Name: chromatin immunoprecipitation (chip) of histone modifications from saccharomyces cerevisiae
Uploaded: 2017-12-29T15:00:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae at JoVE.com

De auteurs bedank leden van de groene lab voor nuttige discussies. Dit werk werd gedeeltelijk ondersteund door subsidies van de NIH R03AG052018 en R01GM124342 naar E.M.G.

1. pre bind-antistoffen tegen magnetische kralen
<ol>
	<li>Meng het eiwit A/G magnetische kralen goed en breng 20 µL van magnetische kralen per één immunoprecipitation (IP) monster tot een 1,5 mL koker. Opmerking: Wanneer pipetteren kralen, wide-boring, lage-retentie tips gebruiken.</li>
	<li>Plaats de buis met kralen in een magnetische stand en laat van parels om te verzamelen aan de kant van de buis. Verwijder het supernatant.</li>
	<li>Wassen van de parels 3 keer met 1 mL koude Tris-gebufferde zoutoplossing (T...

<ol>
	<li>Jaiswal, D., Turniansky, R., Green, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choose+Your+Own+Adventure:+The+Role+of+Histone+Modifications+in+Yeast+Cell+Fate.">Choose Your Own Adventure: The Role of Histone Modifications in Yeast Cell Fate.</a> J Mol Biol. 429 (13), 1946-1957 (2017).</li><li>Suganuma, T., Workman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signals+and+combinatorial+functions+of+histone+modifications.">Signals and combinatorial functions of histone modifications.</a> Annu Rev Biochem. 80, 473-499 (2011).</li><li>Solomon, M. J., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde-mediated+DNA-protein+crosslinking:+a+probe+for+in+vivo+chromatin+structures.">Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures.</a> P Natl Acad Sci USA. 82 (19), 6470-6474 (1985).</li><li>Solomon, M. J., Larsen, P. L., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+protein-DNA+interactions+in+vivo+with+formaldehyde:+evidence+that+histone+H4+is+retained+on+a+highly+transcribed+gene.">Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene.</a> Cell. 53 (6), 937-947 (1988).</li><li>Gilmour, D. S., Lis, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+protein-DNA+interactions+in+vivo:+distribution+of+RNA+polymerase+on+specific+bacterial+genes.">Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes.</a> P Natl Acad Sci USA. 81 (14), 4275-4279 (1984).</li><li>Lefran&#231;ois, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+yeast+ChIP-Seq+using+multiplex+short-read+DNA+sequencing.">Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing.</a> BMC Genomics. 10, 37 (2009).</li><li>Furey, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq+and+beyond:+new+and+improved+methodologies+to+detect+and+characterize+protein-DNA+interactions.">ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions.</a> Nat Rev Genet. 13 (12), 840-852 (2012).</li><li>Déjardin, J., Kingston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+proteins+associated+with+specific+genomic+Loci.">Purification of proteins associated with specific genomic Loci.</a> Cell. 136 (1), 175-186 (2009).</li><li>Byrum, S. D., Raman, A., Taverna, S. D., Tackett, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChAP-MS:+a+method+for+identification+of+proteins+and+histone+posttranslational+modifications+at+a+single+genomic+locus.">ChAP-MS: a method for identification of proteins and histone posttranslational modifications at a single genomic locus.</a> Cell Rep. 2 (1), 198-205 (2012).</li><li>Soldi, M., Bremang, M., Bonaldi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+systems+approaches+for+the+analysis+of+histone+modification+readout.">Biochemical systems approaches for the analysis of histone modification readout.</a> Biochim Biophys Acta. 1839 (8), 657-668 (2014).</li><li>Soldi, M., Bonaldi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ChroP+approach+combines+ChIP+and+mass+spectrometry+to+dissect+locus-specific+proteomic+landscapes+of+chromatin.">The ChroP approach combines ChIP and mass spectrometry to dissect locus-specific proteomic landscapes of chromatin.</a> J Vis Exp. (86), (2014).</li><li>Kuo, M. H., 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=In+vivo+cross-linking+and+immunoprecipitation+for+studying+dynamic+Protein:DNA+associations+in+a+chromatin+environment.">In vivo cross-linking and immunoprecipitation for studying dynamic Protein:DNA associations in a chromatin environment.