Gli autori desiderano ringraziare il Dott. Keji Zhao lab (NIH / NHLBI) per il loro aiuto nel fornire i risultati di sequenziamento. Vorremmo anche ringraziare il progetto genoma UCSC per l&#39;utilizzo di Browser per visualizzare sequenziamento del genoma mappato legge. Questo lavoro è stato sostenuto dal Pathway R00HD055052 NIH al Premio Indipendenza e R01HD065816 dal NICHD, il Lucile Parkard Foundation, e la Johns Hopkins University di start-up fondi per XC

(La procedura intero chip richiede circa due giorni. Preparazione di librerie di chip per alta sequenziamento richiede altri 2-3 giorni.) 1. Sezionare e prepara il tessuto per Experiment ChIP (circa 1 milione di cellule) <ol><li> Dissect tessuto di interesse (ad esempio, 200 paia di Drosophila testicoli) in PBS freddo inibitore della proteasi + 1x (sciogliere 1 pellet di cocktail inibitore della proteasi in 1,5 ml di PBS 1x a 7x ottenendo così la soluzione stock) + PMSF (concentrazione finale 100 pg / mL). Risciacquare tessuto doppio tessuto e risospendere in 200 pl della stessa soluzione di PBS con inibitori. </li><li> Per risolvere il campione aggiungere 5,5 microlitri di formaldeide al 37% (formaldeide calda in bagno d&#39;acqua a 37 ° C per 1 minuto prima di usare). Incubare a 37 ° C per 15 minuti, vortex ogni 5 minuti in mezzo. </li><li> Mettere il campione sul ghiaccio per il tessuto riposare per 2 minuti. Sciacquare 2x con 450 microlitri di PBS (con inibitori della proteasi e PMSF). Esempio può essere ora negoziod a -20 ° C. Ripetere più volte dissezione per ottenere materiale sufficiente per ChIP. </li></ol> 2. Preparare supernatante con la proteina-DNA Coniugazione per l&#39;analisi ChIP <ol><li> Combina campione abbastanza dal punto 1.3 in una provetta eppendorf 1,75 mL. </li><li> Rimuovere dalla PBS campione di tessuto. Aggiungere 200 pl di tampone di lisi (50 mM Tris-HCl, pH 7,6, 1 mM CaCl2, 0,2% Triton X-100 o NP-40, 5 mM butirrato, e 1x cocktail inibitore di proteinasi). Aggiungi fresca soluzione madre PMSF al tampone di lisi (PMSF concentrazione finale di 0,5 mM). Omogeneizzare con il blu omogeneizzatore (Cat Fisher # 749521-1590) fino a quando i tessuti si dissociano completamente senza alcuna aggregazione, incubare a RT (temperatura ambiente) per 10 minuti. </li><li> Sonicazione: microtip sonicatore uso (Misonix HS-XL200) alla potenza 20. Con ultrasuoni per 10 secondi e di riposo in ghiaccio per 50 sec. Ripetere 4-5 volte (tempo di sonicazione ottimale dovrebbe essere determinata empiricamente, come la maggior sonicazione produrrà frammenti più piccoli. Per ChIP con anticorpi contro Histonmodifiche elettroniche, che normalmente ultrasuoni cromatina di circa 200-300 bp; per il fattore di trascrizione o di regolatore di cromatina (ad esempio proteine ​​Polycomb), che normalmente sonicare cromatina a 200-1000 bp. Tuttavia, la dimensione del frammento ottimale deve essere testato empiricamente. Nota: non rimuovere la provetta in ghiaccio ANCHE DURANTE sonicazione PER EVITARE ESEMPIO DA RISCALDAMENTO! </li><li> Diluire il campione con l&#39;aggiunta di 1,8 mL di tampone RIPA (10 mM Tris-HCl, pH 7,6, 1 mM EDTA, 0,1% SDS, 0,1% Na-desossicolato, 1% Triton X-100, con inibitori della proteasi PMSF e alla stessa concentrazione descritta in precedenza). Salvare 40 microlitri come ingresso di controllo sommando 2 microlitri 5M NaCl e incubare a 65 ° C per una notte (O / N) per invertire reticolazione. </li><li> Per anticorpo coniugato a perline, aggiungere 40 ul Proteina A perline in una provetta eppendorf da 1,5 ml, quindi aggiungere 600 microlitri di PBS e roccia a 4 ° C per 2 minuti, perline applicano al magnete e rimuovere supernatante. Aggiungere 100 pl di PBS e anticorpo di interesse (quantità di anticorpo dovrebbe essere determinata empirismocamente). Incubare a RT per 1 ora o 4 ° C per 4 ore. </li><li> Rimuovere il surnatante da perline di magnete e aggiungere 1 ml di estratto di cromatina dal passo 2,4 ai talloni. Ruota a 4 ° CO / N. </li><li> Applicare il campione ChIP al magnete e rimuovere il surnatante. Lavare le perline con