werden für Chromatinimmunpräzipitation nützlich, wenn DNA aus nicht geschert Chromatin ist länger als 10kbp, während DNA aus geschert Chromatin bildet ein Abstrich mit maximaler Signalstärke um 400bp DNA. Wir verwenden routinemäßig 3% (v / v) Formaldehyd für Chromatin Vernetzung in intakten Blättern, aber 1% (v / v) Formaldehyd könnte in den meisten Fällen ausreichend. In unsere Hände, damit eine niedrige Formaldehyd-Konzentrationen für kürzere Zeiten Beschallung und Hintergrund-Signale in Folge PCR-Reaktionen. Allerdings, unzureichende Mengen an Formaldehyd oft in geringen Reproduzierbarkeit der PCR-Signal zwischen unabhängigen Experimenten führen. Dies könnte durch unzureichende Durchdringung der Blätter mit Formaldehyd bei niedrigen Konzentrationen. Achten Sie auf Ihre Formaldehyd Lager häufig austauschen, wie die Verbindung neigt dazu, sich mit der Zeit polymerisieren. Formaldehyd-Lösungen sind nur für etwa einen Monat stabil, und diese Zeit ist kaum von Methanol Stabilisierung verlängert. Es wird empfohlen, verschiedene Formaldehyd-Konzentrationen Test beim Einrichten experimentellen Bedingungen. Betrag von Pflanzenmaterial und Schleifen: Es ist wichtig zu geringe Mengen an Blattmaterial in einem großen Volumen an Puffer zu infiltrieren, um Blätter aneinander kleben zu vermeiden. Blatt kleben oft in unzureichender Infiltration von Formaldehyd. Darüber hinaus wird zu viel Pflanzenmaterial Block Poren der Miracloth Gewebe. Die Mahleffizienz hängt wesentlich mit einem Mörser und Stößel mit einer perfekten Passform. Wir routinemäßig mahlen mit flüssigem Stickstoff gekühlten Mörser in Abwesenheit von zusätzlichen flüssigen Stickstoff dreimal für 50 Sekunden, bis das Material zu einem feinen Pulver gemahlen wird.

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> Protein-A-Agarose </td><td> Roche </td><td> 11 134 515 001 </td><td> abhängig von der Antikörper-Klasse </td></tr><tr><td> Protein-G-Agarose </td><td> Roche </td><td> 11 243 233 001 </td><td> abhängig von der Antikörper-Klasse </td></tr><tr><td> Anti-hyperacetyl-H4 </td><td> Millipore </td><td> 06-946 </td><td> verwendeten wir 5μL </td></tr><tr><td> Anti-Acetyl-H4K5 </td><td> Millipore </td><td> 07-327 </td><td> verwendeten wir 5μL </td></tr><tr><td> Anti-Acetyl-H4K8 </td><td> Millipore </td><td> 07-328 </td><td> verwendeten wir 5μL </td></tr><tr><td> Anti-Acetyl-H4K12 </td><td> Millipore </td><td> 07-595 </td><td> verwendeten wir 5μL </td></tr><tr><td> Anti-Acetyl-H4K16 </td><td> Millipore </td><td> 07-329 </td><td> verwendeten wir 5μL </td></tr><tr><td> Anti-Acetyl-H4K18 </td><td> Millipore </td><td> 07-354 </td><td> verwendeten wir 1μL </td></tr><tr><td> Anti-Acetyl-H3K9 </td><td> Millipore </td><td> 07-352 </td><td> verwendeten wir 5μL </td></tr><tr><td> Anti-Acetyl-H3K14 </td><td> Millipore </td><td> 07-353 </td><td> verwendeten wir 1μL </td></tr><tr><td> Anti-trimethyl-H3K4 </td><td> Diagenode </td><td> PAB-003 bis 50 </td><td> verwendeten wir 5μL </td></tr><tr><td> Anti-dimethyl-H3K4 </td><td> Millipore </td><td> 07-030 </td><td> verwendeten wir 5μL </td></tr><tr><td> Anti-Monomethyl-H3K4 </td><td> Abcam </td><td> ab8895 </td><td> 2,5-10 &amp;mgr; l </td></tr><tr><td> Anti-H3 </td><td> Abcam </td><td> ab1791 </td><td> verwendeten wir 1μL um Histon-Dichte zu überprüfen </td></tr><tr><td> Bioruptor </td><td> Diagenode </td><td> UCD-200 an </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> Komplette Protease Inhibitor </td><td> Roche </td><td> 11836145001 </td><td></td></tr></tbody></table>

