Ce travail est soutenu par l&#39;Université de Virginie, l&#39;Université de Virginie Cancer Center et The F. Thomas et Kate Miller Jeffress Memorial Trust. Nous remercions le James E. Darnell Lab (Rockefeller University), le Laboratoire de Robert Ross (Université Fordham) pour les lignées cellulaires.

PLANIFICATION La veille d&#39;une expérience de ChIP est prévu, les cellules doivent être cultivées de sorte qu&#39;un nombre suffisant est disponible. Supposons que chaque dosage, il faudra ChIP ~ 1 à 1,5 x 10 7 cellules. Prévoyez toujours de faire les deux contrôles négatifs (IgG) et positifs (anticorps pan histones) et des expériences en double. Astuce - Pour les cellules 2FTGH, chaque dosage puce nécessite environ une boîte de culture 15 cm de tissu à 80% de confluence. PARTIE 1: PRÉPARER ET EFFECTUER CHROMATINE ChIP <ol><li> Retirez les milieux de culture et d&#39;induire les cellules pour le temps nécessaire avec 20 ml de 10% de sérum de veau cosmique (CSC) / DMEM contenant de l&#39;IFN-gamma à 5 ng / mL. Remplacez le support sur les plaques non induite avec 20 ml de 10% CCS / DMEM ainsi. </li><li> Dans une hotte, ajouter 540 uL de formaldéhyde 37% à 20 mL de médias (1% en concentration finale) dans le plat de 15 cm. Astuce - plaque inclinable et ajouter le formaldéhyde à la piscine des médias -, puis la plaque de roche pour l&#39;étendre uniformément. </li><li> Permettre la réticulation se dérouler pendant 10 minutes à température ambiante. </li><li> Ajouter à la glycine 0,125 M concentration finale de cesser de réticulation. </li><li> Vide aspirer les médias et laver les cellules une fois avec 10 ml de phosphate de glace froide saline tamponnée (PBS). </li><li> Recueillir les cellules avec un racleur de cellules dans ~ PBS 7 ml et combiner des cellules (à partir de 6 plaques) qui ont été traités de manière identique dans 50 ml tube conique sur la glace. </li><li> Centrifugeuse (Beckman Coutler Allegra 12-R Centrifugeuse) à 1800 rpm à 4 ° C pendant 5 minutes et aspirer le PBS. Astuce - Snap geler la cellule si arrêtant ici et conserver à -80 ° C à granulés </li><li> Reprendre le culot cellulaire dans 4 ml de tampon Gonflement (25 mM HEPES pH 7,8, 1,5 mM MgCl2, 10 mM KCl, 0,1% NP40. Ajouter 1 mM de DTT, les inhibiteurs de protéase complète et refroidir sur glace avant utilisation) et le transfert à un 15 ml tube conique. Astuce - Ce volume tampon gonflement est approprié pour une mise en commun 6 plaques (~ 6-9 x 10 7 cellules). Tampon plus ou moins pourrait changer l&#39;efficacité douncing. </li><li> Incuber la suspension cellulaire sur la glace pendant 10 minutes. </li><li> Transfert de suspension pour une pré-réfrigérée douncer 15 ml (Wheaton) et Dounce 20 coups avec son pilon A. </li><li> Transfert dounced cellules vers un tube 15 ml conique sur la glace. </li><li> Centrifuger à 2500 rpm pendant 5 minutes à 4 ° C et jeter le surnageant. </li><li> Reprendre le nucléaire culot (~ 200 uL de 6 plaques) dans 3 ml (15 volumes granulés) de tampon de sonication (50 mM HEPES pH 7,9, NaCl 140 mM, EDTA 1 mM, 1% Triton X-100, le désoxycholate de sodium à 0,1%, 0,1% SDS. Ajouter les inhibiteurs de protéase complète et refroidir sur glace juste avant l&#39;utilisation). Astuce - Ce volume tampon de sonication est approprié pour une mise en commun 6 plaques (~ 6-9 x 10 7 cellules). Tampon plus ou moins pourrait changer l&#39;efficacité sonication. </li><li> Soniquer suspension de noyaux sur la glace pendant 10 cycles (20 secondes on/40 secondes off / réglage de l&#39;amplitude 8) en utilisant un sonicateur Misonix 3000 équipé de la micropointe. Ce se traduit généralement par des fragments de chromatine en moyenne 200-1000 pb en taille. Astuce - Parce sonicateurs varient, l&#39;efficacité sonication doit être déterminée empiriquement. Pour ce faire, de prendre des aliquotes de 20 uL avant sonication et après chaque cycle de sonication. La chaleur de ces échantillons à 65 ° C pendant 4 heures. Ajouter tampon de chargement de l&#39;ADN et exécuter un standard de 1% d&#39;agarose / TBE gel de visualiser les fragments d&#39;ADN.) </li><li> Transfert soniqué matériel à un tube à centrifuger et centrifuger SS34 pendant 