Questo lavoro è supportato dall&#39;Università della Virginia, l&#39;Università della Virginia Cancer Center e The F. Thomas e Kate Miller Jeffress Memorial Trust. Ringraziamo il James E. Darnell Lab (Rockefeller University), Robert Ross Lab (Fordham University) per linee cellulari.

PIANIFICAZIONE Il giorno prima di un esperimento di ChIP è previsto, le cellule devono essere coltivate in modo che un numero sufficiente è disponibile. Si supponga che ogni saggio ChIP richiederà ~ 1-1,5 x 10 7 cellule. Sempre intenzione di fare sia il negativo (IgG) e positivo (pan anticorpi istoni) controlla e ripetizione degli esperimenti. Suggerimento - Per le celle 2FTGH, ogni saggio ChIP richiede circa un 15 centimetri di tessuto piatto cultura al 80% confluenza. PARTE 1: PREPARAZIONE ED A SVOLGERE CROMATINA ChIP <ol><li> Rimuovere i supporti della cultura e indurre le cellule per i tempi richiesti con 20 ml di 10% siero di vitello cosmica (CCS) / DMEM contenente IFN-gamma a 5 ng / ml. Sostituire i supporti sui piatti uninduced con 20 ml di 10% CCS / DMEM pure. </li><li> In una cappa aspirante, aggiungere 540 microlitri di formaldeide 37% a 20 mL di media (1% di concentrazione finale) nel piatto 15 cm. Suggerimento - piastra Tilt e aggiungere la formaldeide al pool di supporti - poi placca rocciosa a diffondersi in modo uniforme. </li><li> Lasciare reticolazione a procedere per 10 minuti a temperatura ambiente. </li><li> Aggiungi glicina a 0,125 M concentrazione finale per fermare reticolazione. </li><li> Aspirare il vuoto dei media e lavare le cellule una volta con 10 ml di fosfato ghiacciata soluzione salina tamponata (PBS). </li><li> Raccogliere le cellule con un raschietto cellula ~ 7 ml di PBS e combinare le cellule (da 6 piastre) che sono stati trattati in modo identico in tubo da 50 ml su ghiaccio. </li><li> Centrifuga (Beckman Coutler Allegra 12-R centrifuga) a 1800 rpm a 4 ° C per 5 minuti e aspirare il PBS. Suggerimento - Snap congelare il pellet se fermarsi qui e conservare a -80 ° C. </li><li> Risospendere il pellet in 4 mL di tampone gonfiore (25 HEPES mM pH 7,8, 1,5 mM MgCl 2, 10 mM KCl, 0.1% NP40. Aggiungere 1 mM DTT, gli inibitori della proteasi completa e freddo sul ghiaccio prima dell&#39;uso) e trasferimento a 15 mL tubo conico. Suggerimento - Questo volume tampone gonfiore è appropriato per un piatto 6 pooling (~ 6-9 x 10 7 cellule). Tampone più o meno potrebbe cambiare l&#39;efficienza douncing. </li><li> Incubare la sospensione cellulare in ghiaccio per 10 minuti. </li><li> Trasferire la sospensione per un pre-raffreddata 15 ml douncer (Wheaton) e dounce 20 colpi di pestello A. </li><li> Trasferimento dounced cellule di nuovo ad un tubo da 15 ml su ghiaccio. </li><li> Centrifugare a 2500 rpm per 5 minuti a 4 ° C e scartare il surnatante. </li><li> Risospendere il pellet nucleare (~ 200 ul da 6 piastre) in 3 ml (15 volumi pellet) di tampone di sonicazione (50 mM HEPES pH 7,9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, desossicolato di sodio 0,1%, 0,1% SDS. Aggiungi inibitori della proteasi completa e chill sul ghiaccio al momento dell&#39;uso). Suggerimento - Questo volume tampone sonicazione è appropriato per un piatto 6 pooling (~ 6-9 x 10 7 cellule). Tampone più o meno potrebbe cambiare l&#39;efficienza sonicazione. </li><li> Sonicare sospensione nuclei in ghiaccio per 10 cicli (20 secondi on/40 secondi dal / ampiezza impostazione di 8) usando un Sonicator Misonix 3000 equipaggiato con il microtip. Questo genere si traduce in frammenti di cromatina media 200-1000 bps nel formato. Suggerimento - Perché sonicatori varierà, l&#39;efficienza sonicazione deve essere determinata empiricamente. Per fare questo, prendete aliquote di 20 l sonicazione prima e dopo ogni ciclo di sonicazione. Calore questi campioni a 65 ° C per 4 ore. Aggiungi il caricamento del buffer del DNA e pilotare un 1% di agarosio / TBE gel per visualizzare frammenti di DNA.) </li><li> Trasferimento sonicato materiale da un tubo SS34 centrifuga e centrifugare per 20 minuti a 4 ° C a 13000 