Work in the authors' laboratory is supported by a grant from the National Institute of General Medical Sciences (GM41628) and by a grant to the Stowers Institute for Medical Research from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.

1 ATP-dipendenti nucleosoma ritocca saggi Per misurare ATP-dipendente attività nucleosoma rimodellamento, immunopurified INO80 o Sottocomplessi INO80 vengono incubati con ATP e un substrato mononucleosomal, che contiene un singolo nucleosoma posizionato ad una estremità di un frammento di DNA di 216 bp, 32 P-marcato. I prodotti di reazione vengono quindi sottoposti ad elettroforesi in gel di poli-acrilammide native. <ol><li> Per generare il P-marcato, &#39;601&#39; frammento di DNA 32, amplificare da pGEM-3Z-601 4 un frammento di DNA 216 bp contenente un 601 nucleosoma sequenza di posizionamento finale in posizione, utilizzando gli oligonucleotidi 5&#39;-ACAGGATGTATATATCTGACACGTGCCTGG e 5&#39;-AATACTCAAGCTTGGATGCCTGCAG come avanti e retromarce primer. <ol><li> Impostare un 100 microlitri reazione di PCR come descritto nella Tabella 1. </li><li> Eseguire le reazioni PCR in un termociclatore con il seguente programma: 1 min a 96 ° C, followed da 45 sec a 94 ° C, 30 sec a 57 ° C, 60 sec a 72 ° C per 30 cicli; termina con 7 min a 72 ° C. </li><li> Dopo la reazione di PCR, rimuovere i nucleotidi non incorporati facendo passare i prodotti di reazione per due volte attraverso le colonne di spin priva di nucleasi. </li><li> Eseguire 5 ml di prodotto purificato PCR in un gel di agarosio 1,2% per confermare che la reazione di PCR generato il ~ 216 bp frammento di DNA desiderato. </li><li> Diluire 5 ml di prodotto di PCR purificato 20 volte, e misurare la concentrazione di DNA utilizzando uno spettrofotometro UV. La resa media è ~ 40 ng / ml. </li><li> Misurare la radioattività di 1 ml di prodotto purificato PCR in un contatore a scintillazione. Stimare l&#39;efficienza di etichettatura calcolando il cpm / ng. Una reazione di marcatura di successo si prevede di cedere ~ 15.000 cpm / ng di 601 frammento di DNA. </li></ol></li><li> Trasferimento nucleosomi cellule Hela sul marcato 601 DNA utilizzando un metodo di diluizione in serie 5. <ol><li> Mescolare 2 pmol di32 P-601 frammento di DNA marcato con 6 mg di nucleosomi Hela (preparato come descritto 5) in 50 ml di un tampone contenente 1,0 M di NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0,1 mM PMSF, e 1 mM DTT e incubare a 30 ° C per 30 min. </li><li> Sequenziale diluire la miscela a 0,8 M, 0,6 M e 0,4 M di NaCl per diluizione con 12,5 ml, 20,8 ml, e 41,6 ml, rispettivamente, 10 mM Tris-HCl (pH 8,0), 1 mM EDTA, 0,1 mM PMSF, e 1 mM DTT, con 30 minuti di incubazione a 30 ° C dopo ogni diluizione. </li><li> Diluire ulteriormente la miscela a 0,2 M e 0,1 M di NaCl per aggiunta di 125 ml e poi 250 ml dello stesso tampone contenente 0,1% di Nonidet P-40, 20% glicerolo, e 200 mg / ml BSA, con 30 min di incubazione a 30 ° C tra le ultime due diluizioni. Conservare il substrato mononucleosome a 4 ° C per un massimo di 3 mesi. </li></ol></li><li> Eseguire reazioni nucleosoma scorrevole ATP-dipendenti in un volume totale di 10 microlitri. La reaction componenti sono elencati nella Tabella 2. Poiché la concentrazione ottimale di NaCl per INO80 attività nucleosoma rimodellamento è ~ 50 mM NaCl (risultati non pubblicati), regolare la concentrazione totale di NaCl in ogni reazione a 50 mm, tenendo conto della quantità di NaCl contenuta in preparazioni di nucleosomi ed enzimi. <ol><li> Prima di iniziare a creare i saggi, espressi poli-acrilammide gel nativi (18 x 16 cm). Per preparare un singolo gel contenente il 5% di