</a> Methods. 19 (3), 425-433 (1999).</li><li>Meluh, P. B., Broach, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+analysis+of+yeast+chromatin.">Immunological analysis of yeast chromatin.</a> Methods Enzymol. 304, 414-430 (1999).</li><li>Rozen, S., Skaletsky, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer3+on+the+WWW+for+general+users+and+for+biologist+programmers.">Primer3 on the WWW for general users and for biologist programmers.</a> Methods Mol Biol. 132, 365-386 (2000).</li><li>Taylor, S., Wakem, M., Dijkman, G., Alsarraj, M., Nguyen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+RT-qPCR-Publishing+data+that+conform+to+the+MIQE+guidelines.">A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines.</a> Methods. 50 (4), S1-S5 (2010).</li><li>Rodr&#237;guez, A., Rodr&#237;guez, M., C&#243;rdoba, J. J., Andrade, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+primers+and+probes+for+quantitative+real-time+PCR+methods.">Design of primers and probes for quantitative real-time PCR methods.</a> Methods Mol Biol. 1275, 31-56 (2015).</li><li>Briggs, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H3+lysine+4+methylation+is+mediated+by+Set1+and+required+for+cell+growth+and+rDNA+silencing+in+Saccharomyces+cerevisiae.">Histone H3 lysine 4 methylation is mediated by Set1 and required for cell growth and rDNA silencing in Saccharomyces cerevisiae.</a> Gene Dev. 15 (24), 3286-3295 (2001).</li><li>Bernstein, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylation+of+histone+H3+Lys+4+in+coding+regions+of+active+genes.">Methylation of histone H3 Lys 4 in coding regions of active genes.</a> P Natl Acad Sci USA. 99 (13), 8695-8700 (2002).</li><li>Pokholok, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+map+of+nucleosome+acetylation+and+methylation+in+yeast.">Genome-wide map of nucleosome acetylation and methylation in yeast.</a> Cell. 122 (4), 517-527 (2005).</li><li>Krogan, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Paf1+complex+is+required+for+histone+H3+methylation+by+COMPASS+and+Dot1p:+linking+transcriptional+elongation+to+histone+methylation.">The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: linking transcriptional elongation to histone methylation.</a> Mol Cell. 11 (3), 721-729 (2003).</li><li>South, P. F., Harmeyer, K. M., Serratore, N. D., Briggs, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H3K4+methyltransferase+Set1+is+involved+in+maintenance+of+ergosterol+homeostasis+and+resistance+to+Brefeldin+A.">H3K4 methyltransferase Set1 is involved in maintenance of ergosterol homeostasis and resistance to Brefeldin A.</a> P Natl Acad Sci USA. 110 (11), E1016-E1025 (2013).</li><li>Margaritis, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+repressive+mechanisms+for+histone+3+lysine+4+methylation+through+promoting+3'-end+antisense+transcription.">Two distinct repressive mechanisms for histone 3 lysine 4 methylation through promoting 3'-end antisense transcription.</a> PLoS Genet. 8 (9), e1002952 (2012).</li><li>Jezek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histone+methyltransferases+Set5+and+Set1+have+overlapping+functions+in+gene+silencing+and+telomere+maintenance.">The histone methyltransferases Set5 and Set1 have overlapping functions in gene silencing and telomere maintenance.</a> Epigenetics. 12 (2), 93-104 (2017).</li><li>Suka, N., Luo, K., Grunstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sir2p+and+Sas2p+opposingly+regulate+acetylation+of+yeast+histone+H4+lysine16+and+spreading+of+heterochromatin.">Sir2p and Sas2p opposingly regulate acetylation of yeast histone H4 lysine16 and spreading of heterochromatin.</a> Nat Genet. 32 (3), 378-383 (2002).</li><li>Kimura, A., Umehara, T., Horikoshi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+gradient+of+histone+acetylation+established+by+Sas2p+and+Sir2p+functions+as+a+shield+against+gene+silencing.">Chromosomal gradient of histone acetylation established by Sas2p and Sir2p functions as a shield against gene silencing.</a> Nat Genet. 32 (3), 370-377 (2002).</li><li>Rothbart, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Interactive+Database+for+the+Assessment+of+Histone+Antibody+Specificity.">An Interactive Database for the Assessment of Histone Antibody Specificity.</a> Mol Cell. 59 (3), 502-511 (2015).</li></ol>