rpm in un rotore SS34 (Sorvall RC6 Inoltre centrifuga) per far sedimentare ciò che è di solito una piccola quantità di detriti cellulari. </li><li> Con attenzione trasferire il sopranatante in una provetta da 15 ml conica sul ghiaccio. </li><li> Pre-clear con l&#39;aggiunta di 360 microlitri sperma di salmone DNA / proteina A o G agarosio perle di liquami e dondolo a 4 ° C per almeno 30 minuti. Suggerimento - Questo passo si riduce il rumore nel saggio che deriva da eventi legame non specifico. </li><li> Centrifugare a 1000 rpm a 4 ° C per 5 minuti per far sedimentare le perline agarosio. </li><li> Trasferimento pre-cancellati surnatante in un&#39;altra provetta da 15 ml conica sul ghiaccio. </li><li> Misurare la A260 unità di pre-cancellati cromatina e regolare il volume con tampone di sonicazione di quattro (4) A260 unità (0,2 mg / ml assumendo A260 1 unità = 0,050 mg / ml) Suggerimento - Per misurare la A260 unità, diluire 10 ml di pre-cancellati cromatina in 190 microlitri DDW; vuoto spettrofotometro con 10 microlitri di buffer sonicazione in 190 microlitri DDW. </li><li> Risparmiate il 75 microlitri della cromatina (a 4 A260 unità) come il campione di ingresso e conservare a 20 ° C fino al momento di invertire legami crociati. Suggerimento - Questo passo è fondamentale per l&#39;analisi dei dati. Non trascurare di prendere il aliquota di ingresso. </li><li> Aliquota della cromatina pronto a 1,7 mL microcentrifuga su ice aliquote come 1 ml. Suggerimento - Se fermarsi qui, a scatto congelare la cromatina preparato in ghiaccio secco e conservare a -80 ° C. NB: cromatina memorizzati possono degradare, producendo risultati non ottimali, in modo da evitare se possibile. </li><li> Aggiungere il ChIP-grade anticorpi per i campioni indotto e uninduced set in duplice copia. Aggiungi IgG (2 mg) e fondo di anticorpi istone H3 (15 mL), come i controlli negativi e positivi di indotto e uninduced set di campioni. Suggerimento - La quantità di anticorpi da utilizzare deve essere determinata empiricamente o vedere la scheda prodotto del fornitore per importi suggeriti. In genere 1-2 mg di un chip di livello anticorpale funziona bene. Il volume appropriato deve essere determinato empiricamente per gli anticorpi che vengono forniti come siero. </li><li> Roccia tutti i tubi notte a 4 ° C. </li></ol> PARTE 2: COLLECT immunocomplessi <ol><li> Aggiungere 60 ml di sperma di salmone DNA / proteina A o proteina G agarosio liquami perline di tutti i tubi. Usa Proteine ​​Suggerimento A per anticorpi policlonali di coniglio e G proteine ​​per gli anticorpi monoclonali murini. </li><li> Roccia a 4 ° C per almeno 60 minuti. </li><li> Agglomerare le immunocomplessi (perline agarosio) da microfuging gentilmente a 2000 rpm a 4 ° C per 3 minuti. </li><li> Aspirare il sopranatante. </li><li> Lavare le immunocomplessi con 1 ml di ciascuna delle seguenti tamponi di lavaggio per 10 minuti con dondolo a 4 ° C. Microcentrifuga per 3 minuti a 2000 rpm a 4 ° C tra ogni lavaggio e aspirare il surnatante. Conservare i campioni refrigerata sul ghiaccio. Aggiungi PMSF appena prima dell&#39;uso. <ul><li> Sale a basso Wash - 0,1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1 mM PMSF </li><li> Sale alta Wash - 0,1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl, pH 8.0, 500 mM NaCl, 1 mM PMSF </li><li> LiCl Wash - 0,25 M LiCl, 1% NP-40, desossicolato di sodio 1%, 1 mM EDTA, 10 mM Tris-Cl, pH 8.0, 1 mM PMSF </li><li> Lavare TE - due lavaggi con tampone TE -10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 1 mM PMSF </li></ul></li><li> Rimuovere il tampone di lavaggio finale TE e ai talloni, aggiungere 500 microlitri tampone di eluizione (50 mM Tris-Cl, pH 8.0, 10 mM EDTA, 1% SDS). Suggerimento - Il tampone di eluizione devono essere preparate al momento. </li><li> Vortice di mescolare perle di agarosio in tampone di eluizione e calore per 30 minuti a 65 ° C. Suggerimento - vortice ciascun tubo almeno ogni 10 minuti durante il 30 minuti. </li><li> Microcentrifuga per 30 secondi a 2000 rpm e trasferire