detection of histone modifications in plant leaves

Chromatin-Struktur ist für die Regulation der Genexpression in Eukaryoten wichtig. In diesem Prozess spielen die Chromatin-Remodeling, DNA-Methylierung und kovalente Modifikationen an der Amino-terminalen Enden der Histone H3 und H4 wesentliche Rollen 1-2. H3 und H4 Histon-Modifikationen zählen die Methylierung von Lysin und Arginin, Acetylierung von Lysin und Phosphorylierung von Serin-Resten 1-2. Diese Änderungen sind entweder mit Genaktivierung, Unterdrückung oder eine grundierte Stand der Gen, das eine schnellere und eine starke Aktivierung des Ausdrucks unterstützt nach Wahrnehmung der entsprechenden Signale (Mikrobe-associated molecular patterns, Licht, Hormone, etc.) 3-7 verbunden. Hier stellen wir ein Verfahren zur zuverlässigen und empfindlichen Nachweis von spezifischen Chromatin-Modifikationen auf ausgewählte pflanzliche Gene. Die Technik basiert auf der Vernetzung von (modifizierten) Histone und DNA mit Formaldehyd 8,9, Extraktion und Beschallung von Chromatin, Chromatin-Immunopräzipitation (ChIP) mit Modifikation-spezifische Antikörper 9,10 basiert, de-Vernetzung von Histon-DNA-Komplexe und Gen-spezifischen real-time quantitative PCR. Der Ansatz hat sich zum Nachweis von spezifischen Histon-Modifikationen mit C 4 Photosynthese in Mais 5,11 und systemische Immunität in Arabidopsis 3 zugeordnet nützlich.

Watch this Scientific Journal Video about Detection of Histone Modifications in Plant Leaves at JoVE.com

Detection of Histone Modifications in Plant Leaves

Eine zuverlässige und nützlicher Ansatz zur Histon-Modifikationen auf spezifische Pflanzen-Gene erkennen beschrieben. Der Ansatz kombiniert Chromatin-Immunopräzipitation (ChIP) und real-time quantitative PCR. Es ermöglicht den Nachweis von Histon-Modifikationen auf bestimmte Gene mit eine Rolle in verschiedenen physiologischen Prozessen.

Erkennung von Histon-Modifikationen in Pflanzenblättern

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

Research

JoVE Journal

Biology

24.8K Aufrufe.  RWTH Aachen University. Eine zuverlässige und nützlicher Ansatz zur Histon-Modifikationen auf spezifische Pflanzen-Gene erkennen beschrieben. Der Ansatz kombiniert Chromatin-Immunopräzipitation (ChIP) und real-time quantitative PCR. Es ermöglicht den Nachweis von Histon-Modifikationen auf bestimmte Gene mit eine Rolle in verschiedenen physiologischen Prozessen.

Serie: 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.

Erkennung von Post-translationale Modifikationen auf Native Intact Nukleosomen mittels 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 ...

Forschung

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.

Detektion von Protein-Wechselwirkungen in Plant mit einem Gateway kompatibel bimolekularen Fluoreszenz Komplementation (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 ...

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.

Glycan Profiling of Plant Cell Wall Polymers mit 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.

Bezeichnung des Post-translationale Modifikationen von Pflanzenproteinkomplexe

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.

Die Infiltration-Zentrifugation Technik zur Gewinnung von apoplastischen Flüssigkeit aus Pflanzenblättern verwenden<em&gt; Phaseolus vulgaris</em&gt; Als Beispiel

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.

Hohe Auflösung Quantifizierung kristalliner Cellulose Akkumulation in<em&gt; Arabidopsis</em&gt; Roots Gewebespezifische Zellwand Änderungen überwachen

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.

High-throughput CRISPR Vector Konstruktion und Charakterisierung von DNA-Modifikationen durch Erzeugung von Tomaten Hairy 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.

Komplette Workflow zur Analyse von Histon-post-translationale Modifikationen Mit Bottom-up-Massenspektrometrie: Von Histone Extraktion in Datenanalyse

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.

Identifizierung neuartiger Regulatoren der Pflanzentranspiration durch großangelegtes Thermisches Bildgebungsscreening 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 ...

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.

Globale Quantifizierung von posttranslationalen Histonmodifikationen in einem 3D-Zellkulturmodell von Lebergewebe

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