20 minutes à 4 ° C à 13000 rpm dans un rotor SS34 (Sorvall Centrifugeuse RC6 plus) pour obtenir un culot ce qui est habituellement une petite quantité de débris cellulaires. </li><li> Soigneusement transférer le surnageant dans un tube de 15 ml conique sur la glace. </li><li> Pré-clairement en ajoutant 360 uL de sperme de saumon ADN / protéine A ou G agarose perles lisier et bascule à 4 ° C pendant au moins 30 minutes. Astuce - Cette étape réduit le bruit dans le test qui résulterait de la non-événements spécifiques contraignantes. </li><li> Centrifuger à 1000 rpm à 4 ° C pendant 5 minutes pour sédimenter les billes d&#39;agarose. </li><li> Transfert prédédouanés surnageant dans un autre tube de 15 ml conique sur la glace. </li><li> Mesurer la A260 unités de la chromatine prédédouanés et ajuster le volume avec le tampon de sonication à quatre (4) unités A260 (0,2 mg / ml en supposant une unité = 0.050 A260 mg / ml) Astuce - Pour mesurer l&#39;A260 unités, diluer 10 uL d&#39;prédédouanés chromatine dans 190 uL DDW; vierge spectrophotomètre avec 10 uL dans tampon de sonication DDW 190 uL. </li><li> Economisez 75 uL de la chromatine (à 4 unités A260) que l&#39;échantillon d&#39;entrée et de stocker à 20 ° C jusqu&#39;au moment de renverser réticule. Astuce - Cette étape est cruciale pour l&#39;analyse des données. Ne négligez pas de prendre la partie aliquote d&#39;entrée. </li><li> Aliquoter la chromatine préparées à 1,7 ml microtubes de sur iCE que aliquotes de 1 ml. Astuce - Si l&#39;arrêt ici, snap geler la chromatine préparées sur glace sèche et stocker à -80 ° C. NB: la chromatine stockées peuvent se dégrader, produisant moins de résultats optimaux, afin d&#39;éviter si possible. </li><li> Ajouter les anticorps ChIP-grade aux échantillons induits et non induits jeux en double. Ajouter IgG (2 mg) et d&#39;anticorps pan Histone H3 (15 uL) que les contrôles positifs et négatifs induits et non induits aux séries d&#39;échantillons. Astuce - La quantité d&#39;anticorps à utiliser doit être déterminée de manière empirique ou voir la fiche produit du fournisseur pour des montants proposés. Typiquement 1-2 mg d&#39;un anticorps ChIP-grade qui fonctionne bien. Le volume approprié doit être déterminée empiriquement pour des anticorps qui sont fournis que le sérum. </li><li> Tous les tubes rock la nuit à 4 ° C. </li></ol> PARTIE 2: COLLECT immunocomplexes <ol><li> Ajouter 60 ul de sperme de saumon ADN / protéine A ou protéine G billes d&#39;agarose coulis à tous les tubes. Protéines Astuce Utilisez A pour les anticorps polyclonaux de lapin et de protéines G pour les anticorps monoclonaux de souris. </li><li> Rock à 4 ° C pendant au moins 60 minutes. </li><li> Pellet les complexes immuns (billes d&#39;agarose) par microfuging délicatement à 2000 rpm à 4 ° C pendant 3 minutes. </li><li> Aspirer le surnageant. </li><li> Laver les immunocomplexes avec 1 ml de chacun des tampons de lavage suivants pendant 10 minutes avec bascule à 4 ° C. Microfuge pendant 3 minutes à 2000 rpm à 4 ° C entre chaque lavage et aspirer le surnageant. Conserver les échantillons refroidis sur la glace. Ajouter PMSF juste avant l&#39;utilisation. <ul><li> Lavez faible teneur en sel - 0,1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM de Tris-Cl, pH 8,0, NaCl 150 mM, PMSF 1 mM </li><li> Laver en sel élevée - 0,1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM de Tris-Cl, pH 8,0, NaCl 500 mM, PMSF 1 mM </li><li> LiCl Laver - 0,25 M LiCl, 1% NP-40, le désoxycholate de sodium à 1%, EDTA 1 mM, 10 mM de Tris-Cl, pH 8,0, 1 mM de PMSF </li><li> Lavez TE - deux lavages avec du tampon TE -10 mM de Tris-Cl, pH 8,0, EDTA 1 mM, PMSF 1 mM </li></ul></li><li> Retirez le tampon de lavage finale TE et aux billes, ajouter 500 uL de tampon d&#39;élution (50 mM Tris-Cl, pH 8,0, EDTA 10 mM, SDS 1%). Astuce - Le tampon d&#39;élution doit être préparée fraîchement. </li><li> Vortex à mélanger des billes d&#39;agarose dans un tampon d&#39;élution et de la chaleur pendant 30 minutes à 65 ° C. Astuce - Vortex chaque tube au moins toutes les 10 minutes au cours de cette 30 minutes. </li><li> Microfuge pendant 30 secondes à 2000 tpm et le transfert de l&#39;éluant