il seguente tampone a 4 ° C per 10 minuti ciascuno: 2x con 1 ml di tampone RIPA [1,89 mL &#39;tampone RIPA&#39; + 315 pl inibitori della proteasi 7x + 20 pl PMSF]; 2x con 1 ml di tampone RIPA + 0,3 M NaCl [1,89 mL di tampone RIPA + 220 microlitri 3 M NaCl]; 2x con 1 mL di tampone LiCl (0,25 M LiCl, 0,5% NP40, 0,5% NaDOC); 1x con 1 ml di TE 1x + 0,2% Triton X-100; 1x con 1 ml di TE 1x. </li><li> Per invertire reticolazione, risospendere le sfere in 100 microlitri tampone TE + 3 microlitri 10% SDS + 5 pl di proteinasi K (20 mg / mL). Incubare a 65 ° CO / N. </li><li> Applicare perline al magnete e trasferire supernatante in un nuovo tubo. Il supernatante CONTIENE IL CAMPIONE DNA. Lavare perline con 100 microlitri TE + 0,5 MNaCl e combinare le due surnatante. </li><li> Fenolo-cloroformio estrarre il campione di DNA. Aggiungere 200 ul Fenolo: Chlorofrom: IAA (25:24:1), mescolare e vortex. Spin a 14k per 5 minuti a RT. In alternativa, il DNA può essere estratto usando il kit di purificazione PCR Qiagen. </li><li> Trasferire il surnatante / acquoso in una nuova provetta, e aggiungere acrilammide lineare ad una concentrazione finale di 20 ug / mL (In alternativa, 1 pl di glicogeno a 20 mg / ml per ogni 1 ml di surnatante possono anche essere utilizzati. Glicogeno viene utilizzato per qPCR successivo o analisi PCR, mentre acrilammide lineare viene utilizzato per il sequenziamento saggi.) Aggiungere 20 microlitri di 3M NaOAc e 500 microlitri EtOH 100%. Mescolare bene ed incubare a 80 ° C per 10 minuti. Centrifuga alla massima velocità per 20 minuti a 4 ° C. </li><li> Rimuovere il surnatante e lavare con 300 microlitri EtOH 70%. Aria campione secco e risospendere in 50 microlitri tampone TE. Esempio possono essere conservati a -20 ° C. Esempio può essere usato per la PCR quantitativa (qPCR) test (Figura 1). </li></ol> 3 bis. Analizzare ChIP-DNA ed utilizzando qPCR <ol><li> Diluire DNA genomico al seguente concentrazione di una curva standard: non diluito, 1/10, 1/100, 1/1000, 1/5000. </li><li> Per ogni coppia di primer, istituito qPCR in duplice esemplare in una piastra a 96 pozzetti con il DNA genomico da 3a.1 passo, il controllo degli ingressi e ChIP-ed DNA: 10 L 2x PCR SYBR mix (Fermentas, K0222); 1 ul 10 uM innesco in avanti; 1 pl 10 pM primer reverse; x ul-ChIP ed DNA (controllo di input, o DNA genomico); y pl priva di nucleasi H 2 O per regolare il volume di reazione di 20 pl. </li><li> Far girare la piastra a 96 pozzetti con Mini piastra Spinner (Labnet). </li><li> Eseguire qPCR con ABI 7300 PCR in tempo reale di sistema (o altri sistemi in tempo reale PCR) utilizzando la seguente condizione: Fase 1: 50 ° C per 2 min, 1 ciclo; Fase 2: 95 ° C per 10 min, 1 ciclo; Fase 3: 95 ° C per 15 s, 60 ° C per 1 minuto, 40 cycles; Fase 4 (fase di dissociazione): 95 ° C per 15 s, 60 ° C per 1 minuto, 95 ° C per 15 s. </li><li> Controllare la curva di dissociazione: Un picco al tracciato termico dissociazione suggerisce un singolo amplicone dalla reazione PCR. Più di un picco indica non specifico prodotto con la coppia di primer, i dati qPCR non devono essere utilizzati. </li><li> Determinare la fase lineare di amplificazione esponenziale di reazione PCR per ciascuna serie di primer, calcolando la formula che risolve quantità di DNA secondo l&#39;(soglia ciclo) Ct valore, che si basa sulla curva standard utilizzando la serie di diluito DNA genomico in 3a. 1. </li><li> Se il valore di Ct ed ChIP-DNA e controllo di input sono all&#39;interno della gamma lineare del valore Ct, calcolare l&#39;arricchimento di ChIP-DNA rispetto ed input, a seconda del fattore di diluizione della ChIP-DE DNA e controllo di input in 2.4. </li></ol> 3b. Amplifica ChIP-per il sequenziamento del DNA ed elevato throughput. <ol><li> End-riparazione di ChIP-ed mix DNA (UsaEpicentre DNA END-Repair kit), costituita il ghiaccio: 1-34 DNA genomico microlitri da passo 2,12 (da 0,1 a 5 pg) 5 microlitri tampone 10x End riparazione 5 microlitri mix 2,5 mM dNTP 5 microlitri 10 mM ATP x pl H 2 O per regolare il volume di reazione di 49 microlitri 1 pl