Questo lavoro è stato finanziato dal tedesco Science Foundation (DFG) e l&#39;iniziativa eccellenza dei governi tedesco federale e statale.

1. Reticolazione del materiale vegetale <ol><li> Vendemmia 1-2g di foglie di Arabidopsis (6 a 10cm di diametro rosetta) in una da 50 ml tubo di plastica e riempire fino a 40ml di buffer di reticolazione. </li><li> Assicurarsi che lascia soggiorno immerso, ad esempio ripieno un diritto su misura spugna filtro sopra la superficie del buffer. Poi posto i tubi in un essiccatore. </li><li> Vuoto infiltrarsi per 10min. Per aggiungere ogni tubo 2,5 ml di glicina 2M per fermare reticolazione, e invertire i tubi per garantire la dispersione pari della glicina. </li><li> Vuoto infiltrarsi per altri 5 minuti e scartare fluido durante la raccolta foglie su un setaccio. </li><li> Lavare le foglie due volte con 1 litro di acqua in un bicchiere di vetro, poi lascia asciugare con salviette di carta. </li><li> Raccogliere le foglie in un tubo di plastica fresca e congelare in azoto liquido. Conservare le foglie a -80 ° C fino isolamento cromatina. </li></ol> 2. Isolamento di cromatina <ol><li> Foglie a terra con malta e pistillo che sono stati mantenuti in azoto liquido fino al momento dell&#39;uso. </li><li> Trasferimento in polvere ad un 50-ml tubo di plastica e sospendere in tampone di estrazione 30mL # 1. </li><li> Incubare per 15 minuti a 4 ° C su un overhead shaker. </li><li> Sospensione filtrato attraverso quattro strati di miracloth in un nuovo 50-ml tubo di plastica. </li><li> Spin per 20 minuti a 2800 xg a 4 ° C. </li><li> Rimuovere il surnatante e sospendere pellet con un pennello a tampone di estrazione 1mL # 2. In primo luogo aggiungere un piccolo volume di tampone # 2, poi sospendere pellet con un pennello, e quindi aggiungere il resto dei buffer. </li><li> Trasferire la sospensione in una provetta da microcentrifuga 1,5 ml e centrifugare per 10 minuti a 12.000 xg a 4 ° C. </li><li> Nel frattempo, preparare 2 ml-microcentrifuga provette contenenti 1,5 ml di buffer di estrazione # 3. </li><li> Rimuovere il surnatante dal tubo filato (passo 2,7) e sospendere pellet in 300μL di tampone di estrazione # 3. In primo luogo aggiungere un piccolo volume di tampone # 3, sospendere pellet con un pennello, e quindi aggiungere il buffer residuo. </li><li> Con attenzione pipetta precipitato sospeso (passo 2,9) in cima al buffer di 1,5 ml di estrazione n. 3 preparati in fase 2.8. Assicurarsi che le due fasi non si mescolano. Spin per 1h a 16000 xga 4 ° C. </li><li> Rimuovere il surnatante e sospendere cromatina pellet con un pennello in 300μL di buffer di sonicazione. </li><li> Ultrasuoni cromatina con un sonotrodo, o in un bagno a ultrasuoni, a una dimensione del DNA di circa 400bp. Quando sonicating, fare cromatina che non sarà una temperatura superiore di circa 30 ° C per proteggere sensibili al calore legami crociati. La durata di sonicazione deve essere determinato sperimentalmente, perché varia in modo significativo tra i diversi dispositivi di sonicazione. Per la guida, le impostazioni per i due dispositivi differenti: </li></ol> Per sonicazione con un marchio Bandelin sonotrodo esporre cromatina in tubo di 0,6 ml microcentrifuga cinque volte a 20 scatti con impostazioni 25% di energia, ciclo = 50. Così facendo, fresco campione ogni 20 scoppia ° in un bagno di ghiaccio. Per sonicazione con vasca Diagenode Bioruptor ultrasuoni, riempire bagno con acqua e ghiaccio e sonicare dieci volte per 30 secondi in tubi