à un tube de centrifugeuse de nouvelles. Astuce - Lors de l&#39;élution, décongeler vos échantillons d&#39;entrée et ajouter 500 uL de tampon d&#39;élution à chacun. Inclure ces échantillons à toutes les étapes à venir. </li><li> Ajouter 5 M de NaCl à une concentration finale de 200 mM à tous les échantillons et les vortex. </li><li> Incuber à 65 ° C pendant une nuit (ou au moins 4 heures). </li></ol> PARTIE 3: ChIP&#39;d purifier et l&#39;ADN ENTRÉE <ol><li> Microfuge tous les échantillons pendant 30 secondes pour recueillir la condensation sur les paupières. </li><li> Ajouter 1 microlitre de RNAse A (10 U / uL) à chaque tube et incuber à température ambiante pendant 15 minutes. </li><li> Ajouter 10 uL d&#39;EDTA 0,5 M, 40 uL 0,5 M de Tris-Cl pH 6,5, 2 pl protéinase K (10 mg / ml) et incuber à 45 ° C pendant 60 minutes. Astuce Préparer un master mix de ces réactifs et ajouter 52 uL dans chaque tube. </li><li> Extrait d&#39;alcool phénol / chloroforme / alcool isoamylique puis chloroforme / alcool isoamylique extraire les échantillons. </li><li> Ajouter 2 glycogène uL (25 ug / uL) et 0,1 volumes de pH 3M acétate de Na 5,2 et vortex. </li><li> Ajouter 2,5 volumes d&#39;éthanol 100% et le vortex. </li><li> Incuber à 20 ° C la nuit. </li></ol> PARTIE 4: ChIP&#39;d purifier et l&#39;ADN apport continu <ol><li> Échantillons Microfuge pour un minimum de 20 minutes à 13000 rpm à 4 ° C pour précipiter l&#39;ADN / glycogène pellets. </li><li> Laver avec de l&#39;ADN / glycogène boulettes avec 1 ml d&#39;éthanol à 70%. </li><li> Air sec les granulés secs ou rapidement dans l&#39;ADN Savant centrifugeuse sous vide vitesse. Astuce - Ne pas trop sécher les ADN. </li><li> Resuspendre l&#39;ADN / glycogène granulés dans 200 ul de tampon TE. </li><li> L&#39;ADN (1 pi) est prêt pour la quantification par PCR en temps réel. L&#39;ADN magasin reste à 20 ° C. Astuce - Utiliser plus de 1 pl d&#39;ADN peuvent inhiber les réactions PCR en temps réel, afin d&#39;échelle avec prudence. </li></ol> Partie 5: résultats représentatifs: Les échantillons d&#39;ADN et de ChIP entrée sont quantifiées avec Real-Time PCR 7,8 (Applied Biosystems 7500 Fast Real-Time PCR System) utilisant des amorces spécifiques pour le locus du gène d&#39;intérêt. Amorces qui séquence génomique cible connue pour être enrichi ou manquant de la modification des histones être testés peuvent être utilisés comme contrôles positifs et négatifs ainsi. Les données sont présentées comme pourcentage de l&#39;entrée. La procédure de puce devrait être entièrement repeated avec trois biologiques réplique à s&#39;assurer que les changements signalés dans les modifications des histones sont statistiquement significatives. Il ya plusieurs questions importantes à considérer lors de l&#39;utilisation PCR en temps réel pour quantifier l&#39;ADN, y compris la conception d&#39;apprêt, efficacité de la PCR, et la manière appropriée pour calculer les pour cent des procédures d&#39;entrée et de normalisation. Lorsque profilage Occupation modification des histones sur un gène, l&#39;efficacité de PCR doit être cohérente entre tous les couples d&#39;amorces. Le fabricant en temps réel du système de PCR peuvent fournir les informations nécessaires et la formation. La figure 1 montre des résultats représentatifs de la chromatine au cours immunoprécipité STAT1 activation du gène IRF1 9, déclenchée par l&#39;IFN-γ. <img src="/files/ftp_upload/2053/2053fig1.jpg" alt="Figure 1" /> Figure 1. Occupation de STAT1, l&#39;ARN polymérase II et H3K36me3 est dynamique lors de STAT1 activation du gène IRF1. (A, B, C) ​​à copeaux avec α-H3K36me3, α-α-STAT1 et Pol II de la chromatine recueillies à partir des cellules 2FTGH induite par l&#39;IFN-γ pour 30 minutes (carrés rouges), de 1,5 heures (cercles bleus), 5 heures ( triangles verts) ou non induits (losanges noirs). IgG est le contrôle négatif (gris croix). (D) Pan H3 est le contrôle positif et montre l&#39;occupation des histones dans le locus dans l&#39;induit (cercles bleus) et les conditions non induits (losanges noirs). La baisse de -5 pb est dû à l&#39;épuisement des nucléosomes qui se trouve sur les sites de démarrage de la transcription.