acrilammide (acrilammide: bisacrilammide 37.5: 1), 0.5x TBE (Tris 45 mM borato, 1 mM EDTA), 0,01% di ammonio persolfato (APS), e il 0,001% N, N, N, N&#39;-tetramethylethylenediamine (TEMED), mescolare gli ingredienti elencati nella tabella 3. Lasciare polimerizzare il gel per almeno 2 ore a temperatura ambiente. </li><li> Nel frattempo, per ogni reazione da eseguire, unire in un pre-raffreddata siliconato provetta da 1,5 ml microcentrifuga ~ 20 nM INO80 o INO80 subcomplesso (preparato come descritto 3) con una quantità di tampone EB100 ( <strong&gt; Tabella 4) sufficiente ad ottenere un volume di 4,75 ml. Immediatamente ri-congelare tutte le rimanenti frazioni-INO80 contenenti ghiaccio secco in polvere o azoto liquido. </li><li> Impostare un cocktail master con il resto degli ingredienti, scaling up di un fattore &#39;X&#39; (dove X = numero totale di reazioni +3). La quantità di ogni ingrediente necessario per una singola reazione di 10 microlitri è elencato nella Tabella 5; la ricetta dovrebbe essere aumentato a seconda del numero di reazioni da eseguire. Mescolare bene toccando il tubo o pipettando su e giù con un Pipetman e far girare il tubo per alcuni secondi in una microcentrifuga da banco. </li><li> Dispensare 5,25 ml di cocktail master per ciascuna delle provette di reazione istituito nel punto 1.3.2. Mescolare bene pipettando su e giù. Iniziate le reazioni con il trasferimento di provette di reazione in un bagno a 30 ° C blocco di calore o acqua e incubare per 2 ore. </li><li> Nel frattempo, preparare &#39;la rimozione mix&#39; cocktail contenente concorrente DNAe nucleosomi, scaling up di un fattore X (X = numero totale di reazioni + 4). La quantità di ogni ingrediente necessario per preparare 1,5 microlitri rimozione di mix per una singola reazione è elencato nella Tabella 6; la ricetta dovrebbe essere aumentato a seconda del numero di test da eseguire. </li><li> Terminate le reazioni con l&#39;aggiunta di 1,5 ml di miscela rimozione. Mescolare bene, centrifugare, e incubare a 30 ° C per altri 30 min. </li><li> Nel frattempo, pre-eseguire il gel di poliacrilammide nativo in una unità di elettroforesi verticale a 100 V per 30 minuti a 4 ° C, usando TBE 0.5x come tampone di corsa con un ancoretta magnetica all&#39;interno della camera inferiore per mantenere costante la circolazione del buffer. </li><li> Per caricare il campione, aggiungere 2,5 ml di loading dye contenenti 3x TBE, 30% glicerolo, 0,25% blu di bromofenolo, e lo 0,25% xilene Cyanol FF. Mescolare bene, far girare brevemente i campioni, e caricare sul gel con punte di carico. </li><li> Attivare il gel a 200 V per 4,5 ore a 4 ° C con circolazione tampone.</li><li> Per rilevare il segnale, trasferire il gel ad una pila di due fogli di carta da filtro. Avvolgere la carta da filtro con il gel sulla parte superiore con involucro di plastica trasparente, e poi esporlo a uno schermo di archiviazione al fosforo a 4 ° C per il tempo desiderato. </li><li> Scansione lo schermo con un sistema di scanner di imaging isotopi e analizzare i dati utilizzando un software adatto. </li></ol></li></ol> 2 Mononucleosome saggi Binding Per saggiare l&#39;affinità di legame di un dato complesso INO80 per mononucleosomes, effettuare uno spostamento di mobilità elettroforetica Assay (EMSA) utilizzando il substrato mononucleosomal generato nel passaggio 1.2. <ol><li> Impostare la reazione Miscele per saggi di legame come descritto per i test di rimodellamento nucleosoma ma omettere l&#39;ATP e il mix di rimuovere dalle reazioni; incubare a 30 ° C per 30 min. </li><li> Aggiungere 2,5 ml di loading dye per ciascuna miscela di reazione, e si