End-Repair mix enzima (DNA polimerasi T4 + polinucleotide chinasi T4) Incubare per 45 minuti a RT. Purificare reazione con Qiagen reazione MinElute Cleanup kit. Eluire in 30 microlitri tampone di eluizione (EB). </li><li> Aggiungere A-sovrasta all&#39;estremità 3 &#39;: 30 pl di DNA prodotto eluito dalla 3b.1 passo Buffer 5 microlitri NEB 10x # 2 10 pl 1 mM dATP 2 microlitri dH 2 O 3 microlitri 5 U / pl frammento di Klenow (3 &#39;→ 5&#39; Exo-) Incubare per 30 minuti a 37 ° C. Purificare reazione con MinElute kit ReactionCleanup. Eluire in 10 pl EB. </li><li> Solexa legatura linker: 10 pl di DNA eluito dalla 3b.2 passo 10 pl ddh 2 O 2.5 &amp;mu; l 10x tampone di T4 DNA ligasi 1 ml mix oligo Adattatore da Illumina (1/10-diluted dal magazzino) 2,5 microlitri DNA ligasi T4 (400 unità / pl) Incubare per 1 ora a RT. Purificare reazione con MinElute kit ReactionCleanup. Eluire in 20 pl EB. Esempio possono essere conservati a -20 ° C. </li><li> Eseguire il campione eluito da DNA 3B.3 passo utilizzando l&#39;indirizzo e-gel apparecchio. Isolare la banda di 300-500 bp nel gel e purificare mediante il Gel Extraction kit Qiagen (questo passaggio elimina i ~ 125 bp auto-legato linker dalla ligazione dell&#39;adattatore oligo). Eluire in 12 pl EB. </li><li> Amplifica libreria tramite accoppiato-end (PE) primer da Illumina: 10,5 pl di DNA eluito dalla 3B.4 passo 12,5 microlitri di master mix (2X Phusion HF, Finnzymes) 1 ml di primer PCR PE1 1 1 ml di primer PCR PE2 2 PCR condizione: 98 ° C per 30 sec (98 ° C 10 sec, 65 ° C 30 sec, 72 ° C 30 sec), 20 cicli si ripetono 72 ° C per 5 minuti. </li><li> Campioni di 3b.5 passo può essere utilizzato per ChIP-seq dopo aver selezionato il DNA di dimensioni adeguate (300-500 bp) utilizzando lo standard 2% gel di agarosio. Esempio possono essere conservati a -20 ° C. E &#39;meglio isolare ogni campione ChIP su un unico gel per prevenire la contaminazione. I campioni possono essere presentate per high-throughput sequencing. </li></ol> 3c. Solexa pipeline di analisi <ol><li> Il 25-bp sequenziamento legge sono stati ottenuti dal genoma Illumina Analyzer (GA) pipeline. </li><li> Tutte le letture sono state allineate al genoma di Drosophila (dm3) utilizzando l&#39;antilope (Efficient Allineamento locale dei dati Nucleotide) software, che consente fino a due discrepanze con la sequenza di riferimento. </li><li> Letture in modo univoco mappato sono state selezionate per l&#39;analisi a valle. Per letture multiple identici, fino a tre copie sono state conservate per ridurre la possibilità di distorsioni di amplificazione PCR. </li><li> L&#39;uscita del Pipeline GA è stato convertito in dati estendibili del browser (BED) file. </li><li> Per generarei file di parrucca per caricare al browser per la visualizzazione UCSC, abbiamo usato una bisaccia python che è stato descritto in una precedente pubblicazione 3 con 4 bp, come la dimensione della finestra e 160 bp, come la dimensione del frammento di DNA. </li><li> Per classificare i geni nella Drosophila geni silenziosi e espressa, abbiamo utilizzato un numero digitale chiamato RPKM (letture / per kilobasi fusa regione exonic / per milione mappata letture). I geni con RPKM = 0 sono stati classificati come gruppo silenzioso e geni con RPKM ≥ 1 sono stati classificati nei gruppi espresse, che possono essere classificate in tre sottogruppi come i geni con livelli bassi di espressione, moderato e alto, e ogni gruppo ha circa lo stesso numero di geni. </li><li> Per confrontare il livello di modificazione degli istoni e l&#39;espressione genica, abbiamo utilizzato uno script python che è stato descritto in una precedente pubblicazione 3. </li></ol> 4. Risultati rappresentativi Esempi di ChIP-qPCR risultati utilizzando Bam (sacchetto di biglie) mutante<html> testicoli sono mostrati in Figura 1 4. In Bam testicoli, si verifica un errore nel passaggio dal spermatogoni proliferativa a differenziare spermatociti 5, 6. Usiamo bam testicoli come fonte di cellule germinali indifferenziate, che sono arricchiti in questo tipo di tessuto. Differenziazione