De hier beschreven procedure zorgt voor de efficiënte herstel van DNA in verband met gewijzigde histonen in gistcellen door immunoprecipitation. Dit wordt gevolgd door de qPCR gebruikend inleidingen die nadere uitwerking van de regio's van belang zijn voor het bepalen van de lokale verrijking of uitputting van specifieke histone modificaties. Ondanks de bijna twintig jaar geleden als een methode ontwikkeld, blijft chIP de bepalende assay voor het onderzoeken van de status van de wijziging van de Histon op verschillende ...

De dynamische posttranslationele wijziging (PTM) van histones is een belangrijke regelgevende mechanisme voor vele DNA-templated processen, waaronder transcriptie, replicatie en DNA repair1,2. De mogelijkheid om de overvloed en precieze lokalisatie van gemodificeerde histonen gelijktijdig met deze processen is daarom cruciaal te begrijpen hun verordening onder verschillende omstandigheden in de cel. De ontwikkeling van chromatine immunoprecipitation (chIP) als een methode die grotendeels voortvloeide uit de biochemische studies van de interacties tussen eiwitten en DNA, met name in vitro methoden met behulp van chemische crosslinkers, in combinatie met de noodzaak om de dynamiek van eiwit-DNA interacties in vivo en in specifieke regio's van het genoom3,4,5. De vooruitgang van kwantitatieve PCR (qPCR) en sequencing technologieën heeft ook uitgebreid de mogelijkheid om uit te voeren van de experimenten van de chIP met kwantitatieve vergelijkingen en over het geheel genomen, waardoor het een krachtig hulpmiddel voor het ontleden van DNA-eiwit interactie op meerdere niveaus.
ChIP is momenteel een vereiste methode voor elke onderzoeksgroep kochten chromatine-gemedieerde regulering van het genoom zoals er geen vergelijkbare methoden zijn voor het rechtstreeks ondervragen van de fysieke verbinding tussen een gemodificeerde histone en een specifieke genomic locus in vivo. Hoewel er variaties van deze methode met behulp van de volgende generatie sequencing toewijzen histone modificaties gedurende de genoom6,7 beschikbaar zijn, kunnen deze benaderingen betrekking hebben op verschillende wetenschappelijke vragen en hun omvang, kosten en technische middelen kunnen worden beperkt voor sommige onderzoeksgroepen. Bovendien, gerichte chIP-qPCR is noodzakelijk als aanvulling op deze benaderingen door middel van methoden om beide te optimaliseren het chIP-protocol vóór sequencing en valideren van resultaten van de epigenomic datasets. Massaspectrometrie gebaseerde benaderingen voor de identificatie van de volledige aanvulling van Histon merken die zijn gekoppeld aan de genomic regio's ook hebben ontstaan8,9,10,11, echter deze benaderingen hebben sommige beperkingen met betrekking tot die regio's van het genoom kunnen worden bestudeerd en zij vereisen technische expertise en instrumentatie die niet beschikbaar voor alle onderzoeksgroepen is. ChIP blijft derhalve een fundamentele methode voor het analyseren van de omvang en de verdeling van Histon modificaties onder uiteenlopende omstandigheden voor alle onderzoeksgroepen kochten epigenetica, chromatine en de regeling van genomic functies.
Hier beschrijven we een methode voor chIP met behulp van de ontluikende gist Saccharomyces cerevisiae (S. cerevisiae) om te onderzoeken van de verdeling van Histon PTMs op chromatine model. Deze aanpak is afhankelijk van een aantal kernonderdelen van chIP protocollen ontwikkeld in gist en ook toegepast op uiteenlopende model systemen12,13. Interacties tussen gemodificeerde histonen en DNA in de cel worden bewaard door crosslinking met formaldehyde. Na de lysate bereiding, chromatine fragmenten zijn ontbindend in uniform en middelgrote fragmenten door spijsvertering met micrococcal nuclease. Immunoprecipitation van de gemodificeerde histonen is uitgevoerd met commerciële of lab-gegenereerd antilichamen en eventuele bijbehorende DNA is geïsoleerd en geanalyseerd voor verrijking op bepaalde genomic regio's met behulp van qPCR (Figuur 1). Voor vele histone modificaties volstaat de hoeveelheid DNA die dit protocol verkregen voor het testen van meer dan 25 verschillende genomic loci door qPCR.
Deze chIP methode is zeer veelzijdig voor het toezicht op de verdeling van een enkele Histon wijziging op meerdere gemuteerde stammen of milieuomstandigheden, of voor het testen van meerdere histone modificaties in wildtype cellen bij een aantal genomic loci. Bovendien zijn talrijke onderdelen van het protocol gemakkelijk instelbaar tot opsporing van beide zeer - of nederig-overvloedig Histon merken optimaliseren. Ten slotte, uitvoeren van chIP van gemodificeerde histonen in ontluikende gist biedt de mogelijkheid om belangrijke besturingselementen gebruiken voor antilichamenspecificiteit die grotendeels niet beschikbaar zijn in andere systemen. Namelijk, giststammen kunnen worden gegenereerd die puntmutaties in Histon residuen die zijn gericht tot wijziging draagt, en, in sommige gevallen is er slechts een enkele enzym dat wijziging op een bepaalde Histon residu katalyseert (bv. Histon lysine methyltransferases). Daarom kan de chIP worden uitgevoerd in ofwel de Histon mutant of enzym schrapping stammen te assay de mate waarin niet-specifieke binding van het antilichaam kan worden voorkomen en het genereren van vals-positieve resultaten. Dit besturingselement is bijzonder waardevol voor nieuw ontwikkelde antilichamen, en kan zelfs worden gebruikt om te valideren antilichamenspecificiteit voor geconserveerde histone modificaties voorafgaand aan hun gebruik in andere systemen. Deze aanpak vormt een aanvulling op andere methoden om antilichamenspecificiteit test die onderscheid maken tussen verschillende wijziging Staten (zoals mono-, di- en tri-methylering), met inbegrip van sonderen matrices van gemodificeerde peptiden en uitvoeren van westelijke vlekken van histones of nucleosomes met gedefinieerde wijzigingen. Globaal, chIP in de ontluikende gist is een krachtige methode voor de beoordeling van de dynamiek van Histon PTMs in het genoom en het ontleden van de mechanismen inzake hun verordening.