applicano a un gel di poliacrilammide nativo contenente 3,5% di acrilammide (acrilammide: 3 bis7.5: 1), 1% glicerolo, TBE 0.5x, 0,01% APS, e il 0,001% TEMED. </li><li> Utilizzando TBE 0.5x come tampone di corsa, eseguire il gel a 200 V per 2,5 ore a 4 ° C con la circolazione del buffer ed esporre a una schermata di archiviazione fosforo. </li></ol> 3. DNA-e Nucleosome-dipendenti ATPasi saggi Eseguire saggi ATPasi in miscele di reazione 5 microlitri contenenti 20 mM Tris-HCl (pH 7,5), 60 mM NaCl, 6,6 mM MgCl 2, 0.8 mM EDTA, 0,015% Nonidet P-40, 2,5% glicerolo, 0,1 mg / ml di BSA, 1 DTT mM, 0,1 mM PMSF, 2 mM ATP, 2 μCi di [α- 32 P] ATP (3.000 Ci / mmol). Per ogni complesso INO80 o la quantità di complessi INO80 da dosare istituire tre reazioni parallele, uno contenente tampone EB100 per misurare DNA o nucleosoma-indipendente ATPasi, uno contenente chiuso DNA plasmide circolare (5.000 bp, ~ 30 nM) per misurare il DNA ATPasi dipendente, e uno contenente Hela oligonucleosomes (~ 185 nM) per misurare nucleosoma-dipendente ATPasi. Impostare tutte le reazioni su ghiaccio. <ol&gt; <li> Per ogni reazione, combinare 10-50 nM dei INO80 o INO80 Sottocomplessi immunopurified con una quantità di tampone EB100 sufficiente a fornire un volume di 2,2 ml in lubrificati provette da 1,5 ml microcentrifuga pre-refrigerati. Immediatamente ri-congelare eventuali frazioni-INO80 contenenti ghiaccio secco in polvere o azoto liquido. </li><li> Impostare un cocktail master. La quantità di ogni ingrediente necessario per una singola reazione è elencato nella Tabella 7. Scalare la ricetta di scaling up di un fattore 3 (x + 2) +1, dove X = il numero di preparati INO80 da dosare. </li><li> Per preparare contenente tampone &#39;sotto-cocktail&#39; solo, DNA, o nucleosomi, dispensare 2,5 (x + 2) microlitri del cocktail maestro in tre tubi separati. Aggiungere 0,3 (x + 2) ml di entrambi EB100, il DNA plasmide circolare chiuso (1.5 mg / mL), o Hela oligonucleosomes (1.5 mg / mL) e mescolare bene. </li><li> Dispensare 2.8 ml di appropriata sub-cocktail per le provette di reazione-enzima contenente istituiti in vep 3.1. Pipettare delicatamente su e giù per mescolare; evitare di introdurre bolle. </li><li> Per avviare reazioni, trasferire le provette di reazione ad un blocco di calore C a 30 °. </li><li> Dopo 5, 15, 30, e 60 min di incubazione, individuare 0,5 ml di ogni miscela di reazione su una cromatografia su strato sottile di cellulosa polietileneimmina (TLC) piastra (20 x 10 cm) in linea retta di almeno 1,5 cm dal bordo inferiore . Riportare immediatamente provette di reazione al blocco di calore 30 ° C in modo più punti di tempo possono essere presi da un unico tubo. Dopo di macchie, pulire le piastre TLC utilizzando un asciugacapelli. </li><li> Trasferire le piastre TLC ad una camera di vetro contenente abbastanza 0,375 M di fosfato di potassio (pH 3,5) per consentire la parte inferiore 0,5 cm la piastra TLC di essere immersi nella soluzione. </li><li> Coprire la camera, e sviluppare fino alla parte anteriore della fase liquida raggiunge la parte superiore delle piastre TLC. Immediato asciugare le piastre accuratamente con un asciugacapelli. </li><li> Esporre le piastre TLC secchi per uno schermo di archiviazione Phosphor a RT.Scansione lo schermo con un sistema di scanner di imaging isotopo e determinare la quantità di substrato ATP radioattivo e di prodotto ADP. </li><li> Per calcolare la quantità di ATP idrolizzata, moltiplicare ATP% idrolizzato dalla quantità di ATP presenti nella miscela di reazione partendo con la seguente formula: pmol ATP idrolizzata = 10 pmol ATP partendo reazione x [ADP / (ATP + ADP)] </li></ol>