dei geni necessari per la differenziazione degli spermatozoi, come maschio-specifico fattore di trascrizione 87 (mst87F), Don Juan (dj), e la cipolla fuzzy (fzo) non sono espressi in Bam testicoli. Questi geni sono altamente arricchito con la repressione H3K27me3 modifica istoni 7 (Figura 1A), ma sono privi del attivo H3K4me3 modifica istoni 8 (Figura 1B), una firma cromatina abbiamo chiamato 7 &#39;monovalente&#39;. Arricchimento di una o H3K27me3 H3K4me3 è determinata dalla normalizzazione ad una ciclina costitutivamente espressa A (CYCA) del gene 4. contenuto &quot;&gt; ChIP-seq analisi utilizzando lo stesso set di anticorpi (cioè repressiva H3K27me3 e attiva H3K4me3) con bam testicoli mutanti (Figura 2) qPCR convalidato i risultati illustrati nella Figura 1. Per i tre geni testati differenziazione terminale mst87F, dj e fzo , le regioni genomiche sono altamente arricchito con H3K27me3 ma non H3K4me3 (Figura 2A-2C). In contrasto, il gene espresso costitutivamente CYCA ha H3K4me3 significativo ma poco H3K27me3 vincolante in prossimità del suo sito di inizio trascrizione (TSS) (Figura 2D). Inoltre, i profili di ChIP H3K27me3 e H3K4me3 vicino ai TSSS di quattro classi di geni differenzialmente espressi sono coerenti con la funzione di ogni modificazione degli istoni. Come mostrato in figura 3A, l&#39;arricchimento di H3K27me3 valle del TSSS è inversamente correlato al livello di espressione genica. I geni silenziosi hanno il più alto H3K27me3 mentre ilgeni altamente espressi non hanno H3K27me3 vincolanti. Questi dati sono coerenti con il ruolo repressivo di H3K27me3 sull&#39;espressione del gene. Al contrario, l&#39;arricchimento delle H3K4me3 intorno alle TSSS hanno mostrato la correlazione opposta con il livello di espressione genica (Figura 3B), coerente con il ruolo attivo di H3K4me3 sull&#39;espressione genica. <img alt="Figura 1" src="/files/ftp_upload/3745/3745fig1.jpg" /> Figura 1. Analisi qPCR di ChIP-ed utilizzando anticorpi contro il DNA sia il repressiva H3K27me3 modificazione degli istoni (A) o attivo H3K4me3 modificazione degli istoni (B) nei testicoli cellule indifferenziate mutanti arricchito bam. (A) a Bam testicoli, i geni di differenziazione (Mst87F, dj, fzo) sono arricchiti con la modifica H3K27me3 repressiva degli istoni. (B) geni di differenziazione si esauriscono con H3K4me3 marchio attivo. Il livello di DNA ChIP (DNA ChIP / ingresso) al gene bersaglio (Mst87F, dj o fzo) per la prima volta normalizzata la Lev<html> el DNA di ChIP al gene di controllo CYCA. Le barre di errore indicano la deviazione standard da tre repliche indipendenti biologica. <img alt="Figura 2" src="/files/ftp_upload/3745/3745fig2.jpg" /> Figura 2. UCSC Genome Browser istantanee di H3K27me3 e H3K4me3 arricchimento attraverso le intere regioni genomiche di (A) Mst87F (B), dj e (C) fzo, (D) geni CYCA. La A cerchiata H3K27me3 arricchita di regione (D) riflette lo stato della cromatina una sovrapposizione gene CG7264, che si esprime umile bam testicoli (RPKM = 1), ma altamente espresso nel wild-type testicoli (RPKM = 130) 8 (ChIP-seq dati provenienti da 7). Sonde utilizzate in analisi PCR quantitativa dei risultati chip in figura 1 sono classificati nella parte inferiore di ogni parcella. <a href="http://www.jove.com/files/ftp_upload/3745/3745fig2large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . tp_upload/3745/3745fig3.jpg &quot;/&gt; Figura 3. ChIP-seq profili utilizzando H3K27me3 e H3K4me3 in bam testicoli 7. I quattro gruppi di geni (7.509 geni) sono stati classificati in base ai livelli di espressione genica loro basati su RNA-seq 8 risultati. Le classi rappresentative dei geni sono tracciate per l&#39;arricchimento di una particolare modificazione degli istoni, utilizzando sequenze da 3kb 3kb a monte a valle dei siti di trascrizione loro inizio (TSSS). Questo genera un profilo di arricchimento (A) H3K27me3 (K27) e (B) H3K4me3 (K4) ChIP-seq analisi di ciascun gruppo. <a href="http://www.jove.com/files/ftp_upload/3745/3745fig3large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> .

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La versatilità di analisi ChIP discussi in questo protocollo può essere utilizzato su diversi tessuti, che fornisce l&#39;opportunità di studiare lo stato cromatina in un sistema biologicamente rilevante. Esperimenti chip utilizzando cellule coltivate sono sistemi conveniente eseguire a causa grande quantità di cellule può essere ottenuto facilmente. Tuttavia, cellule coltivate non riflette necessariamente cellule in un multi-cellulare ambiente. Con lo sviluppo di questa tecnica usando tessuti sezionati da animali ...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Complete Mini cocktail inibitore della proteasi </td><td> Roche </td><td> 11836153001 </td><td></td></tr><tr><td> La formaldeide (37%) </td><td> Supelco </td><td> 47.083-U </td><td></td></tr><tr><td> PMSF </td><td> Sigma </td><td> 78830 </td><td></td></tr><tr><td> Kontes pellet pestello </td><td> Fischer Scientific </td><td> K749521-1590 </td><td></td></tr><tr><td> PCR Purification Kit </td><td> Qiagen </td><td> 28104 </td><td></td></tr><tr><td> Poliacrilammide lineare </td><td> Sigma </td><td> 56575-1 ml </td><td></td></tr><tr><td> Glicogeno </td><td> Qiagen </td><td> 158930 </td><td></td></tr><tr><td> SYBR green / ROX qPCR Master Mix </td><td> Fermentas </td><td> K0223 </td><td></td></tr><tr><td> Mini Piastra filatrice </td><td> Labnet </td><td> Z723533 </td><td></td></tr><tr><td> Real time PCR System </td><td> Applied Biosystem </td><td> 4351101 </td><td></td></tr><tr><td> Volume piccolo processore ad ultrasuoni </td><td> Misonix </td><td> HS-XL2000 </td><td> Modello interrotto </td></tr><tr><td> Dynabeads, Proteina A </td><td> Invitrogen </td><td> 100-01D </td><td></td></tr><tr><td> Dynamag magnete </td><td> Invitrogen </td><td> 123-21D </td><td></td></tr><tr><td> Fenolo: Chlorofrom: IAA </td><td> Invitrogen </td><td> 15593-049 </td><td></td></tr><tr><td> Epicentre DNA END-Repair Kit </td><td> Epicentre Biotecnologie </td><td> ER0720 </td><td></td></tr><tr><td> MinElute Reaction Cleanup Kit </td><td> Qiagen </td><td> 28204 </td><td></td></tr><tr><td> Klenow Fragment (3 &#39;→ 5&#39; exo-) </td><td> New England Biolabs </td><td> M0212S </td><td></td></tr><tr><td> DNA ligasi T4 </td><td> Promega Corporation </td><td> M1794 </td><td></td></tr><tr><td> Adattatore oligonucleotidi </td><td> Illumina </td><td> PE-400-1001 </td><td></td></tr><tr><td> Abbinato-End Primer 1.0 e 2.0 </td><td> Illumina </td><td> 1001783 1001 784 </td><td></td></tr><tr><td> E-Gel Electorphoresis sistema </td><td> Invitrogen </td><td> G6512ST </td><td></td></tr><tr><td> 2X Phusion HF Mastermix </td><td> Finnzymes </td><td> F-531 </td><td></td></tr></tbody></table>