<table border="0" cellpadding="0" cellspacing="0" width="876">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Yeast Extract</td>
			<td style="width:301px;">Research Products International (RPI)</td>
			<td style="width:164px;">Y20025-1000.0</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Peptone</td>
			<td style="width:301px;">Research Products International (RPI)</td>
			<td style="width:164px;">P20250-1000.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Dextrose</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">BP350-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Glycine</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td>AC12007-0050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Tris</td>
			<td style="width:301px;">Amresco</td>
			<td style="width:164px;">0497-5KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758-500G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">NaCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">BP358-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Nonylphenyl-polyethylene glycol</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">74385</td>
			<td>Equivalent to NP-40</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">MgCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">S25533</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">CaCl2</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">20899-25G-F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">LiCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">AC413271000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Dodecyl Sulfate</td>
			<td style="width:301px;">Amresco</td>
			<td style="width:164px;">M107-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Deoxycholate</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">30970-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Acetate</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">S2889</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">NaHCO3</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">S25533</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>P7626-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Yeast protease inhibitor cocktail</td>
			<td>VWR</td>
			<td>10190-076</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">25 Phenol:24 Chloroform:1 Isoamyl Alcohol</td>
			<td style="width:301px;">VWR Life Science</td>
			<td>97064-824</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Ethanol</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">E7023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Nuclease-Free Water</td>
			<td style="width:301px;">VWR</td>
			<td style="width:164px;">100720-992</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Micrococcal Nuclease</td>
			<td>Worthington Biochemical</td>
			<td>LS004797</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Glycogen</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">R0561</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Proteinase K</td>
			<td>Research Products International (RPI)</td>
			<td>P50220-0.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">RNase A&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>R6513-50MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Bradford Assay Reagent</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">23238</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;BSA Standard 2 mg/mL</td>
			<td style="width:301px;">ThermoScientific</td>
			<td>23210</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H4</td>
			<td>EMD Millipore</td>
			<td>04-858</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H4K16ac</td>
			<td>EMD Millipore</td>
			<td>ABE532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H3</td>
			<td style="width:301px;">Abcam</td>
			<td style="width:164px;">ab1791</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H3K4me2</td>
			<td>Active Motif</td>
			<td>39142</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;High Rox qPCR Mix</td>
			<td>Accuris qMax Green, Low Rox qPCR Mix</td>
			<td>ACC-PR2000-L-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Protein A/G Magnetic Beads</td>
			<td style="width:301px;">ThermoScientific</td>
			<td>88803</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">magnetic stand for 1.5mL tubes</td>
			<td>Fisher Scientific</td>
			<td>PI-21359</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Acid-Washed Glass Beads</td>
			<td>Sigma-Aldrich</td>
			<td style="width:164px;">G8772</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Microtube Homogenizer</td>
			<td style="width:301px;">Benchmark</td>
			<td>D1030</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">2.0 mL screw-cap tubes with sealing rings</td>
			<td>Sigma-Aldrich</td>
			<td>Z763837-1000EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Gel loading tips</td>
			<td>Fisher Scientific</td>
			<td>07-200-288</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Cuvettes</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td style="width:164px;">50-476-476</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Parafilm</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">50 mL conical tubes</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td style="width:164px;">14-432-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">384-Well PCR Plate</td>
			<td>Fisher Scientific</td>
			<td>AB-1384W</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Gyratory Floor Shaker</td>
			<td style="width:301px;">New Brunswick Scientific</td>
			<td style="width:164px;">Model G10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Spectrophotometer</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">ND-2000c</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">&#160;Real-Time PCR Detection System</td>
			<td style="width:301px;">Bio-Rad</td>
			<td style="width:164px;">1855485</td>
			<td></td>
		</tr>
	</tbody>
</table>