il eluente in una provetta da microcentrifuga nuovo. Suggerimento - Durante l&#39;eluizione, scongelare i campioni di ingresso e aggiungere 500 microlitri tampone di eluizione a ciascuno. Includere questi campioni in tutti i passaggi per il futuro. </li><li> Aggiungere 5 M di NaCl ad una concentrazione finale di 200 mm a tutti i campioni e vortice. </li><li> Incubare a 65 ° C per una notte (o almeno 4 ore). </li></ol> PARTE 3: ChIP&#39;d purificare e DNA INGRESSO <ol><li> Microcentrifuga tutti i campioni per 30 secondi per la raccolta della condensa sui coperchi. </li><li> Aggiungere 1ml di RNasi A (10 U / mL) a ciascuna provetta ed incubare a temperatura ambiente per 15 minuti. </li><li> Aggiungere 10 L 0,5 M EDTA, 40 microlitri 0,5 M Tris-Cl pH 6,5, 2 microlitri proteinasi K (10 mg / ml) e incubare a 45 ° C per 60 minuti. Suggerimento Preparare una master mix di questi reagenti e aggiungere 52 microlitri per ogni provetta. </li><li> Fenolo / cloroformio / alcool isoamilico estrarre e poi cloroformio / alcool isoamilico estrarre i campioni. </li><li> Aggiungere 2 glicogeno microlitri (25 mg / mL) e 0,1 volumi di acetato di sodio 3M pH 5.2 e vortice. </li><li> Aggiungere 2,5 volumi di etanolo 100% e vortice. </li><li> Incubare a 20 ° C durante la notte. </li></ol> PARTE 4: ChIP&#39;d purificare e DNA INGRESSO CONTINUATO <ol><li> Campioni microcentrifuga per un minimo di 20 minuti a 13000 rpm a 4 ° C per precipitare il DNA / glicogeno pellet. </li><li> Lavare con DNA / glicogeno pellet con 1 ml di etanolo al 70%. </li><li> Aria secca il pellet o rapidamente a secco in centrifuga DNA vuoto velocità Savant. Suggerimento - Non troppo secco il DNA. </li><li> Risospendere DNA / glicogeno pellet in 200 microlitri tampone TE. </li><li> DNA (1 mL) è pronto per la quantificazione tramite real-time PCR. Conservare il DNA rimanente a 20 ° C. Suggerimento - Utilizzo di più di 1 ml di DNA possono inibire la real-time PCR, in modo da scalare con cautela. </li></ol> Parte 5: I RISULTATI DI RAPPRESENTANZA: Campioni di DNA ChIP e ingresso sono quantificati con Real-Time PCR 7,8 (Applied Biosystems 7500 Veloce Real-Time PCR System) utilizzando primers specifici per il locus genico di interesse. Primer che genomica sequenza bersaglio noto per essere arricchito in o privi di modificazioni degli istoni essere analizzati possono essere utilizzati come controlli positivi e negativi. I dati sono presentati come percentuale di ingresso. La procedura di chip dovrebbe essere interamente repeated con tre repliche biologiche per garantire cambiamenti riportati in modificazioni degli istoni sono statisticamente significative. Ci sono diversi aspetti importanti da considerare quando si utilizzano real-time PCR per quantificare il DNA, compresa la progettazione primer, l&#39;efficienza della PCR, e il modo appropriato per calcolare la percentuale di procedure di ingresso e di normalizzazione. Quando il profiling di occupazione degli istoni modifica attraverso un gene, l&#39;efficienza della PCR devono essere coerenti tra tutte le coppie di primer. Il real-time PCR produttore del sistema in grado di fornire le informazioni necessarie e di formazione. La figura 1 mostra i risultati rappresentante della cromatina durante immunoprecipitato STAT1 l&#39;attivazione del gene IRF1 9, innescato da IFN-γ. <img src="/files/ftp_upload/2053/2053fig1.jpg" alt="Figura 1" /> Figura 1. Occupazione di STAT1, RNA polimerasi II e H3K36me3 è dinamico durante STAT1 attivazione del gene IRF1. (A, B, C) ​​ChIP con α-H3K36me3, α-STAT1 e α-Pol II della cromatina delle cellule raccolte da 2FTGH indotta con IFN-γ per 30 minuti (quadrati rossi), 1,5 ore (cerchi blu), 5 ore ( triangoli verdi) o uninduced (diamanti neri). IgG è il controllo negativo (grigio croci). (D) Pan H3 è un controllo positivo e mostra l&#39;occupazione degli istoni attraverso il locus in indotta (cerchi blu) e le condizioni uninduced (diamanti neri). Il tuffo il -5 bp è dovuto all&#39;esaurimento nucleosoma che si trova nei siti di inizio della trascrizione.