chromatin immunoprecipitation (chip) using drosophila tissue

L&#39;epigenetica rimane un settore in rapida evoluzione che gli studi come lo stato della cromatina contribuisce alla espressione genica differenziale in diversi tipi di cellule a diversi stadi di sviluppo. Regolazione epigenetica contribuisce ad un ampio spettro di processi biologici, tra cui la differenziazione cellulare durante lo sviluppo embrionale e l&#39;omeostasi in età adulta. Una strategia critico negli studi epigenetici è quello di esaminare come le varie modificazioni degli istoni e fattori di cromatina regolare l&#39;espressione genica. Per risolvere questo problema, immunoprecipitazione della cromatina (ChIP) è ampiamente utilizzato per ottenere una fotografia della associazione di fattori particolari con DNA nelle cellule di interesse. ChIP tecnica utilizza comunemente cellule coltivate come materiale di partenza, che può essere ottenuto in abbondanza e omogeneità per generare dati riproducibili. Tuttavia, vi sono diversi avvertimenti: Innanzitutto, l&#39;ambiente far crescere le cellule in piastre di Petri è diversa da quella in vivo, così puònon rispecchiano lo stato endogena cromatina delle cellule in un organismo vivente. In secondo luogo, non tutti i tipi di cellule possono essere coltivate ex vivo. Ci sono solo un numero limitato di linee cellulari, da cui la gente può ottenere abbastanza materiale per l&#39;analisi ChIP. Qui si descrive un metodo per fare esperimenti ChIP utilizzando tessuti di Drosophila. Il materiale di partenza è sezionato da un tessuto animale vivente, quindi in grado di rispecchiare con precisione lo stato endogena cromatina. L&#39;adattabilità di questo metodo con diversi tipi di tessuto permetterà ai ricercatori di affrontare molto più biologicamente questioni rilevanti in materia di regolazione epigenetica in vivo 1, 2. Combinando questo metodo con high-throughput sequencing (ChIP-seq) ulteriormente permetterà ai ricercatori di ottenere un paesaggio epigenomic.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP using Drosophila tissue at JoVE.com

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recentemente high-throughput tecnologia di sequenziamento ha notevolmente aumentato la sensibilità di immunoprecipitazione della cromatina (ChIP) esperimento e ha spinto la sua applicazione con cellule purificate o tessuti sezionati. Qui delineare un metodo per usare la tecnica ChIP con<em&gt; Drosophila</em&gt; Tessuto, che può definire la condizione cromatina endogeno in un sistema ben caratterizzato biologico.

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 ...

chromatin-immunoprecipitation-chip-using-drosophila-tissue

Research

JoVE Journal

Biology

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

25.0K Views.  Johns Hopkins University. Recentemente high-throughput tecnologia di sequenziamento ha notevolmente aumentato la sensibilità di immunoprecipitazione della cromatina (ChIP) esperimento e ha spinto la sua applicazione con cellule purificate o tessuti sezionati. Qui delineare un metodo per usare la tecnica ChIP con Drosophila Tessuto, che può definire la condizione cromatina endogeno in un sistema ben caratterizzato biologico.

Video: Chromatin Immunoprecipitation ChIP using Drosophila tissue

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Patterning di cellule staminali embrionali che utilizzano il chip Bio flip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Ricerca

Biologia

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Un saggio cromatina per tessuto cerebrale umano

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

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 ...

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

Immunoprecipitazione DNA metilato

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

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

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

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

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

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 ...

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 ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Confronto quantitativo di Cis-Regulatory Element (CRE) Attività in transgenici Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Efficiente immunoprecipitazione della cromatina utilizzando Limitare quantità di biomassa

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Generazione di immunoprecipitazione della cromatina Template DNA di alta qualità per high-throughput sequencing (ChIP-Seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

L&#39;approccio ChroP Combina ChIP e Spettrometria di Massa a sezionare Locus-specifiche proteomica Paesaggi della cromatina

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...