chromatin immunoprecipitation (chip) of histone modifications from saccharomyces cerevisiae

Histon posttranslationele modificaties (PTMs), zoals acetylation, methylation en fosforylatie, worden dynamisch geregeld door een aantal enzymen die toevoegen of verwijderen van deze merken in reactie op signalen ontvangen door de cel. Deze PTMS zijn belangrijke bijdragen aan de regulatie van processen zoals gen expressie controle en reparatie van DNA. Immunoprecipitation (chIP) van de chromatine is een instrumentele aanpak voor het ontleden van de overvloed en lokalisatie van vele Histon PTMs in het genoom in reactie op diverse verstoringen naar de cel. Hier is een veelzijdige methode voor het uitvoeren van chIP van post-translationally gemodificeerde histonen uit de ontluikende gist Saccharomyces cerevisiae (S. cerevisiae) beschreven. Deze methode berust op crosslinking van eiwitten en DNA met behulp van formaldehyde behandeling van gist culturen, generatie van gist lysates door parel verslaan, solubilisatie van chromatine fragmenten door micrococcal nuclease en immunoprecipitation van Histon-DNA complexen. DNA die is gekoppeld aan het merk Histon van belang is gezuiverd en onderworpen aan kwantitatieve PCR analyse om te evalueren van de verrijking op meerdere loci gedurende het genoom. Representatieve experimenten sonderen de lokalisatie van de Histon merken H3K4me2 en H4K16ac in wildtype en mutant gist worden besproken om aan te tonen van data-analyse en interpretatie. Deze methode is geschikt voor een verscheidenheid van Histon PTMs en kan worden uitgevoerd met verschillende gemuteerde stammen of in de aanwezigheid van diverse milieu benadrukt, waardoor het een uitstekend hulpmiddel voor het onderzoeken van veranderingen in de dynamiek van de chromatine onder verschillende omstandigheden.