<ol>
	<li>Levy, D., Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling:+Stats:+transcriptional+control+and+biological+impact.">Signalling: Stats: transcriptional control and biological impact.</a> Nature Reviews Molecular Cell Biology. 3, 651-662 (2002).</li><li>Bromberg, J., Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+STATs+in+transcriptional+control+and+their+impact+on+cellular+function.">The role of STATs in transcriptional control and their impact on cellular function.</a> Oncogene. 19, 2468-2473 (2000).</li><li>Hu, X., Ivashkiv, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-regulation+of+signaling+pathways+by+interferon-gamma:+implications+for+immune+responses+and+autoimmune+diseases.">Cross-regulation of signaling pathways by interferon-gamma: implications for immune responses and autoimmune diseases.</a> Immunity. 31, 539-550 (2009).</li><li>Campos, E. I., Reinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histones:+annotating+chromatin.">Histones: annotating chromatin.</a> Annu Rev Genet. 43, 559-599 (2009).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+Modifications+and+Their+Function.">Chromatin Modifications and Their Function.</a> Cell. 128, 693-705 (2007).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Wittwer, C. T., Herrmann, M. G., Moss, A. A., Rasmussen, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+fluorescence+monitoring+of+rapid+cycle+DNA+amplification.">Continuous fluorescence monitoring of rapid cycle DNA amplification.</a> Biotechniques. 22 (130-131), 134-138 (1997).</li><li>Higuchi, R., Dollinger, G., Walsh, P. S., Griffith, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+amplification+and+detection+of+specific+DNA+sequences.">Simultaneous amplification and detection of specific DNA sequences.</a> Biotechnology (N Y). 10, 413-417 (1992).</li><li>Kroger, A., Koster, M., Schroeder, K., Hauser, H., Mueller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activities+of+IRF-1.">Activities of IRF-1.</a> J Interferon Cytokine Res. 22, 5-14 (2002).</li><li>Ross, R. A., Spengler, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neuroblastoma+stem+cells.">Human neuroblastoma stem cells.</a> Seminars in cancer biology. 17, 241-247 (2007).</li></ol>