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The authors declare that they have no competing financial interests.

Per garantire che il rimodellamento dei nucleosomi e le attività ATPasi che osserviamo nei test dipendono l&#39;attività catalitica di complessi INO80, e non sulla contaminazione di rimodellamento e / o enzimi ATPasi, abbiamo nucleosomi regolarmente test rimodellamento e l&#39;attività ATPasica di versioni cataliticamente inattive di complessi INO80, purificata in parallelo con wild type INO80 utilizzando la stessa procedura. Una reazione di controllo negativo manca ATP dovrebbe essere eseguita quando saggiando l&#39...

SNF2 complessi di rimodellamento della cromatina famiglia includono un centro SNF2-come ATPasi subunità 1,2. Alcune funzioni ATPasi SNF2-come come enzimi singole subunità, mentre altri funzionano come la subunità catalitica di grandi complessi multi-subunità. Chiarire i meccanismi molecolari attraverso i quali ciascuna delle subunità dei complessi di rimodellamento della cromatina contribuiscono alla loro attività richiede la capacità di effettuare saggi biochimici che sezionano il processo di rimodellamento. ATP-dipendente rimodellamento dei nucleosomi dal complesso INO80 umano e di altri enzimi rimodellamento della cromatina può essere immaginato come un processo multi-step che inizia con il legame dell&#39;enzima rimodellamento di nucleosomi, seguita dall&#39;attivazione del suo DNA e / o nucleosoma-dipendente ATPasi, traslocazione dell&#39;enzima rimodellamento sul DNA nucleosomal, ed eventuale riposizionamento dei nucleosomi 1,2. Comprendere i dettagli molecolari del ATP-dipendente processo di rimodellamento della cromatina requires dissezione della reazione rimodellamento nelle sue singole fasi e la definizione dei contributi delle singole subunità del complesso rimodellamento della cromatina per ogni fase della reazione. Tali analisi richiedono la capacità di analizzare il rimodellamento dei nucleosomi e altre attività utilizzando substrati molecolari definite in vitro. In un protocollo JOVE precedente, abbiamo descritto le procedure utilizzate per generare INO80 complessi di rimodellamento della cromatina e Sottocomplessi con composizioni subunità definite 3. Qui, vi presentiamo tre saggi biochimici che permettono l&#39;analisi quantitativa del legame, DNA e nucleosomi attivato ATPasi, e le attività di rimodellamento nucleosoma associati a tali complessi nucleosoma.