microcentrifuga 1,5 ml con l&#39;impostazione &#39;alta&#39;. Dopo ogni 30 secondi, i campioni di raffreddare per altri 30 secondi. <ol start="13"><li> Spin sonicato campione per 5 min a 16.000 g a 4 ° C a precipitare materiale non disciolto. Il supernatante possono essere utilizzati direttamente per immunoprecipitazione. In alternativa, i campioni possono essere scossa congelati in azoto liquido e conservati a -80 ° C fino a quando un ulteriore uso. </li></ol> 3. Immunoprecipitazione <ol><li> Preparare 2-mL tubi da microcentrifuga contenente 40μL proteina-A agarosio e 1.8mL di anticorpi di legame buffer. Aggiungere 200μL della preparazione cromatina (passo 2.13). </li><li> Incubare le provette per 1 ora su un overhead shaker. </li><li> Eliminare le proteine-A perline agarosio e l&#39;utilizzo sopranatante per immunoprecipitazione. </li><li> Rimuovere un 40μL-aliquota per determinare la concentrazione della cromatina (&#39;ingresso&#39;). </li><li> Aggiungi 30μL proteina-A agarosio e la quantità di modifica anticorpo specifico che è indicato nella tabella sottostante di reagenti e attrezzature specifiche per 400μL sospensione cromatina sonicato. </li><li> Incubare per 2.5 ore o durante la notte su una lavagna luminosa agitatore a 4 ° C. </li><li> Spin down perline per 2min a 440 x g. </li><li> Lavare tallone pellet con 900μL di ogni tampone di lavaggio (vedi elenco sotto). Per ogni buffer, perline incubare nel buffer per 10 minuti su un agitatore in testa a 4 ° C, poi girare per 2 minuti a 440 xg, scartare il surnatante e aggiungere il buffer successivo. <ol><li> Iposodica tampone di lavaggio </li><li> Alta sale tampone di lavaggio </li><li> LiCl tampone di lavaggio </li><li> TE tampone di lavaggio </li></ol></li><li> Dopo l&#39;ultimo lavaggio, rimuovere completamente qualsiasi residuo di buffer. </li></ol> 4. De-reticolazione degli istoni e del DNA, e la purificazione del DNA precipitato <ol><li> Aggiungere 100μL di de-reticolazione tampone al pellet agarosio e il campione &#39;ingresso&#39; il 40μl dal punto 3.4, mescolare per 5 min su un vortex, campioni di spin, e incubatoribate a 65 ° C durante la notte. </li><li> Purificare il DNA con il DNA commerciale o kit di purificazione PCR. </li><li> Tipicamente 5μl di una diluizione 1:5 del eluato della colonna finale sono sufficienti per eseguire convenzionali locus-specifiche real-time RT-PCR quantitativa (RT-qPCR). </li></ol> 5. Tabella dei buffer <table border="1"><tbody><tr><td colspan="2"> Reticolazione tampone </td></tr><tr><td> Butirrato di sodio </td><td> 10mM </td></tr><tr><td> Saccarosio </td><td> 400mm </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10mM </td></tr><tr><td> β-mercaptoetanolo </td><td> 5mM </td></tr><tr><td> Formaldeide </td><td> 3% (v / v) </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampone di estrazione # 1 </td></tr><tr><td> Butirrato di sodio </td><td> 10mM </td></tr><tr><td> Saccarosio </td><td> 400mm </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10mM </td></tr><tr><td> β-mercaptoetanolo </td><td> 5mM </td></tr><tr><td> Inibitori della proteasi completo </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampone di estrazione # 2 </td></tr><tr><td> Butirrato di sodio </td><td> 10mM </td></tr><tr><td> Saccarosio </td><td> 250mm </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10mM </td></tr><tr><td> β-mercaptoetanolo </td><td> 5mM </td></tr><tr><td> MgCl 2 </td><td> 10mM </td></tr><tr><td> Triton X-100 </td><td> 1% (w / v) </td></tr><tr><td> Inibitori della proteasi completo </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampone di estrazione # 3 </td></tr><tr><td> Butirrato di sodio </td><td> 10mM </td></tr><tr><td> Saccarosio </td><td> 1.7M </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10mM </td></tr><tr><td> β-mercaptoetanolo </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> Inibitori della proteasi completo </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Sonicazione tampone </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 25mM </td></tr><tr><td> NaOH-EDTA, pH 8,0 </td><td> 5mM </td></tr><tr><td> SDS </td><td> 0,5% (w / v) </td></tr><tr><td> Inibitori della proteasi completo </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Anticorpi tampone vincolante </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 50mM </td></tr><tr><td> NaOH-EDTA, pH 8,0 </td><td> 1 mM </td></tr><tr><td> NaCl </td><td> 150mm </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"> Iposodica tampone di lavaggio </td></tr><tr><td> NaCl </td><td> 150mm </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> NaOH-EDTA, 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"> Alta sale tampone di lavaggio </td></tr><tr><td> NaCl </td><td> 500mm </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> NaOH-EDTA, 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 tampone di lavaggio </td></tr><tr><td> Cloruro di litio </td><td> 250mm </td></tr><tr><td> Nonidet-P40 </td><td> 1% (v / v) </td></tr><tr><td> Sodio desossicolato </td><td> 1% (w / v) </td></tr><tr><td> NaOH-EDTA, pH 8,0 </td><td> 1 mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 20 mM </td></tr></tbody></table><table border="1"><tbody><tr><td> TE tampone di lavaggio </td></tr><tr><td> NaOH-EDTA, pH 8,0 </td><td> 10mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 1 mM </td></tr></tbody></table><table border="1"><tbody><tr><td> Decrosslinking tampone </td></tr><tr><td> Tris-HCl, pH 6.8 </td><td> 62,5 mm </td></tr><tr><td> NaCl </td><td> 200mm </td></tr><tr><td> SDS </td><td> 2% (w / v) </td></tr><tr><td> DTT </td><td> 10mM </td></tr></tbody></table> 6. Rappresentante dei risultati: Espressione del gene di Arabidopsis thaliana difesa PR2 che codifica per una β-1,3-glucanasi con attività antimicrobica 12, è stata indotta da benzothiadiazole (BTH), un analogo sintetico del ormone vegetale acido salicilico 3. Figura 1 dimostra che l&#39;attivazione di PR2 espressione è associata ad un aumento della acetilazione degli istoni su residui di lisina H3 e H4 sul promotore PR2. Dal momento che la densità nucleosoma diminuisce durante l&#39;attivazione del gene 13, i valori acetilazione degli istoni sono stati normalizzati per il segnale ottenuto con un anticorpo ad una regione invariante di istone 3. <img src="/files/ftp_upload/3096/3096fig1.jpg" alt="Figura 1"/> Figura 1 BTH induce l&#39;acetilazione degli istoni sul promotore PR2 in Arabidopsis. Le piante sono state trattate con BTH 100μM o un controllo in polvere bagnabile. Dopo 72 ore, le foglie sono state raccolte e cromatina è stato isolato. Il segnale ottenuto dopo precipitazione con acetilazione anticorpi specifici (come indicato in figura) è stata normalizzata per il segnale ottenuto per precipitazione con un anticorpo a un dominio invariante dell&#39;istone H3. Cromatina precipitato è stata quantificata mediante RT-qPCR. I dati sono standardizzati per livelli di modificazione degli istoni, in assenza di trattamento BTH.