Nessun conflitto di interessi dichiarati.

Il protocollo ChIP delineato è stato utilizzato con successo in laboratorio per analisi più di venti differenti modificazioni degli istoni. Inoltre, abbiamo caratterizzato i cambiamenti nella occupazione dei fattori di trascrizione, enzimi che modificano gli istoni, proteine ​​che riconoscono modificazioni degli istoni e la RNA polimerasi II macchinario di trascrizione, in diverse IFN-γ geni indotti. Abbiamo anche utilizzato la procedura per caratterizzare cambiamenti modificazioni degli istoni durante la differe...

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		<th>Comment</th>
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<tr>
<td>37% Formaldehyde</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP531-500</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform/Isoamyl alcohol</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>AC32715-5000</td>
<td>&nbsp;</td>
<tr>
<td>Complete Mini Protease Inhibitors</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1836153</td>
<td>&nbsp;</td>
<tr>
<td>Cosmic Calf Serum</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30087.04N</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30243</td>
<td>&nbsp;</td>
<tr>
<td>DTT</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D0632</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP118-500</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Pharmco</td>
<td>zp1110EP</td>
<td>&nbsp;</td>
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<td>Glycine</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>100149</td>
<td>&nbsp;</td>
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<td>Glycogen</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>901393</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3784</td>
<td>&nbsp;</td>
<tr>
<td>IFN-gamma</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>285-IF-100</td>
<td>&nbsp;</td>
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<td>IgG</td>
<td>&nbsp;</td>
<td>Jackson Immunoresearch</td>
<td> 109-005-003</td>
<td>&nbsp;</td>
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<td>KCl</td>
<td>&nbsp;</td>
<td>MP Biologicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>L4408</td>
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<td>&nbsp;</td>
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<td>M8266</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Fisher</td>
<td>BP333-500</td>
<td>&nbsp;</td>
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<td>Deoxycholic Acid Sodium Salt</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP349-100</td>
<td>&nbsp;</td>
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<td>NaCl</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
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<td>NP40</td>
<td>&nbsp;</td>
<td>US Biological</td>
<td>N3500</td>
<td>&nbsp;</td>
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<td>Anti-Histone H3, CT, pan</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>07-690</td>
<td>&nbsp;</td>
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<td>Phenol/chloroform/isoamyl</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>108-95-2</td>
<td>&nbsp;</td>
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<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
<tr>
<td>PMSF</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>50-230-0316</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>5-Prime</td>
<td>2500140</td>
<td>&nbsp;</td>
<tr>
<td>RNAse A</td>
<td>&nbsp;</td>
<td>5-Prime</td>
<td>2500130</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA/Protein A Agarose</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>16-157</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA/Protein G Agaorse</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>16-201</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP166-500</td>
<td>&nbsp;</td>
<tr>
<td>Tris-Cl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T5941</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Acros</td>
<td>327372500</td>
<td>&nbsp;</td>
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<td>Anti-K36me3</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>ab9050</td>
<td>&nbsp;</td>
<tr>
<td>Anti-STAT1</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-345X</td>
<td>&nbsp;</td>
<tr>
<td>Anti-RNA Polymerase II</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-899</td>
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chromatin immunoprecipitation (chip) to assay dynamic histone modification in activated gene expression in human cells

In risposta ad una varietà di ligandi extracellulari, la STAT (trasduttori di segnale e attivatore della trascrizione) fattori di trascrizione sono rapidamente reclutati dal loro stato latente nel citoplasma ai recettori della superficie cellulare dove vengono attivati ​​da fosforilazione in un singolo residuo tirosina 1. Poi dimerizzano e traslocare al nucleo di guidare la trascrizione dei geni bersaglio, incidono sulla crescita, differenziamento, l&#39;omeostasi e la risposta immunitaria. Non sorprende, dato il loro coinvolgimento nei processi di diffusione delle cellule normali, disregolazione della funzione STAT contribuisce alla malattia umana, in particolare per i tumori 2 e malattie autoimmuni 3. E &#39;ben noto che la trascrizione è regolata da alterazioni al modello cromatina 4,5. Queste alterazioni includono le attività di ATP-dipendente complessi, così come modifiche covalenti degli istoni e la metilazione del DNA 6. Poiché l&#39;attivazione STAT dell&#39;espressione genica è sia rapido e transitorio, richiede meccanismi specifici per la modulazione del modello cromatina a STAT-dipendenti loci genici. Per definire questi meccanismi, ci caratterizzano le modificazioni degli istoni e le attività enzimatiche che li generano a loci genici che rispondono alle STAT segnalazione. Questo protocollo descrive immunoprecipitazione della cromatina, un metodo che è prezioso per lo studio di STAT segnalazione alla cromatina nell&#39;espressione genica attivata.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells at JoVE.com

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Questo protocollo descrive come immunoprecipitazione della cromatina (ChIP) è usato per studiare le alterazioni dinamiche al modello cromatina che regolano la trascrizione indotto da una via di trasduzione del segnale.

Immunoprecipitazione della cromatina (ChIP) per analisi modifica dinamica Histone nell&#39;espressione genica attivata nelle cellule umane

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

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Research

JoVE Journal

Biology

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

28.5K Views.  University of Virginia. Questo protocollo descrive come immunoprecipitazione della cromatina (ChIP) è usato per studiare le alterazioni dinamiche al modello cromatina che regolano la trascrizione indotto da una via di trasduzione del segnale.

Video: Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Immunoprecipitazione della cromatina di cellule staminali embrionali umane

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Ricerca

Biologia

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Robotica e Image Analysis dinamica per gli Studi di espressione genica nei tessuti vegetali

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

Una allele-specifica espressione genica saggio per testare la base funzionale delle Associazioni di genetica

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogenica saggio: cellule aderenti

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Saggio di immunoprecipitazione della cromatina per geni tessuto-specifici utilizzando nella fase iniziale di embrioni di topo

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Ricostituzione in vitro di Active T. castaneum telomerasi

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Immunoprecipitazione della cromatina (ChIP) con Drosophila Tessuto

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Utilizzando Sequence SECM arresto come uno strumento per isolare ribosoma Polipeptidi Bound

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...