<table border="0" cellpadding="0" cellspacing="0" width="839">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Name of Reagent/Material</td>
			<td style="width:212px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:123px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">P8340</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x PCR reaction buffer&#160;</td>
			<td style="width:212px;">Roche Applied Science&#160;</td>
			<td style="width:147px;">11435094001</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roche Taq DNA Polymerase</td>
			<td style="width:212px;">Roche Applied Science&#160;</td>
			<td style="width:147px;">11435094001</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NucAway Nuclease-free Spin Columns&#160;</td>
			<td>Ambion</td>
			<td style="width:147px;">Cat. # AM10070</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ultrapure ATP&#160;</td>
			<td>USB/Affymetrix</td>
			<td style="width:147px;">77241 25 UM</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bovine serum albumin&#160;</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">A9418&#160;</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">N,N,N&#180;,N&#180;-tetramethylethylenediamine (TEMED)</td>
			<td style="width:212px;">Thermo Scientific</td>
			<td style="width:147px;">17919</td>
			<td style="width:123px;">Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40% Acrylamide/Bis 37.5:1</td>
			<td style="width:212px;">Amresco</td>
			<td style="width:147px;">0254-500ML</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicated salmon sperm DNAs&#160;</td>
			<td>GE Healthcare</td>
			<td style="width:147px;">27-4565-01</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% ammonium persulfate (APS)</td>
			<td style="width:212px;">Thermo Scientific</td>
			<td style="width:147px;">17874</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">benzonase&#160;</td>
			<td>Novagen</td>
			<td style="width:147px;">Cat. No. 70664</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">[&#945;-32P] ATP (3000 Ci/mmol)</td>
			<td style="width:212px;">PerkinElmer</td>
			<td style="width:147px;">BLU003H250UC</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">dCTP, [&#945;-32P]- 6000Ci/mmol</td>
			<td style="width:212px;">PerkinElmer</td>
			<td style="width:147px;">BLU013Z250UC</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Equipment</td>
			<td style="width:212px;">Company</td>
			<td style="width:147px;"></td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR thermal cycler PTC 200</td>
			<td>MJ Research</td>
			<td style="width:147px;">PTC 200</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoefer vertical electrophoresis unit</td>
			<td style="width:212px;">Hoefer</td>
			<td style="width:147px;">SE600X-15-1.5</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lubricated 1.5ml microcentrifuge tubes&#160;</td>
			<td style="width:212px;">Costar</td>
			<td style="width:147px;">3207</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Storage Phosphor Screen&#160;</td>
			<td>Molecular Dynamics</td>
			<td style="width:147px;">63-0034-79</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3MM filter paper</td>
			<td>Whatman&#160;</td>
			<td style="width:147px;">28458-005</td>
			<td style="width:123px;">VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Typhoon PhosphorImager&#160;</td>
			<td>GE Healthcare</td>
			<td style="width:147px;">8600</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageQuant software</td>
			<td>GE Healthcare</td>
			<td style="width:147px;">ver2003.02</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">TLC Glass Plates, PEI-Cellulose F</td>
			<td style="width:212px;">Millipore</td>
			<td style="width:147px;">5725-7</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Immobilon-FL Transfer Membrane 7 x 8.4</td>
			<td style="width:212px;">Millipore</td>
			<td style="width:147px;">IPFL07810</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">General purpose survey meter with end-window or pancake GM (Geiger-Mueller) probe</td>
			<td style="width:212px;">Biodex</td>
			<td style="width:147px;">Model 14C</td>
			<td style="width:123px;"></td>
		</tr>
	</tbody>
</table>

biochemical assays for analyzing activities of atp-dependent chromatin remodeling enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Le figure mostrano risultati rappresentativi di saggi biochimici utilizzati per caratterizzare attività INO80, tra cui nucleosoma scorrevole (figura 1) e vincolante (Figura 2) saggi e DNA o saggi ATPasi nucleosoma-dipendente (Figura 3). L&#39;esperimento mostrato in Figura 1 confronta la capacità di complessi INO80 intatti purificato attraverso FLAG-Ies2 o FLAG-INO80E e INO80 Sottocomplessi purificato attraverso sia FLAG-I...

Watch this Scientific Journal Video about Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes at JoVE.com

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Saggi biochimici per le attività di analisi ATP-dipendente rimodellamento della cromatina Enzimi

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

biochemical-assays-for-analyzing-activities-atp-dependent-chromatin

Research

JoVE Journal

Biology

14.7K Views.  Stowers Institute for Medical Research. Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Video: Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

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 ...

Ricerca

Biologia

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafish Cervello iniezione ventricolo

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Fotodecolorazione saggi (FRAP e FLIP) per misurare la dinamica delle proteine ​​della cromatina in Living cellule staminali embrionali

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

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 ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Il prelievo di sangue per l&#39;analisi biochimica in Zebrafish Adult

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Biochimica e High Throughput valutazione microscopica di massa grassa in<em&gt; Caenorhabditis Elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Titolazione biochimica di glicogeno<em&gt; In vitro</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Generazione e Purificazione di umani INO80 rimodellamento della cromatina Complessi e Sottocomplessi

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

L&#39;isolamento di regioni genomiche specifici e l&#39;identificazione di molecole da Associated enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplificazione, sequenziamento di prossima generazione, e DNA genomico mappatura dei siti di integrazione retrovirali

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...