<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>

Le protocole décrit ChIP a été utilisé avec succès dans le laboratoire d&#39;essai de plus de vingt différentes modifications des histones. En outre, nous avons caractérisé les changements dans l&#39;occupation des facteurs de transcription, enzymes de modification des histones, protéines qui reconnaissent modifications des histones et la transcription de l&#39;ARN polymérase II machines, à l&#39;IFN-γ de plusieurs gènes induits. Nous avons également utilisé la procédure de caractériser les changements ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<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>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>100149</td>
<td>&nbsp;</td>
<tr>
<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>
<tr>
<td>IgG</td>
<td>&nbsp;</td>
<td>Jackson Immunoresearch</td>
<td> 109-005-003</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>MP Biologicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LiCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L4408</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Acetate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP333-500</td>
<td>&nbsp;</td>
<tr>
<td>Deoxycholic Acid Sodium Salt</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP349-100</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
<tr>
<td>NP40</td>
<td>&nbsp;</td>
<td>US Biological</td>
<td>N3500</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Histone H3, CT, pan</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>07-690</td>
<td>&nbsp;</td>
<tr>
<td>Phenol/chloroform/isoamyl</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>108-95-2</td>
<td>&nbsp;</td>
<tr>
<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>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>327372500</td>
<td>&nbsp;</td>
<tr>
<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>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

chromatin immunoprecipitation (chip) to assay dynamic histone modification in activated gene expression in human cells