Een belangrijk onderdeel van dit protocol is het optimaliseren van de concentratie van micrococcal nuclease (MNase) gebruikt om te verteren de chromatine in oplosbare fragmenten, zoals beschreven in stap 10. Dit is essentieel voor het verkrijgen van hoge resolutie gegevens met betrekking tot de verdeling van Histon modificaties aan de genomic regio's van belang. Een MNase titratie moet worden uitgevoerd om te bepalen van de meest geschikte concentratie te bereiken vooral mono-nucleosomes ...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae at JoVE.com

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Chromatine Immunoprecipitation (ChIP) van Histone modificaties van Saccharomyces cerevisiae

Hier beschrijven we een protocol voor het chromatine immunoprecipitation van gemodificeerde histonen uit de ontluikende gist Saccharomyces cerevisiae. Immunoprecipitated DNA wordt vervolgens gebruikt voor kwantitatieve PCR te ondervragen de overvloed en lokalisatie van Histon posttranslationele modificaties in het genoom.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

The overall goal of this chromatin immunoprecipation protocol is to determine the abundance and localization of histone modifications at defined regions throughout the budding yeast genome. This method can help answer key questions in chromatin regulation, such as how histone modifications are linked to gene expression control and how these modifications change in response to different environmental conditions. An extremely versatile protocol that can be used to investigate many different histone modifications and multiple E strains at one time.Though this method can provide insight into histone modifications, it can also be applied to investigating other chromatin bonding proteins through the use of epitope tags or available antibodies. Start this procedure by growing the yeast cells inoculate 10 milliliters of YPD medium with a single colony of each appropriate strain, and grow it 30 degrees celsius in a shaking incubator at 220 rpm overnight. On the following day after measuring the optical density at 600 nanometers or OD600 of the culture, dilute the culture to an OD600 of 0.2 in 100 mL of YPD in a new flask.Grow the cells at 30 degree celsius in a shaking incubator at 220 rpm until they reach mid log phase. When the cultures have reached mid log phase, add 2.7 milliliters of 37%formaldehyde directly to the medium in each flask for a final concentration of 1%formaldehyde. Transfer the flasks to a shaker at room temperature, and shake at 50 rpm for 15 minutes.Add five milliliters of 2.5 molar glycine to the medium and continue shaking at 50 rpm at room temperature for five minutes to quench the formaldehyde. Subsequently, spin down the cells and wash them as described in the text protocol. Remove the supernatant after the last spin and proceed to making yeast lysates.Re-suspend the cells in one milliliter of cold chromatin amino precipitation or ChIP Lysis buffer with 1 millimolar PMSF and 1:1000 dilution of a yeast protease inhibitor cocktail. Transfer the suspension to a 2.0 milliliter screw cap tube containing 200 microliters of glass beads. Transfer the cells to a bead-beating apparatus for a high speed agitation at four degree celsius.Bead-beat for 30 seconds and ice for one minute. Bead-beat a total of six times, keeping the cells on ice for at least one minute between each 30 second beat-beating. Using a gel loading tip, transfer the lysate to a 1.5 milliliter tube.Centrifuge the lysate at 15, 500 g at four degree celsius for 20 minutes. Discard the supernatant and re-suspend the pellet in 250 microliters of micrococcal nuclease digestion buffer by gently pipetting up and down. Add 2.5 microliters of micrococcal nuclease to each reaction.Mix gently by inverting four to six times and immediately incubate in a 37 degree celsius water bath for 20 minutes. After 20 minutes, stop the reaction by placing the tubes on ice, adding five milliliters of 0.5 molar EDTA for a final concentration of 10 millimolar EDTA and mixing gently by inversion. Centrifuge at 15, 500 g at four degree celsius for 15 minutes.Then, transfer each supernatant to a new 1.5 milliliter tube. For the most consistent results, it is critical to ensure that the same concentration of protein is used in each amino precipitation. Depending on the number of amino precipitation or IP reactions, make a master mix of each micrococcal nuclease released chromatin fraction and ChIP lysis buffer and allocate one milliliter containing 50 micrograms of total protein to individual tubes.Remove 100 microliters from each IP mix to process as the input sample. Freeze the input samples at 20 degree celsius for later use. It is also important to make sure the same amount of beads bound to the antibody