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	<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. Sci. 25, 99-104 (2000).</li><li>Haring, 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+immunoprecipitation:+optimization,+quantitative+analysis+and+data+normalization.">Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization.</a> Plant Meth. 3, 11-11 (2007).</li><li>Danker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+information+but+not+promoter+activity+controls+the+methylation+state+of+histone+H3+lysine+4+on+two+photosynthetic+genes+in+maize.">Developmental information but not promoter activity controls the methylation state of histone H3 lysine 4 on two photosynthetic genes in maize.</a> Plant J. 53, 465-474 (2008).</li><li>Uknes, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquired+resistance+in+Arabidopsis.">Acquired resistance in Arabidopsis.</a> Plant Cell. 4, 645-656 (1992).</li><li>Workman, J. 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Nessun conflitto di interessi dichiarati.

Passaggi più critici nel protocollo sono la reticolazione del DNA e istoni, il tipo e la quantità di materiale foglia, macinazione del materiale, e la sonicazione della cromatina. Per una discussione più dettagliata di questi problemi, vedere rif. 9 e 10. Sonicazione e reticolazione: E &#39;importante interrompere cromatina durante la sonicazione a frammenti di circa 400bp. Sonicazione esaustivo distruggerà cromatina interrompendo DNA e istoni, sonicazione insufficiente mentre lasceranno frammenti di cromatina lungo. Incidono sulla specificità del metodo, in quanto le modifiche degli istoni distale dal locus testati non possono essere discriminati da modifiche locali. Efficienza sonicazione è meglio testato attraverso la raccolta di cromatina 20μL da campioni prima e dopo la sonicazione, de-reticolazione DNA e istoni, isolare il DNA e determinare la lunghezza del DNA tranciate con elettroforesi su gel di agarosio. RNAse dovrebbero essere aggiunti ai campioni prima di elettroforesi, perché la preparazione della cromatina contiene ancora una grande quantità di RNA in questa fase di purificazione che potrebbero interferire con la visualizzazione del DNA frammentato. I campioni sono utili per immunoprecipitazione della cromatina se il DNA da quelli non tranciati cromatina è più lunga di 10kbp, mentre il DNA da cromatina tranciati forma un striscio con la massima intensità del segnale intorno 400bp di DNA. Noi abitualmente uso il 3% (v / v) di formaldeide per reticolazione cromatina in foglie intatte, ma l&#39;1% (v / v) di formaldeide potrebbe essere sufficiente nella maggior parte dei casi. Nelle nostre mani, basse concentrazioni di formaldeide consentire tempi più brevi sonicazione e segnali di fondo in conseguenti reazioni di PCR. Tuttavia, quantità insufficienti di formaldeide spesso sfociano in bassa riproducibilità del segnale PCR tra esperimenti indipendenti. Questo potrebbe essere dovuto alla scarsa penetrazione delle foglie con formaldeide a basse concentrazioni. Assicurati di scambiare il vostro stock di formaldeide spesso come il composto tende a polimerizzare con il tempo. Le soluzioni di formaldeide sono stabili solo per circa un mese, e questo periodo è appena prolungato dalla stabilizzazione metanolo. Si consiglia di verificare le concentrazioni di formaldeide diversi quando la creazione di condizioni sperimentali. Quantità di materiale vegetale e di rettifica: E &#39;importante per infiltrarsi basse quantità di materiale fogliare in un grande volume di tampone per evitare lascia attaccare gli uni agli altri. Foglia attaccare spesso si traduce in infiltrazione insufficiente di formaldeide. Inoltre, materiale vegetale troppo bloccherà i pori del tessuto miracloth. L&#39;efficienza di macinazione dipende in misura considerevole con un mortaio e pestello con la misura perfetta. Noi abitualmente macinare con un raffreddamento ad azoto liquido pestello e mortaio, in assenza di ulteriori azoto liquido tre volte per 50 secondi fino a quando il materiale viene macinato in polvere finissima.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Proteina-A-Agarose </td><td> Roche </td><td> 11 134 515 001 </td><td> dipende dalla classe di anticorpi </td></tr><tr><td> Proteine-G-Agarosio </td><td> Roche </td><td> 11 243 233 001 </td><td> dipende dalla classe di anticorpi </td></tr><tr><td> Anti-hyperacetyl-H4 </td><td> Millipore </td><td> 06-946 </td><td> abbiamo usato 5μL </td></tr><tr><td> Anti-acetil-H4K5 </td><td> Millipore </td><td> 07-327 </td><td> abbiamo usato 5μL </td></tr><tr><td> Anti-acetil-H4K8 </td><td> Millipore </td><td> 07-328 </td><td> abbiamo usato 5μL </td></tr><tr><td> Anti-acetil-H4K12 </td><td> Millipore </td><td> 07-595 </td><td> abbiamo usato 5μL </td></tr><tr><td> Anti-acetil-H4K16 </td><td> Millipore </td><td> 07-329 </td><td> abbiamo usato 5μL </td></tr><tr><td> Anti-acetil-H4K18 </td><td> Millipore </td><td> 07-354 </td><td> abbiamo usato 1ml </td></tr><tr><td> Anti-acetil-H3K9 </td><td> Millipore </td><td> 07-352 </td><td> abbiamo usato 5μL </td></tr><tr><td> Anti-acetil-H3K14 </td><td> Millipore </td><td> 07-353 </td><td> abbiamo usato 1ml </td></tr><tr><td> Anti-trimetil-H3K4 </td><td> Diagenode </td><td> PAB-003-50 </td><td> abbiamo usato 5μL </td></tr><tr><td> Anti-dimetil-H3K4 </td><td> Millipore </td><td> 07-030 </td><td> abbiamo usato 5μL </td></tr><tr><td> Anti-monometil-H3K4 </td><td> Abcam </td><td> ab8895 </td><td> 2,5-10μL </td></tr><tr><td> Anti-H3 </td><td> Abcam </td><td> ab1791 </td><td> abbiamo usato 1ml per verificare la densità degli istoni </td></tr><tr><td> Bioruptor </td><td> Diagenode </td><td> UCD-200 A </td><td></td></tr><tr><td> Sonotrodo 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> Inibitori della proteasi completo </td><td> Roche </td><td> 11836145001 </td><td></td></tr></tbody></table>

detection of histone modifications in plant leaves

Struttura della cromatina è importante per la regolazione dell&#39;espressione genica negli eucarioti. In questo processo, le modifiche rimodellamento della cromatina, metilazione del DNA, e covalenti sulla code amino-terminali degli istoni H3 e H4 giocano un ruolo essenziale 1-2. Modificazioni degli istoni H3 e H4 comprendono metilazione di lisina e arginina, acetilazione della lisina, e la fosforilazione di residui di serina 1-2. Queste modifiche sono associati sia con l&#39;attivazione del gene, la repressione, o uno stato innescato di gene che supporta l&#39;attivazione più rapida e robusta di espressione dopo la percezione dei segnali appropriati (microbi associati modelli molecolari, luce, ormoni, ecc) 3-7. Qui, vi presentiamo un metodo per il rilevamento affidabile e sensibile di specifiche modificazioni della cromatina sui geni delle piante selezionate. La tecnica si basa sulla reticolazione di (modificato) istoni e del DNA con formaldeide 8,9, estrazione e sonicazione della cromatina, immunoprecipitazione della cromatina (ChIP) con modifica anticorpi specifici per 9,10, de-reticolazione delle istone-DNA complessi, e gene-specifiche real-time PCR quantitativa. L&#39;approccio si è dimostrato utile per la rilevazione di specifiche modificazioni degli istoni associati C 4 la fotosintesi nel mais 5,11 e l&#39;immunità sistemica in Arabidopsis 3.

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

Detection of Histone Modifications in Plant Leaves

Un approccio affidabile e utile per rilevare modificazioni degli istoni sui geni impianto specifico è descritto. L&#39;approccio combina immunoprecipitazione della cromatina (ChIP) e real-time PCR quantitativa. Esso consente il rilevamento di modifiche degli istoni su specifici geni con un ruolo in diversi processi fisiologici.

Rilevamento di modifiche Histone in foglie delle piante

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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JoVE Journal

Biology

24.8K Views.  RWTH Aachen University. Un approccio affidabile e utile per rilevare modificazioni degli istoni sui geni impianto specifico è descritto. L'approccio combina immunoprecipitazione della cromatina (ChIP) e real-time PCR quantitativa. Esso consente il rilevamento di modifiche degli istoni su specifici geni con un ruolo in diversi processi fisiologici.

Video: 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.

Rilevazione di modificazioni post-traduzionali in nativo nucleosomi intatto da 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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Biologia

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.

Rilevamento delle interazioni proteiche in pianta utilizzando un gateway compatibile complementazione fluorescenza biomolecolare (BiFC) Sistema

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

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Analisi di espressione di mammifero Linker-istoni Sottotipi

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

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.

Profiling Glycan dei polimeri cellula vegetale da parete usando i 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.

Identificazione delle modificazioni post-traduzionali di proteine ​​vegetali Complessi

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 tecnica infiltrazione-centrifugazione per l&#39;estrazione di apoplastico fluido da foglie delle piante Utilizzo<em&gt; Phaseolus vulgaris</em&gt; Come un esempio

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.

Risoluzione Quantificazione alta del cristallino cellulosa accumulo in<em&gt; Arabidopsis</em&gt; Roots a Monitor tessuto-specifica delle cellule parete Modifiche

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 vettore Costruzione e caratterizzazione di modifiche del DNA da Generazione di Roots pomodoro Peloso

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.

Flusso di lavoro completo per l&#39;analisi dell&#39;istone modificazioni post-traduzionali Utilizzando Bottom-up Spettrometria di Massa: Da istone estrazione all&#39;analisi dei dati

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

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Un metodo sezionamento migliorato per Recalcitrante tessuti vegetali Campioni

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...