En réponse à une variété de ligands extracellulaires, la STAT (transducteur de signal et activateur de la transcription) des facteurs de transcription sont rapidement recrutés dans leur état ​​latent dans le cytoplasme aux récepteurs de surface cellulaire où ils sont activés par la phosphorylation d&#39;un seul résidu tyrosine 1. Ils ont ensuite dimériser et translocation vers le noyau de conduire la transcription de gènes cibles, affectant la croissance, la différenciation, l&#39;homéostasie et la réponse immunitaire. Il n&#39;est pas surprenant, étant donné leur implication dans les processus généralisé de cellules normales, dérèglement de la fonction STAT contribue à la maladie humaine, en particulier pour les cancers et les maladies auto-immunes 2 3. Il est bien établi que la transcription est régulée par des modifications au modèle de la chromatine 4,5. Ces modifications comprennent les activités de l&#39;ATP-dépendant de complexes, ainsi que covalentes modifications des histones et la méthylation de l&#39;ADN 6. Parce que l&#39;activation de l&#39;expression génique STAT est à la fois rapide et transitoire, elle nécessite des mécanismes spécifiques permettant de moduler le modèle chromatine au locus du gène STAT-dépendante. Pour définir ces mécanismes, nous caractérisons les modifications des histones et les activités enzymatiques qui les génèrent à des loci génétiques qui répondent à STAT. Ce protocole décrit immunoprécipitation de la chromatine, une méthode qui est précieux pour l&#39;étude de la STAT à la chromatine dans l&#39;expression des gènes activés.

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

Ce protocole décrit comment la chromatine immunoprécipitation (ChIP) est utilisée pour étudier les altérations dynamiques du modèle de la chromatine qui régulent la transcription induite par une voie de transduction du signal.

Immunoprécipitation de la chromatine (ChIP) pour doser modification des histones dynamique dans l&#39;expression génique dans les cellules humaines Activé

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

chromatin-immunoprecipitation-chip-to-assay-dynamic-histone

Research

JoVE Journal

Biology

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

28.5K vues.  University of Virginia. Ce protocole décrit comment la chromatine immunoprécipitation (ChIP) est utilisée pour étudier les altérations dynamiques du modèle de la chromatine qui régulent la transcription induite par une voie de transduction du signal.

Vidéo: 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.

Immunoprécipitation de la chromatine des cellules souches embryonnaires humaines

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

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Biologie

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.

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

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.

Robotique et d&#39;analyse d&#39;images dynamiques pour les études d&#39;expression génique dans les tissus végétaux

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.

Aide d&#39;un compteur de cellules automatisé pour simplifier les études d&#39;expression génique: Knockdown siRNA de l&#39;IL-4 dans l&#39;expression des gènes dépendant cellules 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.

Un allèle spécifique Assay expression du gène pour l&#39;épreuve la base fonctionnelle des associations génétiques

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.

Test clonogénique: cellules adhérentes

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.

Assay immunoprécipitation de la chromatine des gènes spécifiques de tissus en utilisant des embryons de souris en début de

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.

La reconstitution in vitro de l&#39;actif T. La télomérase castaneum

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.

Immunoprécipitation de la chromatine (ChIP) à l&#39;aide Drosophila Tissus

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.

Utilisation de la séquence d&#39;arrêt SecM comme un outil pour isoler les polypeptides Ribosome Bound

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