is used for each immunoprecipitation.Using a wide board pipette tip, add 20 microliters of magnetic protein AG beads, pre-bound with antibody to each IP sample. Rotate the samples at eight rpm at four degree celsius for three hours. When the IP is done, place the tubes in a magnetic stand and let beads collect on the side of the tube.Use a pipette to remove the supernatant. Add one milliliter of ChIP lysis buffer to the beads and rotate at eight rpm at four degree celsius for five minutes. Then, place the tubes on the magnetic stand and remove the supernatant.In this manner, wash the beads successively with the following buffers for the number of times indicated. ChIP Lysis Buffer, High Salt Wash Buffer, LiCl/detergent Wash Buffer and TE.With the last TE wash, use 0.5 milliliters of TE to transfer most of the beads to a new tube, and then use another 0.5 milliliters of TE to wash the tube and transfer the remaining beads. Add 250 microliters of freshly made ChIP elution buffer to each tube and vortex the solution briefly.Rotate the samples at eight rpm at room temperature for 15 minutes. Place the tubes in the magnetic stand. Carefully transfer the supernatant to a new tube and avoid the beads.Add another 250 microliters of ChIP elution buffer to the beads. Rotate the samples at eight rpm at room temperature for 15 minutes. Carefully and without disturbing the beads, transfer the supernatant to the tube containing the first elution.Thaw the input samples collected earlier and add 400 microliters of ChIP elution buffer to each input sample. To reverse protein DNA cross links, add to both input and IP samples, 20 microliters of five molar sodium chloride, five microliters of glycogen and 12.5 microliters of Proteinase K.Mix well by inverting or flicking the tubes. Incubate the samples in a 65 degree celsius water bath overnight.To begin this procedure, make a QPCR master mix for each set of primers by combining the appropriate volumes of QPCR mix, the forward primer, and the reverse primer. Make DNA master mixes for each DNA sample where one reaction contains 0.5 microliters of DNA and 4.0 microliters of nuclease free water Add 5.5 microliters of the appropriate primer master mix to each well in a 384 well QPCR plate. Spin the plate at 200 g at room temperature for one minute.Then, add 4.5 microliters of the appropriate DNA master mix to each well. Seal the plate, and spin at 200 g at room temperature for one minute. Perform QPCR using a real time QPCR system with the following conditions, 95 degree celsius for three minutes 40 cycles of 95 degree celsius for 10 seconds and 55 degree celsius for 30 seconds and a melting curve of 65 degree celsius to 95 degree celsius with 0.5 degree celsius increments for five seconds.One key component of this protocol is optimizing the concentration of micrococcal nuclease used to digest the chromatin into soluble fragments. This example result from agarose gel electrophoresis of DNA isolated following digestion of the chromatin pellet with varying amounts of micrococcal nuclease shows that 2.5 microliters of enzyme at 20 units per microliter produced predominantly mononucleosomes. This representative ChIP experiment used antibodies against a histone methyl marker H3K4me2 and histone H3 in wild type and set1 knockout cells.Set1 knockout cells lack the methyl transferase that catalyzes the mark The bars indicate the relative enrichment of H3K4me2 at the indicated low sci. The wild type strain showed clear enrichment for H3K4me2 at the five prime end of two genes known to be direct targets of set1, PMA1 and ERG11 whereas no signal was observed in set1 knockout cells. As expected, there was no enrichment of the H3K4me2 mark at TEL07L, the expression of SPR3 has also been reported to be regulated by set1 but these results indicate the regulation is likely to occur by a different mechanism than at PMA1 or ERG11.Once mastered, this technique can be done within four days if it is performed properly. While attempting this procedure, it's important to remember to carefully test the antibody used for specificity with the proper controls. If available, both positive and negative control regions should be probed in the QPCR.Following this procedure, other methods like GP sequencing can also be performed in order to determine the genome by distribution of histone modifications. After watching this video, you should have a good understanding of how to consistently detect the levels of different histone modifications at unique genomic regions with well controlled experiments.

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Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

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Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription ...